Characterization of the 67 kDa laminin binding protein by Terry Hinz Landowski A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology Montana State University © Copyright by Terry Hinz Landowski (1994) Abstract: In order to successfully complete the process of metastasis, tumor cells must adhere to and invade the extracellular matrix basement membrane. As an essential step in the dissemination of metastatic disease, the adhesive event represents an opportunity for therapeutic intervention. Small synthetic peptides, which mimic the binding domain of an extracellular matrix ligand, have been shown to be effective in blocking the adhesion of tumor cells to basement membrane molecules, thereby terminating the metastatic process. However, before these agents can be effectively utilized in the treatment of human disease, the biological activities of their target proteins must be fully characterized. Cell surface expression of the 67 kDa laminin binding protein has been shown to correlate with the metastatic potential of many solid tumors. Unique structural features of the cDNA deduced amino acid sequence of this protein include the lack of a signal sequence for plasma membrane localization, and the absence of a hydrophobic domain characteristic of transmembrane proteins. In addition, the isolated protein displays an apparent molecular weight of 67 kDa, while the cDNA is sufficient to encode only a 32 kDa protein. The goal of this study was to determine the mode of cell surface association of the 67 kDa laminin binding protein, and to identify the mechanisms responsible for the discrepancy between the predicted and the observed molecular weight. The dhfr mutant CHO cell line, DG44CHO, was utilized as a homotypic overexpression. system in order to obtain sufficient protein for biochemical analyses. This system also provided an opportunity to assess the phenotypic affects of overexpression of the 67 kDa laminin binding protein. Surface expression of the 67 kDa laminin binding protein was found to be independent of mRNA levels in the overexpression system, indicating the possibility of a post translational regulation mechanism. Treatment with endoglycosidases had no apparent affect on the molecular weight of affinity purified protein, indicating that post translational modification with carbohydrates is not likely to be responsible for the molecular weight shift. Transesterification and hexane extraction of the 67 kDa laminin binding protein demonstrated the presence of covalently bound fatty acids. While the quantity of lipids isolated is not likely to be directly responsible for the observed molecular weight shift, they may provide a mechanism for membrane localization.  CHARACTERIZATION OF THE 67 kDa LAMININ BINDING PROTEIN by Terry Hinz Landowski A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana October 1994 D Z rT i s ' a a k 1) Il APPROVAL of a thesis submitted by Terry Hinz Landowski in This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies. Date Chairperson, Graduate Committee Approved for the Major Department Date Approved for the College of Graduate Studies / a y / 3 / f i I 39 of multiple adhesive sites on the 67 kDa laminin binding protein for laminin or other surface structure, possibly including proteoglycans. Peptide 11 and YIGSR A nine amino acid sequence, CDPGYIGSR, located in the P1 fragment of laminin-1, has been identified as a primary binding site for the 67 kDa laminin binding protein (Graf et al., 1987a). This sequence, named peptide 11, represents residues 925-933 of the /?1 chain of laminin-1, and is located within the EGF-Iike repeats of Domain III. Peptide 11 has been shown to inhibit the invasion of B16F10 or B16BL6 mouse melanoma cells through a reconstituted basement membrane matrix in vitro, and to inhibit experimental metastases in a lung colonization assay in vivo (Iwamoto et al., 1987; Ostheimer et al., 1992). In these assays, the amide form of the peptide consistently demonstrated greater inhibitory activity than did a peptide with a free carboxyl terminus, indicating the importance of the positive charge on the side chain on the terminal arginine residue. Anti-angiogenic activity of peptide 11 has also been demonstrated in vivo. In a study by Sakamoto etal. (1991), Peptide 11 was shown to suppress embryonic chorioallantoic membrane angiogenesis in a dose dependent manner. Intravenous administration of Peptide 11 in sarcoma bearing mice resulted in a significant reduction in the growth of the tumor. Clearly, this peptide is capable of specifically interfering with the interactions of tumor cells with basement membrane molecules, and represents a potential therapeutic lead. IThe minimum sequence required for inhibition of tumor cell adhesion to laminin-1 has been shown to reside in the carboxyl terminal 5 residues, YIGSR. This peptide also demonstrates activity in the inhibition of tumor cell invasion of a basement membrane matrix in vitro (Ostheimer et al., 1992), and inhibition of experimental metastasis in vivo (Iwamoto et al., 1987). However, the pentapeptide is not as efficient as the full nine residue sequence in eluting the 67 kDa laminin binding protein from a laminin affinity column (Graf et al., 1987b), presumably due to an inability of the short peptide sequence to maintain the appropriate conformation. Conservative substitutions and glycine-to-alanine substitution studies have demonstrated that the activity of Peptide 11 is highly dependent on the secondary structure of the peptide (Graf et al., 1987b; Ostheimer et al., 1992). The NMR-derived preferred solution structure of Peptide 11 demonstrates an "S" shaped conformation, which appears to be stabilized by a carbonyl interaction between the terminal arginine residue, and the backbone of the tyrosine residue. The substitution of d-alanine for gly-7 (CDGPYI(dA)SR) accommodates this conformation, and the substituted peptide has biological activity equal to that of Peptide 11 (Ostheimer et al., 1992). However, the substitution of l-alanine at the same position (CDPGYI(IA)SR) reduces the biological activity of the peptide to 50% of Peptide 11 activity, and the truncated sequence, IGSR is entirely inactive (Graf et al., 1987b). Likewise, the amino terminal cysteine appears to be required for optimal activity of Peptide 11, as the conservative substitution of serine for cysteine (SDPGYIGSR) is 40 y 11 h 41 inactive (J.R. Starkey, unpublished observations). The cysteine residue may not be a structural requirement, but rather, the cysteine residue may simply physically contact the receptor. Other laminin peptides with bioactivitv Other laminin derived bioactive peptides that have been identified include PDSGR, also in the P1 fragment (Kleinman et al., 1989), F-9, located in the globular Domain IV of the /?1 chain (Charonis et al., 1988), and IKVAV, located in the carboxyl terminal region of fragment ES (Kanemoto et al., 1990; Tashiro et al., 1989). PDSGR has been shown to inhibit lung colonization, although the activity is approximately 20% less than that demonstrated for Peptide 11 (Kleinman et al., 1989, J.R. Starkey, unpublished observations). No specific cell surface receptor has been identified for this sequence. The F-9 peptide, characterized by Charonis et al., was identified as a heparin binding sequence. This peptide was shown to inhibit the adhesion of cells to laminin, presumably by blocking an interaction between cell surface proteoglycan, and laminin. One of the most interesting bioactive peptides derived from the sequence of laminin, which perhaps has a great deal of relevance for tumor metastasis, is the sequence IKVAV. This peptide is located in the carboxyl region of the a chain within the ES fragment. IKVAV has been shown to initiate a signal transduction cascade in various cell types including melanoma (Kanemoto et al., 1990; Royce et al., 1992), and neural cells (Tashiro et al., 1989; Kubota et al., 1992). Cellular responses induced by this peptide include the secretion of Icollagenolytic enzymes by invasive carcinomas and neurite outgrowth, however, the specific receptors involved in the signal transduction by this peptide have not yet been fully characterized. Several proteins have been identified with adhesive activity for the ES fragment of laminin, including the integrins QePv and (Sonnenberg et a!., 1990; Lee et al., 1992), and other proteins with molecular weights in the range of 90-110 kDa which may also be members of the integrin family (Tashiro et al., 1989). However, the IKVAV peptide is but a small portion of the ES fragment, and may not be the specific adhesive site for these receptors, which require the coiled coil structure formed by all three laminin chains (Lissitzky et al., 1992). The 67 kDa laminin binding protein has not been shown to directly attach to this domain. Differential responses to the IKVAV peptide are seen in cells of different origins (Tashiro et al., 1989), indicating a highly regulated interaction between cells and this region of the laminin molecule. As such, it represents a valuable tool for the further investigation of laminin mediated signal transduction, but does not appear to involve the 67 kDa laminin binding protein. 42 Preview to the Experiments General project goals The general goal of the project which includes this study is the development and assessment of specific anti-metastatic therapies. Since the adhesion of tumor cells to the extracellular basement membrane is considered 43 to be critical to successful completion of the metastatic cascade, interference with this step represents an opportunity for therapeutic intervention. Small synthetic peptides which mimic the binding domain of a receptor-ligand interaction can specifically inhibit such an adhesive advent. As previously described, peptides of this design have been shown to be effective in reducing tumor growth and lung colony formation in experimental animals. These molecules have the added advantage of being small enough to evade immune detection, and so, do not elicit an antigenic response in the host. The major challenge in the design of synthetic peptides as therapeutic agents is their susceptibility to serum proteases, which results in a very short half life in vivo. Additionally, as small fractions of a much larger molecule, the synthetic peptide may experience minimal structural constraints, and may not stably represent the conformation of the amino acid sequence in its native context. If the peptide is to effectively mimic the binding domain of a ligand, and so, block the receptor- ligand interaction, tertiary structure is likely to be of key importance. The determination of the three dimensional structure when it is associated with its target receptor will allow the synthesis of organic compounds locked into the appropriate conformation. This approach has the potential to alleviate the problem of instability, as well as extend the in vivo half life of the molecule. However, care must be taken to ensure that the interaction of the synthetic peptide with its target receptor does not have unexpected adverse affects. Since cell surface receptors are typically used by cells to communicate 11 with their environment, any receptor-ligand interaction may have the potential to initiate a signal transduction cascade, evoking a cellular response. Many cellular activities are governed by the highly controlled interactions of molecules on the cell surface with those of the extracellular matrix. If the ligand-derived peptides physically interact with the receptor molecule in such a manner as to activate the cell, rather than simply blocking the adhesion, the results could be disastrous. Thus, it is imperative that the activities and mechanisms of the target molecule be well characterized before these agents can become fully useful in the treatment of human disease. As detailed in earlier sections of this paper, expression of the 67 kDa laminin binding protein has been shown to directly correlate with the metastatic potential of human tumors. Peptide 11, a specific laminin derived binding sequence for this protein, represents a potential anti-metastatic agent through its ability to interfere with the adhesion of tumor cells to a laminin substrate. In a collaborative project with investigators in the biochemistry department, studies are progressing to determine the secondary structure of peptide 11 in association with the 67 kDa laminin binding protein. This section of the study is intended to address the structural features of the 67 kDa laminin binding protein, and to begin to understand its biological activities. Unpublished results Since the metastatic character of the B16BL6 mouse melanoma cell line is well documented, this cell line was initially selected as an experimental model. 