Scholarship & Research
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/1
Browse
17 results
Search Results
Item Structural characterization of the Csa3/cA4 complex - a nexus for class 1 CRISPR-Cas immune response coordination & establishing a cure for highly efficient galectin expression(Montana State University - Bozeman, College of Letters & Science, 2024) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin Lawrence; This is a manuscript style paper that includes co-authored chapters.Though Class I CRISPR-Cas systems, primarily Type I and Type III, are the most abundant CRISPR systems in archaea and bacteria, mechanisms driving their immune response regulation are not well understood. Csa3 family transcription factors, composed of N-terminal CARF and C-terminal winged helix-turn-helix domains, are frequently encoded within Type I CRISPR-Cas systems. Csa3 transcription factors are hypothesized to bind cyclic oligoadenylate (cOA) second messengers produced by Type III interference complexes, likely modulating their DNA-binding activity. Therefore, we investigated the interaction between Csa3a and cyclic tetra-adenylate (cA4). Isothermal titration microcalorimetry showed S. solfataricus Csa3a binds cA4 at biologically relevant concentrations in an entropically driven interaction. Ring nuclease assays revealed Csa3a lacks self-regulatory phosphodiesterase activity exhibited by other CARF domain proteins. We crystallized and solved the structure of the Csa3/cA4 complex, which revealed conserved motifs are responsible for cA4 binding and illuminated significant conformational changes induced by the interaction. We also identified an 18-bp palindromic motif, which we designated CAPPa, that is conserved in the 27 sequenced members of the order Sulfolobales, and shows synteny with Csa3a and acquisition genes in these genomes. We found Csa3a binds CAPPa in a nonspecific, cooperative, and cA4-independent manner. These characteristics suggest a more complex method of transcriptional regulation than previously hypothesized. However, the interaction between Csa3a and cA4 confirmed here signifies a nexus between Type I and Type III systems; we thus propose a model in which this interaction coordinates the two arms of an integrated immune system to mount a synergistic, highly orchestrated, adaptive immune response. We applied the workflow designed to produce significant protein quantities for crystallographic studies of Csa3a to the study of Homo sapiens galectin proteins, a family of beta-galactoside-binding proteins. Here, we identified a putative autoinhibitory mechanism affecting traditional IPTG-induction methods by characterizing IPTG-binding capabilities of galectins and quantifying basal protein expression over various IPTG concentrations. To bypass this predicted feedback loop, we employed a highly efficient and approachable autoinduction method, resulting in a 7-fold increase in protein expression. Much of this work was done in the context of a course-based undergraduate research experience with great success.Item When a lectin binds a sugar, and other sweet tales(Montana State University - Bozeman, College of Letters & Science, 2019) Bernhard, Samuel Pruitt; Chairperson, Graduate Committee: Mary J. Cloninger; Mackenzie S. Fricke was an author and Katharina Achazi, Paul Hillman, Willy Totten, Rainer Haag and Mary J. Cloningerwere co-authors of the article, 'The toxicity, uptake, and impact on galectin-3 mediated apoptosis of lactose functionalized dendrimers' submitted to the journal 'Biomolecules Special Issue: Moving Forward with Dendrimers' which is contained within this dissertation.The current state of chemotherapy and cancer treatment leaves much to be desired. Treatment is generally non-specific and relies on high dosage to achieve therapeutically relevant concentrations at target sites. Glycopolymer-drug conjugates, featuring targeting molecules and therapeutic prodrug on a water-soluble polymeric scaffold, offer a solution to these contemporary problems. Here, the complexity of glycopolymer design is explored through the lens of a biologically significant carbohydrate-binding receptor. In particular, galectin-3 is a complex Beta-galactoside binding lectin that experiences altered expression in many cancer pathologies and is implicated in metastasis, angiogenesis and poor overall prognosis. Galectin-3 mediates undesired cancer promoting processes through carbohydrate binding and oligomerization. A more complete understanding of the role galectin-3 plays in cancer progression will guide development of methods in the therapeutic intervention of these processes. In the interest of understanding galectin-3 and using it as a targeted receptor, its binding characteristics have been assessed through fluorescence lifetime and dynamic light scattering measurements. Employment of carbohydrates and glycopolymers including mannose, lactose, and lactose functionalized poly(amidoamine) (PAMAM) dendrimers, dendritic polyglycerols (dPG), and linear polymers (LP) provided insight into the carbohydrate binding avidity of galectin-3 and its propensity to oligomerize or form micron scale aggregates. A relationship between scaffold