44 L I i i I Isolation of the 67 kDa laminin binding protein was carried out by the method of Wewer et ai (1986), and resulted in a pure preparation of high affinity laminin binding protein of approximately 65-67 kDa as determined by silver stained SDS- PAGE. Yield of this protein was approximately 10 //g/ml packed cell volume, and was increased to approximately SO/vg/ml when isolated from cells which had been previously selected for their ability to traverse an EHS basement membrane barrier (B16BL6/EHSx3). Amino acid analysis of the affinity purified 67 kDa laminin binding protein by HPLC of PITC labelled hydrolysates by the method of Heinrickson and Meredith (1984), indicates a composition compatible with the sequence published by Yow etal. (Table 1.1). This protein is clearly not the same as that identified by Mecham et al. (1989), nor is it consistent with the amino acid composition of the laminin binding protein isolated from muscle cells by Lesot et al. (1983). Replicate HPLC analysis of protein hydrolyzed for 24, 48, or 72 hours demonstrated complete hydrolysis at 48 hours. All samples were noted to contain several large peaks that eluted at a high concentration of organic solvent, and most probably represent non-protein material. At least one of these peaks is likely to represent excess PITC. Others may represent non-protein molecules associated with the 67 kDa laminin binding protein. Computer comparison of the cDNA predicted amino acid sequence with other proteins having well characterized structural domains, was carried out to identify domains of the 67 kDa laminin binding protein which might indicate 45 Table 1.1: Amino Acid Ratios for Various Laminin Binding Proteins Aspartactin Hall et al. Muscle LB-68 Lesot et al. Elastin binding Mecham et al. Colon cDNA Yow et al. B16BL6 67 kDa receptor Ser/Gly 0.69 1.05 1.03 0.93 0.97 Ala/His 3.0 2.6 2.2 9.25 ND* Ala/Asp 0.26 0.84 1.09 2.45 2.45 Val/Glu 0.54 0.184 0.16 0.76 0.98 *Histidine not separated 11 It biological activities of the protein. The ALIMAT program, developed by Argos (1987), identifies regions within compared protein sequences with similar specific structural characteristics. These characteristics have been shown, on an empirical basis, to indicate similar functional domains. Using this program, several regions were identified which may be of functional significance. When combined with the MOTIF program comparing the laminin binding protein sequence with functional motifs in the Prosite database, the ALIMAT homologies gained additional significance in some cases. Previously published reports have indicated that the 67 kDa laminin binding protein may interact with the cytoskeleton, specifically with actin (Brown et al., 1983; Cody and Wicha, 1986). The amino acid sequence was therefore compared to several known actin binding proteins. Most of these proteins showed no homologies with the laminin binding protein sequence. However, the sequence did show regions of strong structural homology with both chicken alpha-actinin and plasma gelsolin. The 75 amino-terminal residues of the laminin binding protein sequence align with residues 669-743 of alpha-actinin, with 14 identical and 13 conserved residues. This region of alpha-actinin is known to be required for dimerization of the molecule during the polymerization of F-actin (Simonidze et al., 1988), and is also the domain known to contain the region which interacts with the cytoplasmic domain of the # integrin chain (Otey et al., 1990). Gelsolin is an actin modulating protein. It also demonstrated an 85 47 U-Il 48 residue region with a high degree of similarity to the laminin binding protein. Gelsolin has been shown to contain two actin-binding domains, one which is calcium dependent and one which is not (Kwiatkowski et al., 1986). The non­ calcium dependent actin-binding region has been identified as residues 421-738. This domain encompasses the region of homology with the laminin binding protein, residues 470-540, suggesting the possibility that the laminin binding protein may also be capable of interacting with actin through a similar structural domain. A strong structural homology was found with th e # integrin chain, aligning residues 203-251 of the laminin binding protein sequence with residues 195-243 of the chicken # sequence. This domain lies some 15 residues to the carboxyl side of a conserved site in the p integrins which has been shown to cross-link the RGD motif of the ligand b y # (gpllla) (D’Souza et al., 1988). T h e # integrin chain and the laminin binding protein are oppositely oriented, resulting in the homologous regions of both proteins being located extracellularly. In the laminin binding protein sequence, the homologous region encompasses a highly charged domain predicted to form an alpha helix, which is demonstrated in this study to interact with laminin. Phofbol esters have been shown to enhance the laminin binding activity of mast cells (Thompson et al., 1990), NIH 3T3 cells (Kato et al., 1988), and neutrophils (Yoon et al., 1987), as well as to stimulate the metastatic capacity of tumorigenic cells (Gopalakrishna and Barsky, 1988; Nishizuka, 1984). 49 Furthermore, one of the well characterized responses to phorbol esters is that of protein phosphorylation via activation of nucleotide binding proteins (Parker et al., 1986). We therefore compared the sequence of the 67 kDa laminin binding protein to several kinases, including some known to be stimulated by phorbol esters. These included protein kinase C, lactic dehydrogenase, alcohol dehydrogenase, and adenylate kinase. The degree of homology seen with each of these proteins was not particularly striking. However, what was significant was the observation that residues 1-50 of the laminin binding protein consistently aligned with the specific region known to contact the nucleotide in each of these proteins. In an analysis of the critical residues required for orientation of the nucleotide, all consensus residues are satisfied. Several potential phosphorylation motifs are identified by comparison with the Prosite database. Threonine28 in the laminin binding protein sequence is a potential phosphorylation site in a casein kinase phosphorylation motif (Pinna, 1990), serine 43 is predicted as a potential c-AMP dependent phosphorylation site, and tyrosine47 was predicted to be a tyrosine kinase phosphorylation site. Comparison of the laminin binding protein sequence to the Pz integrin subunit of platelets (gpllla) by the ALIMAT program demonstrated a strong structural homology of residues 1-50 with the cytoplasmic tail of/?3, which has been shown to be phosphorylated on tyrosine in response to ligand binding (Elmore et al., 1990). 50 No significant similarities were detected with proteins chosen to represent several other classes of cell surface proteins, including some with well characterized transmembrane domains, members of the EGF-Iike and CAM families of receptors, and mammalian lectins (Table 1.2). Table 1.2: Representative proteins compared to the deduced amino acid sequence of the 67 kd high affinity laminin binding protein which demonstrated no significant structural homology Transmembrane Receptors Lectins M13 Coat protein (J02461) Bacteriorhodopsin (M11720) Porin (E.coli) (M74489) Cytochrome b (X12783) Calcitonin (J00109) Glucagon (X05388) ELAM-1 (M24736) ICAM-1 (M31585) I CAM-2 (X15606) PDGF receptor (M34480) gpllb (M34480) gpllla (M35999) Macrophage lectin (M35368) Rat hepatic lectin (K02817) Galactoside binding (X16074) Numbers in () are Genbank/EMBL accession numbers (releases 69/27) 52 References Albelda, S.M. (1993). Role of integrin and other cell adhesion molecules in tumor progression and metastasis. Lab. Invest. 68, 4-17. Argos, P. (1987). A sensitive procedure to compare amino acid sequences. J. Mol. Biol. 193, 385-396. Aumailley, M., Nurcombe, V., Edgar, D., Paulsson, M., and Timpl, R. (1987). 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This adhesion and invasion is accomplished by the use of cell surface receptors specific for basement membrane components (for reviews, see Aznavoorian etal., 1993; Stetler-Stevenson etal., 1993). Several laminin binding receptors have been identified, one of which is a high affinity laminin binding protein that migrates with an apparent molecular weight of 67 kDa on SDS-PAGE. This 67 kDa high affinity laminin binding protein was initially isolated from a highly metastatic breast carcinoma by immunoprecipitation with a function blocking antibody (Barsky et al., 1984). Using this antibody as a probe, a partial cDNA sequence was identified in a human endothelial cell expression library. The sequence was found to completely overlap that of a mRNA which was preferentially over expressed in highly metastatic colon carcinoma as compared to adjacent normal colonic epithelium (Yow et al., 1988). This 67 kDa laminin binding protein has since been shown to be expressed at high levels on lung carcinoma (Satoh et al., 1992), melanoma (Vacca et al., 1993), breast carcinoma (IVIartignone et al., 1992), and several other invasive solid tumors. The 67 kDa laminin binding protein is expressed at 65 66 relatively low levels in benign and normal tissues, and its level of expression correlates very well with the metastatic phenotype of the tumor (Mafune et al., 1990). Since adhesion to the basement membrane is considered to be a critical step in the metastatic cascade, the laminin binding function of this protein makes it a potential target for therapeutic intervention in metastatic disease as well as a useful prognostic indicator. The full length nucleotide sequence of the high affinity laminin binding protein is sufficient to encode a protein with predicted molecular weight of approximately 32 kDa, while the laminin affinity isolated protein migrates with an apparent molecular weight of 67 kDa on reduced SDS-PAGE. The mechanism of this apparent molecular weight shift is not readily apparent, and is somewhat controversial. Although the original data have not been published, one group has reported that an E. coli expression product of a full length clone fails to bind to a laminin affinity column (discussed in Mecham, 1991). The 67 kDa laminin binding protein, when isolated from tumor cell membrane extracts, initially binds with high affinity to a Iaminin-Sepharose column. However, following laminin affinity purification, the protein does not re-bind to laminin with the same high affinity (M. Sobel and H. Kleinman, personal communication, this manuscript). More recently, another group has reported that a fusion protein of the 67 kDa laminin binding protein cDNA with TrpE which was expressed in E coli did bind to a laminin-sepharose affinity column (Siyanova, 1992). However, it is not clear whether the TrpE secretory signal sequence was present or not, or what 67 molecular weight the affinity isolated product displayed. Taken together, these data would imply that there are unidentified structural characteristics of the laminin binding protein which play a critical role in its adhesion to laminin. Rao et al. (1989) have reported the existence of a 37 kDa precursor in mouse NIH3T3 cells which can be chased into a 67 kDa product. These proteins were immunoprecipitable with an antibody raised to a synthetic peptide corresponding to the amino terminal region of the cDNA sequence. However, they were unable to identify the mechanisms underlying the molecular weight shift. Although there are 14 serine and 23 threonine residues in the putative amino acid sequence of the cDNA clone which might be modified with O-Iinked gIycosylation, there are no consensus sites for N-Iinked carbohydrates. Analysis of the predicted amino acid sequence of the protein reveals no simple hydrophobic domain characteristic of a transmembrane region, leading to some speculation that the protein may not actually be a cell surface receptor (Grosso et al., 1991). Several groups, in independent, and often unrelated studies, have identified proteins in yeast (Davis et al., 1992), hydra (Keppel and Schaller, 1991), and Drosophila (Melnick et al., 1993), with extensive sequence similarity to the 67 kDa laminin binding protein. These proteins are not cell surface proteins, but are apparently components of the translational machinery. They have not been shown to possess post translational modifications, and are associated with ribosomes. While the cDNA sequence homology of these proteins with the mammalian laminin binding protein is very high in the amino 68 terminal half of the molecule, the carboxyl terminal domain, which has previously been identified to contain the region important for association with laminin, is much less highly conserved. In the present paper, we hypothesize that the 67 kDa laminin binding protein has two functions in mammalian cells. One function may be intracellular, perhaps similar to that seen in lower species. The second function, which may be dependent on post translational modifications, is responsible for its surface localization and laminin binding characteristics. Recently, many proteins have been identified with post translational modifications in which lipid moieties are covalently attached to the protein (for review see Schmidt, 1989; Mcllhinney, 1990). These acylproteins have varied functions in the cell, including playing key roles in adhesion and signalling. Several mechanistic roles for the lipid modifications have been proposed, including localization of the protein to a lipid bilayer membrane. Lipid modifications have also been shown to modify proteimprotein interactions. Finally, acyl modifying groups may be active in generating a second messenger in signal transduction cascades (reviewed in Magee, 1990; Chow et al., 1992). We report here that the 67 kDa high affinity laminin binding protein is acylated by the fatty acids palmitate, oleate and stearate. These fatty acids appear to be covalently associated via an ester or thioester linkage and, in the absence of other known mechanisms, are likely to be responsible for targeting the protein to the cell surface, where it can participate in the adhesion of cells I I 69 to the extracellular matrix. From our results, we also postulate that associations with other molecule(s) are required to mediate high affinity ligand binding. Materials and Methods Expression of the laminin binding protein A cDNA clone of the laminin binding protein, isolated from a hamster expression library, was kindly provided by Dr. James Strauss of the California Technical Institute in the pcDNAI/neo vector (Invitrogen) (pcLR). This expression vector utilizes a CMV promoter and G418 selectable marker (Wang et al., 1992). An expression vector for the dihydrofolate reductase (dhfr) gene, under the control of an SV40 promoter, was obtained from ATCC (Rockville, MD). Both plasmids were simultaneously transfected by calcium phosphate precipitation (Chen and Okayama, 1988) into DG44CHO cells which were obtained from Dr. Lawrence Chasin of Columbia University. These cells are double negative mutants for the dhfr gene, and rely on the presence of exogenous hypoxanthine and thymidine (HT) in the culture media for proliferation (Urlaub and Chasin, 1980). Cotransfection of the pcLR and dhfr plasmids was carried out at 1:1, 5:1, 10:1, and 20:1 ratios of pcLR:dhfr with a total of 20 //g of DNA per 10s cells. Untransfected cells were grown in crMEM (Sigma) containing 10% FBS (Intergen) and supplemented with MT, 5 //g/ml bovine insulin, 10 mM L-glutamate, and 100 U/L each of penicillin and streptomycin. Selection of transfectants was carried 70 out in the same medium without MT, containing 10% dialyzed FBS1 and 400 //g/ml G418 (Gibco). Methotrexate treatment of dhfr/pcLR transfected cells was initiated at a concentration of 0.03 ^m, and increased by 0.02-0.05 increments approximately every 5th cell passage. Amplification of pcLR expression was monitored by Northern analysis and FACScan (Landowski et a!., manuscript submitted). For metabolic labelling of gIycosyl phosphatidylinositol (GPI) modified proteins, 14C-ethanolamine (NEN) was added to the culture medium at a concentration of 0.1 //Ci/ml. Labelling of cells was carried out for 16 hours at 37°C. Laminin binding protein isolation The laminin binding protein was extracted by the method of Wewer et al. (Wewer et al., 1986) with minor modifications. DG44CHO cells, selected for expression of the pcLR and dhfr plasmids, were harvested by rinsing twice with Puck’s CMF Saline G solution, approximately 10 minutes each at 37°C, and a third time with the same solution containing 0.01 mM EGTA until the cells detached. This harvesting technique was used to avoid the use of trypsin and preserve membrane proteins. Cells were centrifuged for 10 minutes at 500 x g, the pellet resuspended in 10 volumes of CMF-Dulbecco’s PBS (v/v), and centrifuged again under the same conditions. Included in this, and all subsequent buffers, were the protease inhibitors 50 //g/ml PMSF, 1mM N- ethylmaleamide, and 5 mM benzamidine. Cells were then suspended in 2 71 volumes of ice cold 25mM Tris/0.3 M sucrose, pH 7.4 (v/v), and sonicated with 4-5 x 5 second bursts on ice using a Fisher Sonic Dismembrator, Model 50 at 50% power. Nuclei, cytoskeletal proteins and unbroken cells were pelleted by centrifugation at 500 x g for 10 minutes at 4°C, the supernatant collected, and the pellet resuspended in 5 volumes of Tris/sucrpse buffer. Sonication was repeated, and the supernatant again collected and added to the first supernatant. These steps were repeated three times, or until the vast majority of the cells appeared lysed under microscopic examination. The membrane fraction was then collected on a sucrose cushion by centrifugation at 143,000 x g for 90 minutes at 4°C, and adjusted to a protein concentration of approximately 1 mg/ml with 25 mM Tris/150 mM NaCI/1 mM CaCI2ZS mM MgCI2, pH 7.4 buffer. An equal volume of the same buffer containing 1% NP-40 detergent was added, and the solution was rotated end over end for 12-16 hours at 4°C. Insoluble material was pelleted by centrifugation at 200,000 x g for 60 minutes. The supernatant was combined with a laminin-sepharose column which had been pre-equilibrated with 10 column volumes of 25 mM Tris/150 mM NaCI/1 mM CaCI2ZS mM MgCI2 + 0.05% NP-40 followed by the same buffer containing 400 mM NaCI. Laminin- sepharose affinity columns were prepared by coupling EHS laminin (Gibco) to CnBr-activated Sepharose 4B at a concentration of 0.5 mg/ml according to the procedure recommended by the manufacturer (Pharmacia). After application of the detergent extract, the column was washed with 50 mM Tris/0.1 M NaCI, pH 7.4 and the UV absorbance was monitored at 214 nm until it returned to 72 baseline. Approximately one column volume of 50 mM Tris/1.0 M NaCI, pH 7.4, was added and the column allowed to stand 15-20 minutes prior to elution of the high affinity binding protein with the latter elution buffer. Isolated laminin binding protein was labelled with 125I by the Iactoperoxidase method using Enzymobeads (Bio-Rad), and labelled protein was separated from free iodine by chromatography on a BioGeI P6 column with 1 M NaH2PO4, pH 7.4. Specific activity of the labelled protein was 1.5 x 106 cpm/mg. Antibody production and western blotting A peptide corresponding to residues 205-229 of the amino acid sequence of the 67 kDa laminin binding protein (RDPEEIEKEEQAAAEKAVTKEEFQG) was synthesized using standard Fmoc chemistry on a Milligen 9050 automated peptide synthesizer. Following purification by reverse phase HPLC and assessment of purity with electrospray mass spectrometry, the peptide was conjugated to KLH (Sigma) by glutaraldehyde crosslinking with 0.2% glutaraldehyde for 2 hours at room temperature. The solution was dialyzed against Dulbecco’s PBS for 48 hours with 4 changes of dialysate to remove glutaraldehyde and free peptide prior to immunization. New Zealand white rabbits were injected with 1 mg of peptide-KLH and Freund’s complete adjuvant at multiple subcutaneous sites, and boosted every two weeks with peptide-KLH and incomplete Freund’s. Thirty days later a test bleed was obtained and the anti-peptide activity titered by ELISA using KLH, peptide-KLH, and peptide conjugated to ovalbumin as target antigens. The polyclonal antiserum was purified on a Protein A column (Pierce) and the majority of the anti-KLH activity was removed with a KLH-Sepharose column. Affinity isolated laminin binding protein was electrophoresed on a 10% SDS-PAGE gel and transferred to PVDF membrane (Bio-Rad) using a semi-dry blotter (Ellard Instrumentation). We found that the 67 kDa protein did not adhere well to PVDF membrane, therefore, the filter was fixed in Dulbecco’s PBS containing 0.02% glutaraldehyde for 2 hours at room temperature prior to washing and blocking with 5% non-fat dry milk and 0.1% Tween-20 in PBS. To ensure that the glutaraldehyde fixation was not introducing false positive results, fixation in 10% acetic acid/20% methanol was also used, and found to give identical results. Antibody detection was performed using alkaline phosphatase conjugated goat anti-rabbit antibody (Bio-Rad) and BCIP/NBT chromogenic substrate (Kirkegaard Perry Inc.). Determination of molecular weight Determination of the molecular weight of the affinity isolated laminin binding protein was performed by Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI TOF-MS) on a Vestec instrument. One //I of 0.5 mg/ml laminin binding protein solution in 50 mM Tris/1.0 M NaCI was applied to a sample well, along with 1 //I of a saturated water solution of the UV sensitizer, sinapinic acid (Aldrich). Samples were air dried and subjected to 5 ns flashes from a 366 nM nitrogen laser. External mass calibration was carried out with cytochrome c, bacteriorhodopsin, and ovalbumin. IL-I 74 Lipid Characterization Affinity purified 67 kDa laminin binding protein was frozen and Iyophilized to dryness. Fifty //g of dry weight protein was then dispersed in 200 //J of Methyl Prep Il (AIItech) and transesterified at 65°C for 60 or 90 minutes. The reaction was quenched by the addition of 200 //I of double distilled H2O, and 100 //I of methanol containing 20//g/m l butylated hydroxytoluene, and the fatty acid methyl esters were extracted with hexane. The Methyl Prep Il reagent converts esterified fatty acids to methyl esters, and any non-esterified fatty acids that may be present partition into the aqueous phase as (m-trifluoromethylphenyl) trimethylammonium salts (McCreary et al., 1978). The hexane extracted fatty acid methyl esters were then dried down under a stream of argon, and redissolved in 20 //I of iso-octane. Gas chromatographic analyses were performed with a Hewlett-Packard Model 5890 Series Il gas chromatograph equipped with a split injector and flame ionization detector, using a DB-8 column. Temperatures used were: injector, 250°C; column, 100qC to SOO0C at S0CZmin. The affinity isolation buffer plus NP-40 was similarly treated as a control for external lipid contamination. In separate assays, Iyophilized laminin binding protein samples were first washed with methylene chloride, and the wash was treated with Methyl Prep Il and analyzed by gas chromatography as described above. The methylene chloride extraction was carried out to test for the presence of non-covalently associated lipids. I I W 75 Glvcosidase treatments Affinity purified 67 kDa laminin binding protein was treated with Endo-F (Boehringer-Mannheim), Neuraminidase (Boehringer-Mannheim) and O- Glycanase (Genzyme) under conditions recommended by the manufacturers. Endo-F treatment was carried out at 37°C in 10 mM NaHPO4 with 1 U enzyme per mg protein. For O-Glycanase treatment, 125I labeled 67 kDa laminin binding protein was dialyzed into 10 mM calcium acetate/20 mM sodium cacodylate buffer. Duplicate samples of 5 0 //I each (0.5 mg/ml) were denatured with 0.1% SDS at SO0C for 5 minutes, and a 6 fold excess of NP-40 was added to reduce the concentration of SDS prior to treatment with 0.35 U Neuraminidase at 37°C for 2 hours. O-glycanase was added to a concentration of 0.1 U///g protein, and the incubation carried out for an additional 2 hours or overnight. Triton X-114 solubility To assess the potential membrane association of the protein, the method of Bordier (Bordier, 1981) was used to determine Triton X-114 solubility. Affinity purified, 125I-IabeIIed laminin binding protein was dialyzed to 10 mM Tris/150 mM NaCI, pH 7.4, and dissolved in precondensed 1.0% Triton X-114 (Sigma) in the same buffer at O0C. The samples were then warmed to SO0C for 5 minutes and the phases separated by centrifugation at 10,000 x g on a 6% sucrose cushion at room temperature. The upper aqueous phase was removed and brought to 0.5% Triton X-114, cleared on ice, and again brought to 30°C for five minutes. This sample was reapplied to the 6% sucrose cushion, and centrifuged to 76 separate the phases. Aqueous and detergent-rich phases were then analyzed by electrophoresis on 10% SDS-PAGE and the radioactive components were visualized on a Molecular Dynamics™ phosphorimager using ImageQuant™ software. Reconstitution of laminin binding Affinity purified 125I labelled laminin binding protein was dialyzed back to the laminin affinity column binding buffer, 25 mM Tris/150 mM NaCI/ 3 mM MgCI2ZI mM CaCI2, pH 7.4. In one experiment, this solution was brought to 0.05% NP-40 and reapplied directly to a laminin-sepharose column. In a second experiment, the solution was reconstituted with fractions 1 and 4 of the 50 mM Tris/0.1 M NaCI wash from a previous laminin binding protein extraction. These fractions were chosen because fraction 1 contains the majority of the NP-40 soluble cellular proteins that do not bind to laminin directly, while fraction 4 includes a "shoulder" which was consistently noted on the low ionic strength peak, and may include components which bind to laminin with intermediate affinity. The reconstituted solution was brought to 0.05% NP-40, and reapplied to a laminin-sepharose affinity column equilibrated as for the initial purification. Column elution was carried out exactly as for the de novo isolation procedure for the high affinity laminin binding protein. 77 Results Expression and identification of the laminin binding pro te in Since other labs have reported difficulties in obtaining a high affinity laminin binding product in artificial expression systems, we postulated that post translational modification may be required to obtain a fully functional protein. Post translational modifications may be species specific, or may be determined by the non-coding sequences in the transfected plasmid. We therefore chose to transfect CHO cells with the hamster cDNA clone for the laminin binding protein. The hamster protein was identified by Wang et al. as a cell surface receptor for the Sindbis virus (Wang et al., 1992). The cDNA shows 99% identity with the mouse cDNA (Rao et al., 1989) and 96% identity with the human cDNA (Yow et al., 1988). Only two amino acids differ with each species. Northern analysis of transfected cell populations demonstrated two mRNA products, one an endogenous product which was also found in untransfected controls, and a second at a slightly higher molecular weight consistent with the expected product of the transfected plasmid (Landowski et al, manuscript submitted). Although the mechanism is not well understood, it has been shown that methotrexate causes reduplication of transfected dhfr plasmids and simultaneous amplification of any cotransfected plasmid (Kaufman and Sharp, 1982). Using this protocol, we were able to achieve amplification of the transfected gene and to increase expression of the transfected plasmid by approximately 10 fold as measured by quantitative analysis of densitometry. The protein product isolated by laminin affinity from these transfected cell lines was also increased by approximately 10 fold, resulting in a yield of approximately 300 //g of purified laminin binding protein per 8 ml of packed cell volume (Fig. 2.1a). The identity of the laminin binding protein was confirmed by Western blot using a sequence specific polyclonal antiserum (Fig. 2.1b). This antibody stained the 67 kDa laminin affinity isolated protein from the transfected cell lines, as well as laminin affinity isolated proteins from the EHS tumor and B16BL6 mouse melanoma cells. Antibody binding on Western blots could be completely inhibited by the presence of 1 mg/ml of the immunizing peptide. The antiserum failed to react with bovine serum albumin, ovalbumin, or any other protein tested. The MALDI-TOF MS measurement of molecular weight is compatible with the apparent MW observed bv SDSrPAGE Since SDS-PAGE provides apparent molecular weights, and mobility artifacts are common with membrane proteins, the true molecular weight of the affinity isolated material was determined by MALDI-TOF mass spectrometry. Mass spectra of the protein indicated the major molecular species at a mass of 66.7 kDa. Minor peaks were identified at 33 kDa, 133 kDa, and 201 kDa (Fig. 2.2). The calibration of the mass spectrometer was with external standards, and the accuracy of the molecular weight determination is expected to be in the range of + 0.2% (Aitken, 1992). To determine if disulfide bonding is involved in the shift in molecular weight from the cDNA predicted 32 kDa to the 67 kDa apparent MW on SDS-PAGE, 125I labelled laminin binding protein was subjected 78 79 a. 94 kDa L i 67 kDa 45 kDa 32 kDa i ‘ » •» I I f » * I • 29 kDa b. 140 kDa 87 kDa -I •. ■ -VV-IUSWik': 48 kDa ' # 33 kDa « ' - y # 29 k D a # 21 kDa I K,ii: ■ -i" ■•' V • i S S t :IiiE f l : . . Figure 2.1. a) Silver stained SDS-PAGE demonstrates that the 67 kDa laminin binding protein is isolated in pure form by elution from a Iaminin-Sepharose column by high ionic strength solution. Lane 1, molecular weight standards (Pharmacia); Lane 2, starting membrane detergent extracts applied to the Iaminin-Sepharose column; Lane 3, high salt eluate of DG44CHO pcLR/dhfr detergent extracts from Iaminin-Sepharose column. b) Polyclonal antiserum raised to a synthetic peptide derived from the deduced amino acid sequence of the 67 kDa laminin binding protein recognizes the affinity isolated product on a Western blot. Lane 1, prestained molecular weight standards (Bio-Rad); Lane 2, 67 kDa laminin binding protein probed with the sequence specific anti-peptide 205-229 antibody. 80 300- 13JI<6.6 200 - 100- 31202 49413 71793 m/z Figure 2.2. Affinity purified 67 kDa laminin binding protein was subjected to MALDI-TOF mass spectrometry for accurate molecular weight determination, and found to be 66.7 kDa. The accuracy of this method is +0.2%, with external calibration carried out using cytochrome c, bacteriorhodopsin, and ovalbumin. The insert demonstrates the relative quantities of the minor molecular weight species seen at 33 kDa, 133 kDa, and 201 kDa. .81 to reduction with 1 M DTT at SO0C for 1 hour. This treatment did not affect the apparent molecular weight of the protein on SDS-PAGE (Fig. 2.3). This finding is consistent with the report by Rao etal. (1989). Treatment of the material with 10 mM DTT followed by MALDI-TOF MS also did not significantly change the relative quantities of the four peaks obtained (data not shown). The 67 kDa laminin binding protein is covalently associated with lipids, but does not appear to be modified by carbohydrates The treatment of affinity isolated 67 kDa laminin binding protein with O- Glycanase, or O-Glycanase and Neuraminidase had no effect on the apparent molecular weight of 125I-IabeIIed protein (Fig. 2.4). Consistent with the absence of consensus sites for N-Iinked glycosylation, no molecular weight shift was seen when the affinity isolated protein was treated with Endo-F (data not shown). Affinity isolated laminin binding protein was dialyzed against double distilled H2O to remove buffer salts and reduce detergent remaining from the extraction procedure prior to lyophilization. The Iyophilized material was analyzed on SDS- PAGE and showed the same apparent molecular weight as the starting material (data not shown). Samples of 50 //g of protein (dry weight) were transesterified with the alkaline methanolic reagent Methyl Prep II, The resulting fatty acid methyl esters were hexane extracted and subjected to GC and GC-MS. Comparison of peaks observed in electron ionization GC-MS with a library of mass spectrometry data enabled us to identify three lipid moieties: palmitate, stearate, and oleate (Figs. 2.5 and 2.6). Semi-quantitative comparison of the lipids extracted from the 67 kDa laminin binding protein with external standards 82 140 kDa I 5 y 29kDa .^!H SriJr1 4 MW Figure 2.3. Reduction of 67 kDa laminin binding protein with /?-mercaptoethanol or dithiothreitol has no effect on the apparent molecular weight of the protein on 10% SDS-PAGE. 125I-IabeIIed laminin binding protein isolated by Iaminin- Sepharose affinity chromatography is used in all cases. Lane 1, /?-ME treatment for 2 hours at SO0C . Lanes 2 and 3, DTT treatment for 2 hours at room temperature and 80°C. Lane 4, starting material. 83 IHli 140 kDa 87 kDa 48 kDa 33 kDa 29 kDa 1 2 3 4 5 6 7 MW Figure 2.4. The molecular weight shift from a cDNA predicted polypeptide of 32 kDa to the observed 67 kDa of affinity isolated laminin binding protein does not appear to be mediated by gIycosylation. Treatment with Neuraminidase, or Neuraminidase and O-Glycanase has no effect on the apparent molecular weight of affinity purified 67 kDa laminin binding protein. 125I-IabeIIed laminin binding protein is used in all cases, and experiments are analyzed by SDS-PAGE. Lanes 1 and 2, Neuraminidase treatment for 60 minutes (1) or 2 hours (2); Lanes 3 and 4, Neuraminidase followed by O-Glycanase treatment for 2 hours (3) or for 16 hours (4); Lanes 5 and 6, 67 kDa laminin binding protein denatured at SO0C with 0.1% SDS to control for detergent effects; Lane 7, untreated starting material. The positions of prestained molecular standards were marked with 14C ink prior to exposure of the phosphorimaging screen. 84 indicated approximately 0.05 //g of palmitate, 0.04 //g of stearate, and 0.02 //g of oleate per 50 //g protein sample. Transesterification at room temperature resulted in a significantly lower yield of palmitate, and the stearate and oleate were not detected in all experiments under these conditions. Organic solvent extraction and esterification of the extracts of the isolated protein yielded no detectable fatty acid methyl esters. Analyses of Iyophilized laminin affinity elution buffer containing NP-40 were entirely negative for fatty acids, as were methylene chloride wash extracts. While our data are in fairly good agreement with a 1:1 molar ratio for each lipid species identified, this acylation is not likely to be sufficient to account for the entire molecular weight shift from the predicted 32 kDa to the observed 67 kDa. Lipids identified with the 67 kDa laminin binding protein are not associated via a alvcosvl-phosphatidvlinositol (GPh structure Lipid modifications of plasma membrane bound proteins are frequently found to be attached by a GPI moiety. Two separate assays were used to determine whether the lipids we identified were present as part of a glycosyl- phosphatidylinositol structure. Temperature-induced phase separation in Triton X-114 showed that the 125I labelled laminin receptor partitioned primarily into the aqueous phase (Fig. 2.7). This finding is compatible with the predicted hydrophobicity index of the cDNA deduced amino acid sequence (Rao et al., 1989), but unlikely for a GPI-tailed protein. Metabolic labelling of the 67 kDa laminin binding protein with 14C-EthanoIamine in the DG44CHO pcLR/dhfr overexpression system did not lead to incorporation of detectable levels of 85 TIC: TLLR5-1.DMHindance 450000 400000 ■ 350000 - 300000 - 250000 - 20 0 00 0 ■ 150000 100000 - 50000 - 30.0028.0026.0024.002 2 . 0 02 0 . 0 0rime -> 18.00 Figure 2.5. Affinity isolated protein was transesterified by alkaline methanolysis, the fatty acid methyl esters extracted with hexane, dried down under argon, and redissolved in iso-octane for GC analysis. Peak A was identified as palmitate (Figure 2.6, panel A), Peak B was identified as oleate (Figure 2.6, panel B) and Peak C was shown to be stearate (Figure 2.6, panel C) The two small peaks indicated by * were identified as plasticizers. Scan 1538 (20.278 ram): TLLR 5-I. D (*) 9000 8000 7000 - 6000 - 5000 4000 3000 1000 Abundance Scan 1834 (23.598 min): TLLR5-I.D (*) 9000 8000 7000 6000 5000 4000 3000 2 000 1 000 60 80 100 120 140 160 180 200 220 240 260 280j / Z - > Scan 1887 (24.192 min): TLLR5-1.D (*) 9000 8000 - 7000 5000 4000 3000 2 000 1000 100 120 140 160 180 200 220 240 260 280 300M/Z -> Figure 2.6. Mass spectrometry identification of fatty acid methyl esters. Peak A, palmitate; Peak B, oleate; Peak C, Stearate. 87 1 2 3 4 MW Figure 2.7. Partitioning of affinity purified laminin binding protein into the aqueous phase of a Triton X -114 solubility assay indicates that it is not likely to be modified by a glycosyl-phosphatidylinositol tail. 125I-IabeIIed protein was subjected to temperature induced phase separation at O0 and 30°C, and separated on a sucrose cushion. Lane 1, 1st detergent phase; Lane 2, 2nd detergent phase; Lane 3, aqueous phase; Lane 4, untreated protein; M, prestained molecular weight markers (Bio-Rad) marked with 14C ink. 88 radioactive label into the affinity isolated product. Extended exposure of the phosphorimage screen indicated that, while specific activity of the labelling was relatively low, it was sufficient for detection. Non-laminin binding fractions of the affinity purification protocol showed a low level of radioactivity derived from the 14C-ethanolamine, some of which was clearly associated with a protein of approximately 40-45 kDa on SDS-PAGE (data not shown). High affinity laminin binding is modulated bv accessory factors When 125I-IabeIIed laminin binding protein was reapplied to a Iaminin- Sepharose column under conditions identical to its initial purification, approximately one half of the applied activity was recovered in the unbound column effluent (Fig. 2.8). The remaining bound activity was entirely eluted from the column with a low ionic strength buffer. During the initial purification of the 67 kDa laminin binding protein, this low ionic strength buffer is used to remove all loosely associated material, and is found to contain many proteins with a wide range of molecular weights. The high affinity products are then eluted with a high ionic strength buffer. Upon repurification, no detectable level of 125I-IabeIIed 67 kDa protein was recovered in the high ionic strength elution. The addition of fractions 1 and 4 of the low affinity eluate to the purified 67 kDa laminin binding protein solution resulted in an elution profile very similar to that seen on the initial isolation. SDS-PAGE' analysis of the eluates demonstrated a significantly higher proportion of the 67 kDa laminin binding protein in the high affinity fractions of the reconstituted rebinding experiments (data not shown). 89 1 2 3 4 140 kDa 87 kDa 48 kDa 33 kDa 29 kDa Figure 2.8 Fractions collected from a Iaminin-Sepharose affinity column following application of purified, 125I-IabeIIed 67 kDa laminin binding protein indicated that the majority of the applied material did not rebind, but was present in the column effluent. All samples were analyzed by SDS-PAGE. Lane 1, unbound fraction; Lane 2, first fraction of low ionic strength column wash; Lane 3, fourth fraction of low ionic strength column wash; Lane 4, high ionic strength column eluate. Molecular weight standards are as marked. ( L 90 Discussion The 67 kDa laminin binding protein has been shown to play an important role in the adhesion and extravasation of tumor cells during the metastatic dissemination of cancer (reviewed in Sobel1 1994). However, neither the structure, nor the ligand binding mechanism of this protein is understood in any detail. Considerable confusion in the literature has been generated by the fact that a number of laminin binding proteins of similar apparent molecular weight have been described, and frequently assumed to be the same molecule (reviewed in Mecham, 1991). Subsequent sequencing has identified some of these proteins as 5’-nucleotidase (IVIisumi etal., 1990), aspartactin (Clegg etal., 1989), and a splice variant of /?-galactosidase (Hinek et al., 1993). Antibodies to these various laminin binding proteins have been shown to cross react with each other, possibly due to common epitope structures (Risse et al., 1989). The isolation of the cDNA clone for the laminin binding protein failed to clarify the issue, as the cDNA clone was sufficient to encode only a 32 kDa protein. Furthermore, the deduced protein sequence contained no consensus sites for N-Iinked gIycosylation which could account for the molecular weight discrepancy, and no obvious transmembrane region is apparent from the sequence (Yow e ta l., 1988). Classical biochemical analyses such as peptide mapping and direct amino acid sequencing have been limited by insufficient material for standard assays. Reticulocyte lysate in vitro translation studies (Castronovo et al., 1991; Rao et al., 1989) were reported to result in a 37 kDa 91 protein. In those studies, anti-synthetic peptide antibodies which immunoprecipitated the 67 kDa product did not always recognize the putative 37 kDa precursor. In the current study, we have utilized a homotypic eukaryotic over-expression system to produce the 67 kDa laminin binding protein in its physiologically functional form. Using the technique of MALDI-TOF mass spectrometry, we were able to demonstrate that the molecular weight of the protein is consistent with its SDS- PAGE migration. It is attractive to speculate that the 33 kDa species seen on the MALDI TOF-MS could represent the native gene product with the 66 kDa species representing a dimer, and the 133 and 201 kDa representing 4x and 6x subunits, respectively. Treatment with disulfide reducing agents did not affect the molecular weight, either on SDS-PAGE or in the mass spectra, indicating that, if this protein does exist as a dimer or higher multimer, the association is not dependent on disulfide bonds. If the high molecular weight peaks identified on the TOF MS represent nonspecific aggregates of a 33 kDa monomer, one would also anticipate the presence of 3x and 5x multimers, which were not detected. However, it should be emphasized that with the methods employed here, it is not possible to definitively distinguish between a singly protonated monomer (MH+1) of 33 kDa, and a doubly protonated monomer (MH2+2) of 66 kDa which would also appear at M/Z 33 kDa. Therefore, the cDNA predicted product of 32 kDa has not been unambiguously rationalized with the principal 67 kDa observed species. Further studies, such as mass spectrometry of tryptic 92 digest, or cyanogen bromide, cleavage products will be required to definitively determine the monomeric unit of this macromolecule, and these studies are underway in our laboratory. Castronovo et al. reported that N-glycosidases had no effect on the molecular weight of isolated 67 kDa laminin binding protein (Castronovo et al., 1991), and there are no consensus sites for N-Iinked carbohydrates. They were also unable to metabolically label a transfected construct in COS-7 cells, with 3H- glucosamine or galactosamine. Our results, using endoglycosidase digestion of affinity purified laminin binding protein, support the conclusion that neither N-, nor O-Iinked gIycosylation is responsible for the molecular weight shift from the cDNA predicted product of 32 kDa to the affinity isolated 67 kDa product. It is conceivable that there are carbohydrates associated with the molecule in a manner not recognized by commonly used endoglycosidases. However, glycoproteins typically show some degree of heterogeneity in gIycosylation. The MALDI-TOF spectra show sharp peaks, with no evidence of gIycosylation induced heterogeneity. The lack of heterogeneity in molecular weight is strong evidence against the 67 kDa laminin binding protein being a glycoprotein. Therefore, modifications with non-carbohydrate moieties are a more likely explanation for the discrepancy between the predicted and observed molecular weights. Acylation of proteins has recently been shown to be relevant to the structure and function of numerous mammalian proteins (reviewed in Chow et 93 al., 1992). Three primary mechanisms of fatty acid attachment have thus far been described: 1) ester or thioester linkages to an internal amino acid; 2) amide linkages to an amino-terminal glycine residue; 3) a phosphodiester linkage to a gIycan moiety, forming a gIycosyl phosphatidylinositol (GPI) tail on the carboxyl terminus of the protein. The fatty acid most commonly identified with an ester linkage is palmitate, whereas cysteine residues in a CAAX consensus sequence are frequently modified with isoprenoids through a thioester linkage. We have identified the covalent association of palmitate, stearate, and oleate with the affinity isolated 67 kDa laminin binding protein. These lipids may provide a mechanism for membrane association of the molecule on the cell surface, as there is no standard transmembrane domain apparent within the predicted amino acid coding sequence of the cDNA. Lipid modified proteins are known to be associated with both the extracellular and cytoplasmic surfaces of plasma membranes. The majority of the known acylproteins on the extracellular I surface are associated with the membrane by lipids covalently bound to the protein through a gIycosyl phosphatidylinositol linkage. Using two separate assay methods, we did not find any evidence for the presence of a standard GPI linkage. Triton X-114 solubility is a technique commonly used to separate amphipathic proteins, and to identify transmembrane proteins. This technique is based on the ability of the non-ionic detergent Triton X -114 to partition into two distinct phases at 30°C; a detergent rich phase and an aqueous phase. 94 Amphipathic proteins with a transmembrane domain tend to partition predominantly (>80%) into the detergent rich phase, while hydrophilic proteins generally partition into the aqueous phase. Proteins which are anchored in the membrane with GPI tails have been shown to partition into the detergent phase even when associated with a very hydrophilic protein (Hooper, 1992). However, the 67 kDa laminin binding protein remained predominantly in the aqueous phase. In a second assay for GPI association of the identified lipids, we attempted to metabolically label the 67 kDa laminin binding protein with 14C- Ethanolamine. We failed to detect any 14C labelling in isolated 67 kDa laminin binding protein. Ethanolamine is a specific label for GPI moieties, but would result in only a 1:1 molar ratio under optimum biosynthetic conditions. It is possible that the specific activity used was below the limits of detection in this system, however, radioactivity was detected in a non-laminin binding fraction of the cell extract, so this is considered unlikely. The conditions required to release the covalently bound lipid from the protein were rather stringent, relative to standard fatty acid analyses (McCreary et al., 1978). These conditions also precluded the recovery of the intact protein from the reaction mix, so the effects of deacylation could not be evaluated. Transesterification under milder conditions resulted in significantly lesser quantities of methyl esters, and, in fact, the oleate and stearate were not always detectable under the milder experimental conditions. Acid hydrolysis also failed to release fatty acids, indicating the linkage mechanism is not likely to be an amide bond (unpublished observations). The most common mechanism for palmitate attachment to proteins has been shown to be an ester bond with the hydroxy group on serine or threonine (Mcllhinney1 1990). Our data are compatible with this chemistry. Hydroxyester linkage of stearate and oleate are also a possibility. As these two lipids are common components of the phospholipid bilayer, great care was taken to ensure their presence was not due to contamination of the protein preparations. Given the harsh conditions required for isolation of these fatty acids, we are confident they are, in fact, covalently associated with the protein. However, we were unable to definitively identify the linkage chemistry. On a molecular basis, we did not find the quantity of lipid to be sufficient to account for the total molecular weight shift from the cDNA predicted 32 kDa to the observed 67 kDa. However, as discussed earlier, given the experimental conditions required to release the lipid, complete transesterification may not have been achieved. One possible mechanism for the shift from 32 kDa to 67 kDa would be a dimerization of the native gene product which may be stabilized by Iipkklipid association. With an error range o f+0.2%, the MALDI-TOF molecular weight measurement compared to the cDNA predicted molecular weight could accommodate up to 2 long chain fatty acids per 32 kDa molecule, or 4 per 67 kDa molecule., Lipid modification of the protein may be responsible Tor plasma membrane association of the 67 kDa laminin binding protein in the apparent absence of a hydrophobic membrane anchor polypeptide domain. Alternatively, 95 96 the lipids may promote an association with a second protein. Castronovo et al. (1991) have proposed a covalent linkage with a second protein molecule, and our results do not eliminate that as a possibility. Indeed, we show evidence in this paper, that the high affinity ligand binding function is modulated by an, as yet, unidentified factor in detergent extracts of tumor cells. The ligand binding activity of many cell surface receptors is known to be modulated by local environmental factors. The ligand binding activity of the integrin receptor OtJJ2 has been shown to be enhanced by an, as yet, unidentified lipid factor, which is suggested to function as an allosteric activator (Hermanowski-Vosatka et al., 1992). Neutrophil activation is required for the localization of this factor, which appears to be a single, low molecular weight anionic species. Ligand specificity of the oJJ3 integrin is modulated by the lipid composition of the surrounding membrane (Conforti et al., 1990). Isolated CTxJff3 receptors incorporated into liposomes composed solely of phosphatidylcholine bound specifically to vitronectin, while those incorporated into a mixed vesicle of phosphatidylcholine and phosphatidylethanofamine also bound fibronectin and von Willebrand factor. We show evidence in this paper that reconstitution of the laminin binding protein with cell detergent extracts restores the original avid laminin binding ability of the affinity isolated 67 kDa laminin binding protein. It is possible that the plasma membrane components in the extracts provide the proper hydrophobic environment for a conformation dependent binding. However, M I 97 detergent alone was not sufficient to restore the high affinity binding of the isolated protein, and it seems more likely that a second protein or lipid factor is required for proper conformation or orientation. Expression of the 67 kDa laminin binding protein has been shown to correlate with expression of the cyfr, integrin receptor on small cell lung cancer cell lines (Pellegrini et al., 1994). These authors speculated that the 67 kDa laminin binding protein did not bind laminin directly, but functioned as an accessory molecule for This integrin receptor is thought to bind to the ES fragment of laminin-1, which is located in the distal region of the long arm (Sonnenberg et al., 1990). The 67 kDa laminin binding protein has been shown to interact with a five amino acid sequence, YIGSR, located in the proximal region of the short arm (Graf et al., 1987). This finding, however, was not supported by rotary shadowing experiments, which showed isolated 67 kDa laminin binding protein associated with the long arm of laminin-1 (Cioce et al., 1993). Coordinated binding by the two receptors, aJ3v and the 67 kDa laminin binding protein, could reconcile these discrepant observations. Since the laminins are large, multi-domain proteins with multiple functions, it is also possible that two or more adhesive sites exist within the laminin molecule, whose activities are modulated by environmental or conformational factors. We suggest a working model where, in the presence of accessory factors, possibly molecules such as Cr6Zi1, or other non-proteinaceous factors, a high affinity binding site is accessible and used. In the absence of such factors, perhaps the 67 kDa 98 laminin binding protein utilizes a lower affinity binding site. Additionally, the expression of both laminin binding proteins and laminin isoforms are known to be developmentally regulated (reviewed in Kleinman et al., 1993). It would be reasonable to expect that the determination of binding sites available for specific laminin binding proteins is also influenced by the developmental stage of the tissue. Since neoplasia is, by definition, an unregulated growth, one would anticipate a loss of coordination of cell surface receptor functions with their extracellular matrix ligands. The present work has implicated cofactors in the high affinity adhesion of the 67 kDa laminin binding protein with laminin. Further definition of the surface form and adhesive characteristics of laminin binding proteins should clarify our understanding of their mechanisms of action and so, facilitate the design of highly specific therapeutics for the treatment of metastatic disease. References Aitken, A. (1992). Structure determination of acylated proteins. In Lipid Modifications of Proteins: A Practical Approach. N.M Hooper and A.J. Turner, eds. (Oxford: IRL Press at Oxford U. Press), pp. 63-88. Aznavoorian, S., Murphy, A.N., Stetler-Stevenson, W.G., and Liotta, LA. (1993). Molecular aspects of tumor cell invasion and metastasis. Cancer. 71,1368-1383. Barsky, S.H., Rao, C.N., Hyams, D., and Liotta, LA. 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Clegg, D.O., Helder, J.C., Hann1 B.C., Hall, D.E., and Reichardt, L.F. (1989). Amino acid sequence and distribution of mRNA encoding a major skeletal muscle laminin binding protein: An extracellular matrix-associated protein with an unusual COOH-terminal polyaspartate domain. J. Cell Biol. 107, 699-705. Conforti, G., Zanetti, A., Pasquali-Ronchetti, I., Quagliano, DJr., Neyroz, P., and Dejana, E. (1990). Modulation of vitronectin receptor binding by membrane lipid composition. J. Biol. Chem. 265, Davis, S.C., Tzagoloff, A., and Ellis, S.R. (1992). Characterization of a yeast mitochondrial ribosomal protein structurally related to the mammalian 68-kDa high affinity laminin receptor. J. Biol. Chem. 267, 5508-5514. Graf, J., Ogle, R.C., Robey, PA., Sasaki, M., Martin, G.R., Yamada, Y., and Kleinman, H.K. (1987). A pentapeptide from the laminin BI chain mediates cell adhesion and binds the 67000 laminin receptor. Biochem. 26, 6896-6900. Grosso, L.E., Park, P.W., and Mecham, R.P. (1991). Characterization of a putative clone for the 67-kilodalton elastin/laminin receptor suggests that it encodes a cytoplasmic protein rather than a cell surface receptor. Biochem. 30, 3346-3350. Hermanowski-Vosatkai A., VanStrijp, J.A.G., Swiggard, W.J., and Wright, S.D. (1992). Integrin Modulating FaCtor-1: A lipid that alters the function of leukocyte integrins. Cell 68, 341-352. 100 Hinek, A , Rabinovitch, M., Keeley, F., Okamura-Oho, Y., and Callahan, J. (1993). The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of B-galactosidase. J. of Clin. Invest 91 1198-1205. Hooper, N.M. (1992). Identification of a glycosyl-phosphatidylinositol anchor on membrane proteins. In Lipid modifications of Proteins: A Practical Approach. N.M Hooper and AU. Turner, eds. (Oxford: IRL Press at Oxford U. Press), pp 89-116. Kaufman, RU. and Sharp, PA . (1982). Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. U. Mol. Biol. 159, 601-602. Keppel, E. and Schaller, H.C. (1991). A33kDa protein with sequence homology to the "laminin binding protein" is associated with ,the cytoskeleton in hydra and mammalian cells. U. Cell Sci. 100, 789-797. Kleinman, H.K., Weeks, B., Schnaper, H.W., Kibbey, M.C., Yamamura, K., and Grant, D.S. (1993). The laminins: Afamily of basement membrane glycoproteins important in cell differentiation and tumor metastases. Vit. and Hormones 47, 161-186. Mafune, K., Ravikumar, T.S., Wong, U.M., Yow, H., Chen, L.B., and Steele, G.D.,Ur. (1990). Expression of a MW 32,000 laminin-binding protein messenger RNA in human colon carcinoma correlates with disease progression. Cancer Res. 50, 3888-3891. Magee, A l. (1990). Lipid modification of proteins and its relevance to protein targeting. U. Cell Sci. 97, 582-584. Martignone, S., Pellegrini, R., Villa, E., Tandon, N.N., Mastroianni, A., Tagliabue, E., Menard, S., and Colnaghi, M.l. (1992). Characterization of two monoclonal antibodies directed against the 67 kDa high affinity laminin receptor and application for the study of breast carcinoma progression. Clin. Exp. Metas. 10, 379-386. McCreary, D.K., Kossa, W.C., Ramachandran, S., and Kurtz, R.R. (1978). A novel and rapid method for the preparation of methyl esters for gas chromatography: Application to the determination of the fatty acids of edible fats and oils. U. Chromat. Sci. 16, 329-331. 101 Mcllhinney, R.A.J (1990). The fats of life: the importance and function of protein acylation. TIBS 15, 387-391. Mecham, R.P. (1991). Receptors for laminin on mammalian cells. FASEB J. 5, 2538-2546. Melnick, M.B., Noll, E., and Perrimon, N. (1993). The Drosophila stubarista phenotype is associated with a dosage effect of the putative ribosome-associated protein D-p40 on spineless. Genetics 135, 553-564. Misumi, Y., Ogata, S., Hirose, S., and lkehara, Y. (1990). Primary structure of rat liver 5’-nucleotidase deduced from the cDNA. J. Biol. Chem. 265, 2178-2183. Pellegrini, R., Martignone, S., Menard, S., and Colnaghi, M.l. (1994). Laminin receptor expression and function in small-cell lung carcinoma. Int. J. Cancer Suppl.8, 116-120. Rao, C.N., Castronovo, V., Schmitt, M.C., Wewer, U.M., Claysmith, A.P., Liotta, LA., and Sobel, M.E. (1989). Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor. Biochem. 28, 7476-7486. Risse, G., Stochaj, U., Elsasser, K., Dieckhoff, J., Mannherz, H.G., and von der Mark, K. (1989). Structural comparison of the 68 kDa laminin binding protein and 5’-nucleotidase from chicken muscular sources: Evidence against a gross structural similarity of both proteins. Biochim. Biophys. Acta. 994, 258-263. Satoh, K., Narumi, K., Isemura, M., Sakai, T., Abe, T., Matsushima, K., Okuda, K., and Motomiya, M. (1992), Increased expression of the 67kDa-laminin receptor gene in human small cell lung cancer. Biochem. and Biophys. Res. Comm. 182, 746-752. Schmidt, M.F.G. (1989). Fatty acylation of proteins. Biochim. Biophys. Acta. 988, 411-426. Siyanova, E.Y. (1992). Expression of LBP 32/67 kD human gene in E. coli and analysis of its binding with laminin. Bull. Exp. Biol. Med. 113, 70-72. Sobel, M.E. (1994). Differential expression of the 67 kDa laminin receptor in cancer. Semin. Cancer Biol. 4, 311-317. 102 Sonnenberg, A , Linders, C J ., Modderman, P.W., Damsky, C.H., Aumailley, M., and Timpl, R. (1990). Integrin recognition of different cell-binding fragments of laminin (P1,E3,E8) and evidence that aJ3^ but not a^3A functions as a major receptor for fragment ES. J. Cell Biol. 110, 2145-2155. Stetler-Stevenson, W.G., Aznavoorian, S., and Liotta, LA. (1993). Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu. Rev. Cell Biol. 9, 541-573. Urlaub, G. and Chasin, LA. (1980). Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity^ Proc. Natl. Acad. Sci. USA 77, 4216-4220. Vacca, A., Ribatti, D., Roncali, L , Lospalluti, M., Serio, G., Carrel, S., and Dammacco, F. (1993). Melanocyte tumor progression is associated with changes in angiogenesis and expression of the 67-kilodalton laminin receptor. Cancer. 72, 455-461. Wang, K-S., Kuhn, R.J., Strauss, E.G., Ou, S., and Strauss, J.H. (1992). High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. J. Virol. 66, 4992-5001. Wewer, U.M., Liotta, LA., Jaye, M., Ricca, GA ., Drohan, W.N., Claysmith, A.P., Rao, C.N., Wirth, P., Coligan, J.E., Albrechtsen, R., Mudry, M., and Sobel, M.E. (1986). Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin. Proc. Natl. Acad. Sci. USA 83, 7137-7141. Yow, H., Wong, J.M., Chen, H.S., Lee, C., Steele, G .D.Jr., and Chen, L.B. (1988). Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein. Proc. Natl. Acad. Sci. USA 85, 6394-6398. 103 CONTROL PATHWAYS OF THE 67 kDa LAMININ BINDING PROTEIN: SURFACE EXPRESSION AND ACTIVITY OF A NEW LIGAND BINDING DOMAIN Introduction The greatest challenge to clinicians in the treatment of neoplasia is the threat of distant metastases. Metastasis is described as a multi-step process which is modulated by both the tumor cells and the host tissues (reviewed in Liotta et al., 1991; Stetler-Stevenson et al., 1993). Since the extracellular basement membrane represents the most formidable barrier to metastatic dissemination, tumor cell interaction with basement membrane molecules is considered a critical determinant of the tumor’s ability to invade adjacent tissues and spread to distant sites. A major class of basement membrane glycoproteins is the laminins. Large, heterotrimeric molecules with multiple structural domains, the laminins have been shown to contain multiple adhesive sites for other matrix molecules and cellular receptors (reviewed in Kleinman et al., 1993). Invasion of the extracellular basement membrane is mediated in part by the adhesion of tumor cells to laminins via specific cell surface receptors. Understanding the biology of these receptors promotes the ability of the clinician to predict the course of metastatic disease in the patient. Furthermore, definition of their ligand binding sites and associated signal transduction pathways should enable the design of highly effective anti-metastatic therapies. One of the laminin binding proteins whose expression has been shown to CHAPTER 3 104 correlate with the metastatic potential of solid tumors is the 67 kDa LBP. This non-integrin protein was initially identified on the surface of highly metastatic breast carcinomas (Barsky et al., 1984). Using immunohistochemical and molecular techniques, the 67 kDa LBP has since been identified in many cancer tissues, including melanoma (Kleinman et al., 1989), lung carcinomas (Martignone et al., 1992), and colonic adenocarcinoma (IVIafune et al., 1990). A strong positive correlation has been demonstrated, with expression of the 67 kDa LBP significantly increased in highly metastatic tumors as compared to non- malignant tissues and non-invasive tumors of similar histologic origins (Martignone et al., 1992; Mafune et al., 1990; Marques et al., 1994; D’Errico et al., 1991; Vacca et al., 1993). In the present study, we evaluate the metastatic phenotype of Chinese hamster ovary (CHO) cell lines which overexpress the 67 kDa LBP. Translational regulation of the expression of the 67 kDa laminin binding protein has been proposed by several groups (Rao et al., 1989; Makrides et al., 1988). These predictions were based on consensus sequences found in the 5’ untranslated region of the cDNA of the mouse sequence. In this manuscript, we demonstrate that translational regulation is, indeed, a likely mechanism used by cells to control the surface expression of the 67 kDa laminin binding protein. Transfected cells which expressed high levels of the 67 kDa LBP mRNA did not always express correspondingly elevated levels of the 67 kDa protein on their ---------------- 11 — ' ' ‘ ' I ------ ---------------- I I M ! I l _ _ I i n [ >: I surface. JL I j: We have also used the CHO overexpression system to investigate the laminin binding site of the 67 kDa LBP. A nine amino acid sequence located in the p chain of laminin-1 has been identified as a binding site for the 67 kDa LBP. This sequence, CDPGYIGSR, known as peptide 11, has been shown to block the adhesion of tumor cells to laminin in vitro, and to interfere with the metastatic cascade in vivo. The corresponding ligand binding domain of the 67 kDa laminin binding protein has been shown to reside in the carboxyl terminal half of the protein by blocking with an antibody raised to the truncated product (Wewer et al., 1986). Castronovo et al. have demonstrated that a synthetic peptide corresponding to residues 161-180 (Peptide G) of the 67 kDa LBP blocked the adhesion of tumor cells to cultured endothelium (Castronovo et al., 1991b). Adhesion via this domain of the 67 kDa LBP was later shown to be dependent on heparin, another basement membrane macromolecule which also binds laminin (Guo et al.,. 1992). In the present study, we demonstrate biological activity of a second synthetic peptide, corresponding to residues 205-229 of the LBP, in direct interaction with laminin, and in modulating tumor cell invasion of basement membrane matrix and tumor lung colonization. For this peptide, interaction with laminin could be inhibited with peptide 11. Materials and Methods Animals and tumor cell lines The highly invasive and metastatic B16BL6 murine melanoma cell line 105 106 was isolated originally by Dr. LR. Hart (Poste et al., 1980) and was obtained from the Mason Research Institute, Worcester, MA. B16BL6 cells were propagated in RPMI 1640 medium (Sigma) with 10% fetal bovine serum (Intergen), 5 /zg/ml insulin and penicillin/streptomycin (complete medium). The CHO mutant cell line (DG44) was kindly provided by Dr. Lawrence Chasin of Columbia University. These cells are double negative mutants for the dhfr gene, and rely on the presence of exogenous hypoxanthine and thymidine (HT) in the culture medium for proliferation (Urlaub and Chasin, 1980). Untransfected DG44CHOs (DG44wt) were maintained in crMEM (Sigma) with 10% dFBS, and supplemented with HT1 5 //g/ml insulin, and cover antibiotics. Selection of dhfr and pcLR transfected DG44CHOs was carried out in crMEM containing 10% fetal bovine serum (Intergen) extensively dialyzed against CMF saline (dFBS) and 400 //g/ml G418 (Gibco). Female C57BI/6J mice, 8 to 12 weeks of age, were used in these experiments. All mice were raised in the Montana State University Animal Resources Center from breeding stock obtained from the Jackson Laboratories, Bar Harbor, ME. Expression of the laminin binding protein A cDNA clone of the laminin binding protein (pcLR), isolated from a hamster expression library, was kindly provided by Dr. James Strauss, of the California Technical Institute, in the pcDNA1/neo vector (Invitrogen). This expression vector utilizes a CMV promoter and G418 selectable marker. A dihydrofolate reductase (dhfr) expression vector, under the control of an SV40 promoter, was obtained from ATCC (Rockville, MD). DG44CHO cells were simultaneously transfected with both plasmids at ratios of 5:1, and 20:1 pcLR:dhfr by calcium phosphate precipitation (Chen and Okayama, 1988). Methotrexate (CaIbioChem) was added to amplify the dhfr expression vector, and thus, co-amplify the pcLR vector. Methotrexate treatment was initiated at a concentration of 0 .03 //M and increased by three-fold increments approximately every 5th passage, to a maximum concentration of 120 //M . Expression of the laminin binding protein was assessed by Northern and FACScan analysis using the anti-LBP peptide 205-229 polyclonal antiserum described below. Laminin binding protein isolation The laminin binding protein was extracted essentially by the method of Wewer et. al. (1986). DG44CHO cells, selected for expression of the pcLR and dhfr plasmids, were harvested by rinsing twice with Puck’s calcium and magnesium-free Saline G solution, approximately 10 minutes each at 37°C, and a third time with the same solution containing 0.01 mM EGTA, until the cells detached. This harvest technique was used to avoid the use of trypsin and thus optimally preserve membrane proteins. Cells were then centrifuged for 10 minutes at 500 x g, the pellet resuspended in a 10x volume (v/v) of calcium and magnesium-free Dulbecco’s PBS, and centrifuged again under the same conditions. Included in this and all subsequent buffers were the protease inhibitors PMSF at 50 //g / ml, N-ethylmaleamide at 1mM, and benzamidine at 5 107 mM. Cells were then suspended in 2 volumes of ice cold 25mM Tris/0.3 M sucrose, pH 7.4, and sonicated on ice with 4-5 x 5 second bursts using a Fisher Model 50 Dismembrator at 50% power. Nuclei, cytoskeletal proteins, and intact cells were pelleted by centrifugation at 500 x g for 10 minutes at 4°C, the supernatant collected, and the pellet resuspended in 5 volumes of Tris/sucrose buffer. Sonication was repeated, and the supernatant again collected and pooled with the first supernatant. This was repeated three times, or until the majority of the cells appeared to be lysed on microscopic examination. The membrane fraction was then collected on a 0.3 M sucrose cushion by centrifugation at 143,000 x g for 90 minutes at 4°C, and adjusted to a protein concentration of approximately 1 mg/ml with 25 mM Tris/150 mM NaCI/1 mM CaCI2ZS mM MgCI2, pH 7.4 buffer. To this was added an equal volume of the same buffer containing 1% NP-40 detergent, and the solution was rotated end over end for 12-16 hours at 4°C. The insoluble material was pelleted by centrifugation at 200,000 x g for 60 minutes, and the supernatant combined with a laminin-sepharose column which had previously been pre-equilibrated with 10 volumes 25 mM Tris/150 mM NaCI/1 mM CaCI2ZS mM MgCI2 + 0.05% NP-40 followed by 1 volume of the same buffer containing 400 mM NaCI. The solution was then rotated for 8-12 hours at 4°C. For isolation of high affinity laminin binding proteins, the column was first washed with 50 mM Tris/0.1 M NaCI, pH 7.4 and UV absorbance monitored until it returned to baseline. Approximately one column volume of 50 mM Tris/1.0 M NaCI1 pH 7.4, was added and allowed 108 109 to stand 15-20 minutes at room temperature prior to elution of high affinity binding proteins. Peptide 11 elution of laminin binding proteins was carried out in the same manner, except that a 1 mg/ml solution of peptide in 50 mM Tris/0.1 M NaCI, pH 7.4, was added and allowed to stand 15-20 minutes before elution of proteins. This treatment was followed by 50 mM Tris/1.0 M NaCI, pH 7.4 to elute any remaining high affinity laminin binding proteins. Structure prediction and homology studies Using the predicted amino acid sequence for the 67 kDa LBP published by Yow et a/., secondary structure predictions, as well as sequence and functional homologies were determined. Structure prediction programs utilized were a) the program developed by Finer-Moore and Stroud (1984) with default parameters suggested by the authors for a membrane bound protein; and b) the Michigan State University program MSEC. Structural homology comparisons were done using the ALIMAT program developed by Argos (1987). This program combines two distinct scoring processes in comparing protein sequences including (1) the Dayhoff related ness odds matrix for amino acid exchange, and (2) a selection of five physical characteristics for amino acid residues: hydrophobicity, turn preference, refractivity index, residue bulk, and anti-parallel strand preference. The sequence was also searched for known functional motifs using the MOTIFS program (Devereux et al., 1984) to examine the ProSite database release 7.0 (Bairoch, 1991), running on the Pittsburgh Supercomputing Center (PSC) Vax system. Protein sequences for comparison 110 were obtained from Genbank/EMBL and NBRF/PIR databases at PSC. Peptide synthesis and competitive binding studies All peptides were synthesized on a Milligen 9050 automated peptide synthesizer employing standard Fmoc chemistry on polystyrene (PAL) resin (Milligen-Biosearch). Crude peptides were purified by preparative HPLC, and purity determined to be >90% by analytical reverse-phase HPLC and FAB and electrospray mass spectrometry. A highly charged region, predicted by the Finer-Moore-Stroud and MSEQ protein structure prediction programs to form a helical structure (residues 205- 229), exists within the carboxyl-terminal half of the cDNA derived amino acid sequence for the 67 kDa laminin binding protein. Since the laminin binding domain of the 67 kDa laminin binding protein has been localized to the carboxyl- terminal half of the protein (Wewer et al., 1986), a peptide including this sequence was synthesized (LBP 205-229; RDPEEIEKEEQAAAEKAVTKEEFQG) and examined for its ability to bind to whole laminin in a plate assay. For this assay, 1 mg laminin was tritiated by reaction with 500 //Ci [3H]-acetic anhydride (Amersham) in a buffer consisting of SOmM Tris-HCI/0.2M NaCI/SmM CaCI2, pH 7.4, for 2 hours at room temperature. Unbound radioactivity was removed by extensive dialysis against the same buffer. The laminin binding protein-derived synthetic peptide was dissolved at 1 mg/ml in neutral buffer, 0.5 ml of the peptide solution was applied to each well of positively charged 24-well Primaria plates (Falcon), and the plates dried under vacuum for 48 hours at room 111 temperature. Just before use in the binding assay, the peptide-coated plates were washed twice with Tris buffered saline (20 mM Tris/500 mM NaCI, pH 7.5) to remove dried buffer residue, and treated with 10% non-fat milk dried blocking solution for 30 minutes. The binding assay was performed in the same buffer used for laminin binding protein isolation on the laminin affinity column, with the addition of 0.05% Tween-20 to favor only highly specific binding. [3H]-laminin (25 //g laminin, 5000 cpm per well) was added, with or without 500 //g peptide 11, in 250 fj\ binding buffer to each peptide coated well and to control wells which had not been treated with synthetic peptide. The plates were incubated for 2 hours at 37°C with intermittent shaking. Unbound laminin was then removed with five washes of binding buffer. The material remaining on the plate was harvested using an incubation with 0.2ml 2%SDS for 2 hours at 37°C, and the SDS solution was then measured by scintillation counting in Aquassure (Amersham) to quantitate the amount of bound laminin. The association of peptide 11 with LBP peptide 205-229 was also monitored by following the transit time of 2 mg of [3H]-gly-peptide 11 through an affinity column composed of sepharose-coupled synthetic LBP peptide. 20 mg of the LBP 205-229 synthetic peptide was coupled to 10 ml CnBr-activated Sepharose using the methods described by the manufacturer (Pharmacia). A radioactive sample of peptide 11, CDPGYIGSR, was synthesized using [3H]- glycine (New England Nuclear) to label the peptide. The radioactive glycine was mixed with cold glycine and derivatized with the Fmoc group using standard 112 protocols. The [3H]-glycine preparation was used in both glycine cycles during peptide synthesis, resulting in 11,000 cpm/mg specific activity of the peptide 11. Transit time for the [3H]-gly- peptide 11 was assessed with and without competition from 2 mg cold peptide 11. The matrix was washed with phosphate buffered saline and collected in 2 ml fractions until radioactivity could no longer be detected. The ability of the LBP 205-229 derived synthetic peptide to bind [3H]- Iaminin was also assayed by this protocol. In this case, 2 mg of [3HJ-Iaminin was applied to the LBP peptide column, and unlabelled peptide 11 added as competitor. A non-related, highly charged peptide derived from the sequence of cytochrome c was used as a negative control. Tumor cell invasion of basement membrane matrix For experiments using B16BL6 cells, an 8 micron pore size polycarbonate separation filter between upper and lower chambers in a 6.5 mm Transwell (Costar) was impregnated with a 1:20 dilution of Matrigel in serum free medium as described by Repesh (1989). Matrigel was prepared in our lab from freshly excised EHS tumor tissue by the method of Kleinman et al. (1986). Preliminary experiments with DG44CHO cells indicated that these cells invaded the matrix so rapidly that it was necessary to add additional type IV collagen (Sigma) to the Matrigel matrix in order to slow down invasion and see differential effects. The minimum supplemental concentration of type IV collagen required to sufficiently retard invasion by the DG44CHO cells was determined to be 2.5 mg/ml, and this 113 concentration was added to the Matrigel for both DG44CHOwt cells, and DG44CHO pcLR/dhfr cells. 5 x 1 0 " monodispersed tumor cells were added to the upper chamber (Transwell insert) in 0.2 ml complete medium. 0.8 ml complete medium was added to the base well. Both chambers contained 100 A#g/ml of the specific peptide, and were fed daily with fresh peptide-containing medium. No chemoattractant was used in the lower well. At the end of one week, the loosely adherent cells were washed from the bottom of the inserts into the lower wells, the inserts removed, and colonies allowed to develop. Because of superior performance of Nunc plastic when growing B16BL6 cells from very low numbers, Costar transwells were used in Nunc 24 well plates. Colonies which developed in the lower well were washed, fixed in methanol and stained with hematoxylin for counting. Control experiments indicated that, for cell lines, such as B16BL6, which have very high cloning efficiencies, colony counting gave identical results to those obtained using radiolabelled cells (data not shown). DG44CHO pcLR/dhfr cells appear to have a much longer doubling time than untransfected DG44CHO wt cells. Therefore, for the CHO cell lines, individual cells invading through the Matrigel barrier to the lower chamber were counted on a daily basis to reduce experimental variation resulting from differences in proliferation rates. The day 4 results are shown in figure 3.6. Control experiments indicated that none of the peptides used in these experiments grossly affected cell viability or proliferation rates. Methotrexate was not included in any of the media for invasion assays. 114 Northern analysis Total RNA was extracted from pcLR/dhfr transfected cells and untransfected control cells by the guanidine-isothiocyanate method of Chomczynski ef a/. (Chomczynski and Sacchi1 1987), separated in a 1.2% agarose gel containing 29% formaldehyde, blotted to nitrocellulose (Schleicher and Schuell), and probed with the EcoRI laminin binding protein insert previously labelled with 32P by nick translation. Blots were prehybridized for 2-4 hours at 42°C in 20xSSC, 50% Denhardt solution, 1 M NaH2PO4, 1 mg/ml yeast tRNA, 10% dextran sulfate, and hybridized under the same conditions with 2 x 106 cpm/ml probe, which had been previously denatured by boiling for 15 minutes and cooled on ice (Sambrook et al., 1989). Visualization and quantitation of the blots was performed with a Molecular Dynamics Phosphorimager™ using ImageQuant™ software. Sample loading was assessed by stripping the blots with boiling water and probing under the same conditions with a human cardiac yff-actin probe which had been similarly labelled. Genomic DNA extraction and Southern blot analysis Genomic DNA was extracted from untransfected DG44CHO cells, and transfected DG44CHO pcLR/dhfr cells using SDS and proteinase K to liberate the DNA, followed by purification by phenol-chloroform extraction. The DNA was digested to completion with EcoRI and Smal, and electrophoretically resolved in I a 0.8% agarose gel. Following denaturation for 45 minutes in 0.5 N NaOH, and depurination in 0.2 N HCI, DNAs were neutralized for 30 minutes in 1 M Tris/1.5 M NaCI, pH 7.4. DNA was subsequently transferred to a nitrocellulose filter (Schleicher & Schull) by capillary transfer in 20x SSC for 16 hours (Sambrook et al„ 1989). The DNA was fixed to the filter by UV crosslinking. Probing and visualization of the blot were done as for the Northern analysis. Antibody production and Western blotting The LBP peptide 205-229 (described above) was conjugated to KLH (Sigma) by crosslinking for 2 hours at room temperature in 0.2% glutaraldehyde, and the solution then dialyzed against Dulbecco’s PBS for 48 hours with 4 changes of dialysate to remove glutaraldehyde and free peptide prior to immunization. New Zealand white rabbits were immunized with 1 mg of peptide- KLH and Freund’s complete adjuvant at multiple subcutaneous sites, and boosted every two weeks with peptide-KLH and incomplete Freund’s adjuvant. Thirty days later, a test bleed was obtained and the anti-peptide activity titered by ELISA with KLH, peptide-KLH, and peptide conjugated to ovalbumin used as target antigens. The polyclonal antiserum was purified using a protein A-column (Pierce), and the majority of the anti-KLH activity removed with a KLH- Sepharose affinity column. Laminin-affinity isolated 67 kDA LBP was separated on a 10% SDS-PAGE gel, and transferred to PVDF membrane (Bio-Rad) using a semi-dry blotter (Ellard Instrumentation). We found that the protein did not adhere well to PVDF membrane. Therefore, it was necessary to fix the membrane in 0.02% glutaraldehyde for 2 hours at room temperature prior to blocking with 5% nonfat 115 116 dry milk in DPBS. To ensure that the glutaraldehyde was not introducing false positive results, fixation in 10% acetic acid/25% methanol was also used, and shown to give identical results. Antibody detection was performed using alkaline phosphatase conjugated goat anti-rabbit antibody (Bio-Rad) and BCIP/NBT chromogenic substrate (Kirkegaard Perry Inc.). FACScan analysis Cells were harvested for FACScan analysis with three washes of Tyrode’s CMF saline solution, followed by very brief trypsin exposure, and rapid trypsin inactivation in serum containing medium. Following recovery in 50:50 Tyrode’s CMF:complete medium, the cells were washed with 10 volumes of 50:50 Tyrode’s CMF:serum free medium. Unpermeabilized cells were incubated with the primary antibody or PBS in the presence of NaN3 for 60 minutes on ice. The primary antibody used was Protein A purified rabbit anti-LBP 205-229 peptide immunoglobulin fraction. After pelleting the cells and resuspending them in Dulbecco’s PBS, the secondary antibody, FITC labelled goat anti-rabbit Ig (Sigma Immunochemical), was added and allowed to react for 60 minutes on ice in the presence of azide. The cells were then washed by centrifugation, were resuspended in phosphate buffered saline, and filtered through nylon mesh immediately prior to analysis. FACScan analysis was performed on a Becton Dickinson FACScan. In order to evaluate the effects of subculturing the CHO cells on LBP surface expression, transfected cells were harvested by incubating for 10 minutes at 37°C in Tyrode’s CMF saline, followed by a brief exposure to 117 Tyrode’s CMF containing 0.05% trypsin, and plated in selection medium. Surface LBP expression was measured by FACScan analysis at 3, 6, 14, and 24 hours post plating. The effects of cell density on LBP expression were measured using cells from subcultures made at various split ratios. All cells were assayed at 16 hours post plating, and split ratios of 1:4, 1:8 and 1:16 were used. Quantitative lung colony assay v Tumor cells were harvested from subconfluent (60%-80% confluency) cultures using minimal trypsin exposure as described above for FACScan analysis. The cells were harvested in complete medium to allow for serum inactivation of trypsin, then washed three times and resuspended in serum-free medium. Despite the lack of reports in the literature, we have found that mice injected intravenously with excesses of some matrix derived peptides can show evidence of intravascular embolization. Therefore, where possible, we use a modified protocol to avoid injecting excess free peptide. Tumor cell suspensions, harvested as described above, were first incubated with 2 mg/ml peptide for 20 minutes at 37°C. The cells were then pelleted, the supernatant containing excess peptide discarded, the cells washed in buffer consisting of 50% Tyrode’s CMF saline: 50% complete RPMI medium, then counted and diluted in the same buffer. After removal from peptide-containing solutions, the cells were held on ice. Prior to injection, the animals were warmed at 37°C for 30 minutes. Tumor cells, 5 x 104, in 0.2 ml were injected per mouse via the 118 lateral tail vein. Where the experiment required co-injection of tumor cells with peptide, cells were prepared as indicated above without the preincubation step. 1 mg peptide dissolved in the injection buffer was mixed with the aliquot of tumor cells immediately prior to injection to give a total volume of 0.2 ml. Three weeks after injection, the animals were sacrificed and autopsied. All tissues with suspect tumor colonies were rinsed in a balanced saline solution and fixed for 3 days in Bouin’s fixative for gross and histological examination. The number of superficial nodules in the Bouin’s-fixed tissues was determined using a dissecting microscope. Results Messenger RNA levels of the 67 KDa LBP do not directly reflect the surface expression of the protein Since Chinese Hamster Ovary (CHO) cells have been shown to express the 67 kDa LBP on their surface (Graf et al., 1987a; Wang et al., 1992), we elected to utilize a homotypic expression system to assess the phenotypic affects of overexpression of this protein. The cell line DG44CHO, which is deficient in dihydrofolate reductase (dhfr), was selected as a methotrexate amplifiable expression system. DG44CHO cells were transfected with the hamster 67 kDa LBP expression vector pcLR, and co-transfected with a dhfr expression plasmid. Although the mechanism is not well understood, it has been shown that methotrexate treatment of such dhfr transfected cell lines results in reduplication of the dhfr plasmid, and concurrent amplification of cotransfected 119 plasmids. Two populations of cells were selected from the transfection and propagated for further study. The first population, designated "flats", were transfected at a pcLR:dhfr ratio of 20:1, and were initially selected in HT deficient medium containing G418, but without methotrexate treatment to induce amplification. These cells are characterized by a flattened morphology, form close cellxell associations, and a majority of the cells display a cobblestone like appearance (Fig. 3.1). The DG44CHO pcLR/dhfr "flats" appeared to have longer doubling times than the parental cell line or the other transfected cell lines. FACScan analysis of the "flats" using the sequence specific anti-LBP peptide 205-229 antibody, revealed the presence of a subpopulation of the cells which showed a substantial surface expression of the 67 kDa LBP protein, which was further increased following later treatment with methotrexate (Fig. 3.2a-c). Northern analysis with a cDNA probe identified two species of 67 kDa LBP mRNA in the "flat" cells, one endogenous product of approximately 1.0 kb, and one slightly larger transcript consistent with the expected product from the pcLR plasmid (Fig. 3.3). The second population of cells, designated "5:1" were transfected at a pcLR:dhfr ratio of 5:1, and were morphologically similar to the DG44CHOwt parental cell line, which contains many poorly adherent, rounded cells, as well as a population of "fibroblastic" cells. Methotrexate treatment of the transfected cells was initiated at 0.03 /jM methotrexate, and increased in three-fold 120 Figure 3.1. Phase contrast photomicrographs of a) DG44CHOwt cells, and b) DG44CHO cells transfected with pcLR and dhfr at a ratio of 20:1. These cells were designated "Flats" due to their spread, cobblestone-like appearance. Magnification is 150x. 121 log fluorescence intensity Figure 3.2. FACScan analysis of the surface expression of the 67 kDa LBP. Cells were treated with anti-LBP peptide 205-229 antiserum, and stained with FITC conjugated goat anti-rabbit secondary antibody, a) endogenous expression of the 67 kDa LBP in untransfected cells; b) DG44CHO cells transfected with the pcLR and the dhfr plasmid; c) DG44CHO cells transfected with pcLR and the dhfr plasmid, and treated with 1 //M Methotrexate; d) DG44CHO cells transfected with pcLR and the dhfr plasmid showing adaptation to methotrexate. A constant vertical scale is used to show the relative size of different peaks. 122 1 2 pcLR Endogenous 3 4 Actin Figure 3.3 Northern analysis of DG44CHO mRNA probed with the coding region of the 67 kDa LBP cDNA. Arrows indicate the transcript of the endogenous gene product, and the transfected plasmid product, a) untransfected DG44CHO cells; b) pcLR/dhfr transfected DG44CHO cells; c) pcLR/dhfr transfected DG44CHO cells treated with 1 /j M Methotrexate; d) pcLR/dhfr transfected DG44CHO cells treated with 12 yvM Methotrexate. 123 increments approximately every 5th passage. Southern analysis' of 5:1 cells which were at a methotrexate treatment level of 0.3 //M demonstrated an increased copy number of the pcLR plasmid, as expected (Fig. 3.4). Treatment of the 5:1 transfected cells with increasing levels of methotrexate resulted in an amplification of the plasmid derived mRNA, while levels of the endogenous transcript remained constant (Fig. 3.3). FACScan analysis of the unpermeabilized 5:1 cell line using the anti-LBP peptide antibody showed that surface expression of the 67 kDa laminin binding protein was initially increased after growth in methotrexate. However, after the cells apparently adapted to higher levels of methotrexate, surface expression of the protein decreased and again approached the levels seen in untransfected cells (Fig. 3.2d). Incremental increases of the level of methotrexate resulted in the 5:1 cells transiently displaying the flattened phenotype, however, following several additional passages in the higher level of methotrexate, the cells again regained the characteristic morphology of the parental cells, and the 67 kDa LBP surface expression was concomitantly decreased. Such cells were designated "methotrexate adapted". mRNA levels remained increased in a methotrexate dose dependent manner even in "methotrexate adapted" cells whose surface expression of the 67 kDa LBP had returned to basal levels, as shown by FACScan analysis (Figs. 3.2 and 3.3). Both late passage transfected CHO cells and methotrexate "adapted" CHO cells expressed surface levels of LBP comparable to untransfected cells. However, when the same cultures were 124 A B C D E Figure 3.4 Southern analysis of DG44CHO transfected cell lines. Genomic DNA was digested with EcoRI (lanes A-C) or Smal (lanes D and E) and probed with the coding region of the 67 kDa LBP cDNA. Lane A, untransfected DG44CHO cells; Lanes B and D1 DG44CHO cells tranfected with pcLR and dhfr at a ratio of 5:1; Lanes C and E, DG44CHO cells transfected with pcLR and dhfr at a ratio of 5:1, and treated with Methotrexate to 0.3 //M . 125 sparsely plated and less than 24 hours old, a substantial proportion of the population expressed high levels of the 67 kDa LBP. The largest proportion of cells expressing high levels of the LBP was observed at 14 hours after subculture (Fig. 3.5, Panel A). Cell density was also found to effect the proportion of high LBP expressing cells. Only 5% of the cells subcultured at a 1:4 split ratio were high expressors, whereas 40% of the cells subcultured using a 1:8 split ratio, and 60% of the cells subcultured using a 1:16 split ratio were high expressors (Fig. 3.5, Panel B). Cells exposed to methotrexate for extended periods of time express a 37 kDa laminin binding protein The 67 kDa laminin binding protein, isolated from membrane extracts of "5:1" DG44CHO pcLR:dhfr cells, was eluted from a laminin-sepharose column with high ionic strength salt buffers and shown to be a single band at 67 kDa by silver stained SDS-PAGE. This product was specifically recognized by the anti- LBP peptide antibody in a Western blot. Furthermore, antibody binding in the Western blot was completely inhibited by excess LBP peptide (data not shown). However, extraction and affinity purification of laminin binding protein under identical conditions from "methotrexate adapted" 5:1 pcLR/dhfr cells resulted in the isolation of a laminin binding protein with apparent molecular weight of 37 kDa on reduced SDS-PAGE (data not shown). This protein was isolated in significantly lesser quantities than the 67 kDa product, and was never found in the same preparation along with the 67 kDa protein. However, laminin binding affinity appeared to be similar to that of the 67 kDa protein, as evidenced by \ 126 A B log fluorescence intensity Figure 3.5. FACScan analysis of 67 kDa LBP expression at various times and densities post plating. DG44CHO "flats" cells were harvested with Tyrode’s CMF and Tyrode’s CMF with 0.05% trypsin, and plated in selective medium. The effects of time and density were assayed after staining with anti-LBP peptide 205-229 antiserum, and FITC conjugated goat anti-rabbit secondary antibody. A constant vertical scale is used to show the relative size of the peaks. Panel A: a) 3 hours post plating; b) 6 hours post plating; c) 14 hours post plating; d) 24 hours post plating. Panel B: a) high cell density; b) low cell density after subculturing at a split ratio of 1:4; c) split ratio of 1:8; and d) split ratio of 1:16. elution with high ionic strength buffers. The 37 kDa species was also shown to be specifically eluted from the laminin-sepharose affinity column by the laminin derived peptide 11. A new Iiaand binding domain in the 67 kDa LBP Peptide 11 has been previously shown to block the adhesion of tumor cells to laminin-1, presumably, by interfering with the interaction of the 67 kDa laminin binding protein with laminin (Graf et al., 1987a; Graf et al., 1987b). Computer analysis of the predicted amino acid sequence of the 67 kDa LBP using the Stroud and MSEQ structural prediction programs (Finer-Moore and Stroud, 1984), allowed us to identify a region of highly charged amino acids in the carboxyl terminal half of the molecule (residues 205-229) which are predicted to form an alpha helical structure. We hypothesized that the highly charged nature of this domain could contribute to a direct interaction with laminin. A synthetic peptide with a sequence corresponding to this region did show laminin binding activity in a plate assay. This activity was directly competed by peptide 11 (Table 3.1), however, as expected for peptide:peptide interactions, direct interaction between the LBP peptide 205-229 and the laminin derived peptide 11 proved to be very difficult to demonstrate. When radiolabelled peptide 11 was added to a LBP peptide-Sepharose column, the transit time for radiolabelled peptide was only marginally affected by the presence of unlabelled peptide 11. Slightly over 10% of the peptide 11 counts appeared in earlier fractions of the , column eluate when cold additional Peptide 11 was present (data not shown). 127 128 Table 3.1. Laminin bound per well per mg synthetic peptide1 2 Laminin bound in the absence of peptide 11 13.9 + 1 .0 7 //g Laminin bound in the presence of peptide 11 0.97 + 0.5 //g 1WeIIs were coated with peptide 205-229 and evaluated for their ability to bind tritiated laminin. 500 //g peptide 11 and 25 //g laminin were used per well, and the data shown are averaged from 4 replicate wells. The experiment was run in duplicate. 2Residues 205-229 from the laminin receptor predicted sequence. However, the binding of whole laminin to the LB peptide-Sepharose column was clearly decreased by the presence of peptide 11, (Table 3.2) indicating there is, at least, a destabilizing interaction between these two molecular domains. No significant effect was seen with an unrelated, highly charged peptide. These data were highly reproducible, and indicate that the highly charged LBP domain (residues 205-229) is involved in the association of the 67 kDa LBP with laminin. 129 Table 3.2. Binding of laminin to 67 kDa LBP peptide 205-229 1 Peptide added 3H-Iaminin bound by 2 mg peptide 205-229 coupled to a sepharose matrix % of control c.o.m. bound None 100.0 25,182 Peptide 11 50.5 12,723 Control peptide 84.9 21,400 1140 //g initiated laminin were incubated with 2 mg peptide 205-229 in the presence and absence of 2 mg peptide 11 or an unrelated peptide, KISSWGKlKEC derived form the sequence of cytochrome c. The sepharose was washed until no unbound radioactivity was apparent, then the matrix bound counts were quantitated using scintillation counting in 20 ml Aquassure. Extended exposure of B16BL6 melanoma cells to LBP 205-229 enhances their metastatic capability The LBP peptide showed biological activity both in vivo and in vitro. In a tumor lung colony assay, injection of the peptide along with the mouse melanoma B16BL6 tumor cells resulted in a reduction of experimental metastasis to 66% of the control value (Table 3.3). However, when the peptide was preincubated with the tumor cells and excess peptide removed prior to injection, lung colonization was enhanced to 156% of the control. This is in contrast to the inhibitory effects of the laminin derived peptide 11, which were seen whether the tumor cells were pretreated with the peptide, or co-injected. 130 Table 3.3. Effect of synthetic peptides on tumor lung colonization by B16BL6 melanoma cells Peptide used: Preincubated with tumor cells Injected with tumor cells Average number of tumor lung colonies per mouse (% of control) None — — 100 LBP peptide 205-229 yes — 156 (p=0.08)2 LBP peptide 205-229 yes 66 (p=0.06) Laminin peptide 11 yes — 63 (p=0.04) Laminin peptide 11 — yes 60 (p=0.007) 1 Mice were injected intravenously with tumor cells as described in "Methods". After 3 weeks, the animals were killed and tumor lung colonies enumerated. Data shown are from groups of mice consisting of 7-13 individuals per data point, and represent the results of 6 separate experiments. 2 Statistical differences between the experimental and control groups were evaluated using the Mann Whitney 2-tailed test. 131 In vitro, the LBP peptide enhanced the invasion of the B16BL6 mouse melanoma tumor cells through a basement membrane matrix (Fig.. 3.6). The invasion of the B16BL6 cells was increased by four fold over control values. In contrast, the invasion of DG44CHO and DG44CHO pcLR/dhfr "flats" cells was not enhanced by the LB peptide, but rather, both cell lines were inhibited. Peptide 11 inhibited the invasion of Matrigel by B16BL6 melanoma and both DG44CHO cell lines. Figure 3.6. Peptide Modulation of Tumor Cell Invasion of Basement Membrane Matrix Legend B16BL6 DG44CHOwt DG44CHO pcLR/dhfr 500 400 Peptide Treatment Percentage of Contn 133 Discussion Elevated expression of the 67 kDa LBP in highly metastatic tumors has been well documented. Early studies by Liotta et a!., using monoclonal antibodies raised to laminin-affinity purified 67 kDa LBP from human metastatic breast cancer, demonstrated an increase in expression of the protein as compared to adjacent normal tissues (Hand et al., 1985). More recently, production of a monoclonal antibody to the 67 kDa LBP by immunizing mice with a panel of highly metastatic human cell lines has been reported (Martignone et al., 1992). Immunoperoxidase staining of surgical specimens with this antibody showed a clear difference between highly invasive carcinomas, and in situ or benign lesions. Sequence specific antibodies, raised to synthetic peptides derived from the deduced protein sequence of the 67 kDa laminin binding protein, have also been useful in characterizing 67 kDa LBP expression. This approach has been used to demonstrate high levels of protein expression on invasive tissues of colon cancer (Cioce et al., 1991), melanoma (Castronovo et al., 1991b), and breast cancer (Castronovo et al., 1990). Additional data supporting the relevance of the 67 kDa LBP in tumor metastasis has been provided by mRNA analyses of surgical specimens. Mafune et al. (1990) examined 67 kDa LBP mRNA from twenty-one surgical specimens of primary colon carcinoma and six liver metastases of colon carcinoma. Fifteen of the twenty-one primary tumors demonstrated mRNA levels 134 at over 150% of adjacent normal tissue, and only one patient sample was found to have LBP mRNA levels in the tumor tissue that did not exceed the adjacent colonic epithelium. A similar study by Satoh et al. analyzed expression of the 67 kDa laminin binding protein mRNA in lung cancer tissues and lung cancer cell lines (Satoh et al., 1992). Small cell and oat cell carcinomas, typically highly aggressive tumors, demonstrated the highest levels of mRNA expression, with up to 7-fold increases over normal adjacent lung tissue. Although the clinical data demonstrates a strong positive correlation between the expression of the 67 kDa LBP and the metastatic phenotype of the tumor, very little is known about the regulation of the expression of this protein. Several groups have hypothesized a translational control of expression of the 67 kDa laminin binding protein. Makrides et al. (1988) identified a consensus sequence within the 5’ flanking region of the cDNA which has been implicated in translational control of protein expression. Additionally, a computer generated prediction of the secondary structure of the mouse mRNA sequence has been reported to be consistent with translational control (Rao et al., 1989). In the present study, we have shown that, in a homotypic overexpression system, the surface expression of the molecule is not entirely regulated by the level of mRNA transcription. Transfection of DG44CHO cells with the pcLR expression vector initially resulted in significantly elevated cell surface expression of the 67 kDa LBP. At later time points, the surface expression of the protein was seen to be decreased, while mRNA levels remained elevated. 135 With the overexpression system we have used, it is possible that the expression of the native gene product exceeded the capacity of cellular enzymes required for post translational modifications. However, this is not likely, since early after transfection, and/or methotrexate pressure, high surface levels of the protein, proportional to the mRNA levels are seen. Wang et al. (1992) also reported a similar instability of surface expression of 67 kDa LBP in CHO cells transfected with the pcLR plasmid. The existence of a 37 kDa precursor protein has been previously reported (Rao et al., 1989; Castronovo et al,, 1991a), and studies in our lab have identified a putative mechanism for post translational modifications required to shift the molecular weight to the 67 kDa product (Landowski et al., manuscript submitted). These post translational modifications or molecular associations may be required for cell surface expression, as has been suggested by Castronovo et al. (Castronovo et al., 1991a). Although we did not quantitate the laminin binding affinity of the 37 kDa species, it was isolated under conditions that would imply an affinity nearly equivalent to that of the 67 kDa LBP. The relatively small quantities, which were found in our detergent extracts, most likely reflect a cytoplasmic form of the nascent polypeptide chain which may not be stable or soluble in detergents. Recently, several proteins have been identified which autoregulate their own expression by binding to specific sequences of their own mRNA (reviewed in Hentze, 1994). Several of these proteins are dinucleotide binding enzymes whose dinucleotide binding fold overlaps the region that contacts the RNA. Comparison of the structural characteristics of the 67 kDa LBP predicted amino acid sequence to several enzymes with kinase activity identified a region with structural similarity to the dinucleotide binding domains of lactic dehydrogenase, alcohol dehydrogenase, and adenylate kinase. Alignment of residues 1-50 of the 67 kDa LBP with the dinucleotide binding domain of these proteins indicates that it would be possible for the 67 kDa LBP protein to form a dinucleotide fold structure (Fig. 3.7). Proteins with extensive cDNA sequence homology to the 67 kDa LBP have been identified which are cytoplasmic, and have been found to be associated with ribosomes (Davis et al., 1992; Auth and Brawerman, 1992). We propose a model where the 67 kDa laminin binding protein has a dual function which may be influenced by the developmental stage of the tissue, or by the proliferation rate of the cells. In undifferentiated cells, or those undergoing a high rate of proliferation, the protein may be more highly expressed on the cell surface, where it participates in the interaction of the cells with basement membranes. In more differentiated cells, or those that are dividing at a lower rate, when high affinity laminin binding abilities are not required, the protein may remain in the cytoplasm, where it could associate with the RNA and function as a translational regulator. Three recent reports would support the transcriptional level of the 67 kDa LBP mRNA as being related to the proliferation status of the cell. Demeter et al. (1992) reported that in situ hybridization of cervical 136 Figure 3.7. ALIGNMENT OF THE PUTATIVE NUCLEOTIDE BINDING SITE IN THE HIGH AFFINITY 67 KD LAMININ BINDING PROTEIN WITH THE NUCLEOTIDE BINDING REGIONS OF LACTIC DEHYDROGENASE 20 30 L K F L A A G T H L G G T N L D F Q M E Q Y + I 11 + Q K I T V L G V R Q V G M A C G S S I L M 40 50 I Y K R K(S)D G I Y I I N L K R + + + I I I I K S L A D Q L A L L D A M E * ft u>