size and receptor recruitment was observed, which sheds light into multivalent binding motifs initiated by these glycopolymers and establishes a threshold for minimum requisite lactose functionality on lactose functionalized dendritic polyglycerols. In vitro cell based glycopolymer studies with AlexaFluor 647 and lactose functionalized PAMAM dendrimers revealed size-dependent uptake and demonstrated that accumulation occurs within the lysosome. Cellular aggregation experiments revealed that lactose functionalized LPs and dPGs influence galectin-3 mediated homotypic cellular aggregation and, in fact, augment this aggregation through receptor recruitment and cross-linking. The results reported here have provided a more fundamental understanding of galectin-3 binding interactions and have laid the groundwork for optimized glycopolymer-drug conjugate design.Item Cancer processes probed by multivalency: investigations with galectin-3 and lactose functionalized dendrimers(Montana State University - Bozeman, College of Letters & Science, 2019) Fricke, Mackenzie Sue; Chairperson, Graduate Committee: Mary J. Cloninger; Samuel P. Bernhard was an author and Willy Totten, Katarina Achazi, Paul Hillman, Rainer Haag and Mary J. Cloninger were co-authors of the article, 'The toxicity, uptake, and impact on galectin-3 mediated apoptosis of lactose functionalized dendrimers' submitted to the journal 'Biomolecules special issue: moving forward with dendrimers' which is contained within this thesis.; Kyle Tweedy and Mary J. Cloninger were co-authors of the article, 'Lactose functionalized dendrimers impact galectin-3 mediated cancer cell migration in vitro' submitted to the journal 'ACS chemical biology' which is contained within this thesis.Cancer has become a prevalent disease that is the second leading cause of death in the United States. Various cancers have been identified as either over or under expressing a sugar binding protein: galectin-3. The target of this research is to investigate cancerous events that are impacted by galectin-3 and mediate these events through the use of a multivalent binding partner to galectin-3. This binding partner is lactose functionalized PAMAM dendrimers. Apoptosis has been reported as another phenomenon that galectin-3 impacts. By using a reporter assay, viability, cytotoxicity and apoptosis were observed for cancer cell line A549 in the presence of exogenously added galectin-3 and/or lactose functionalized dendrimers. It was found that exogenous galectin-3 and glycodendrimers did not have any significant impact on these cell viability tests. Therefore, glycodendrimers can be used to probe multivalent effects without threat of toxicity. Metastasis was investigated through a modified in vitro scratch assay. By monitoring the migration of cancer cells, it was found that exogenously added galectin-3 retarded cell migration. When glycodendrimers were included, migration was partially restored. This revealed the implications of exogenous galectin-3 regarding the metastatic potential of carcinomas. When the implications of the domains of galectin-3 were investigated, it was found that the truncated galectin-3 containing only the carbohydrate recognition domain (CRD) was unable to replicate the same effects observed in full length galectin-3. Immunofluorescence microscopy was used to locate the multivalent binding partner and galectin-3 in the assay. While endocytosis of galectin-3 was observed, no colocalization with the multivalent binding partner was observed intracellularly, supporting the hypothesis of an extracellular interaction mediating the results. Multivalent interactions between glycodendrimers and galectin-3 impacted cellular migration. Angiogenesis revealed that exogenous galectin-3 induced neovascularization. Glycodendrimers impacted galectin-3 mediated angiogenesis. Glycodendrimers alone could elicit effects either enhancing or negating angiogenesis depending on the dendrimer generation. Fluorescent tags revealed glycodendrimer accumulation on or inside the cells and galectin-3 on the surface of cell groups. Overall, these studies show that glycodendrimers can interact multivalently and affect cellular processes.Item Multivalently presented carbohydrates can be used as drug delivery vehicles and to study protein carbohydrate interactions(Montana State University - Bozeman, College of Letters & Science, 2018) VanKoten, Harrison Wesley; Chairperson, Graduate Committee: Mary J. Cloninger; Wendy M. Dlakic, Robert Engel and Mary J. Cloninger were co-authors of the article, 'Synthesis and biological activity of highly cationic dendrimer antibiotics' in the journal 'Molecular pharmaceutics' which is contained within this thesis.; Rebecca Moore, Coleen Murphy and Mary J. Cloninger were co-authors of the article, 'Probing the LEC-1 and LEC-10 oxidative stress pathway in Caenorhabditis elegans using GALBeta1-4FUC dendrimers' which is contained within this thesis.Dendrimers in general excel as drug delivery vehicles since there are many different ways they can be assembled and different ways to tailor them to the system being studied. Glycodendrimers are generally nontoxic and can be further developed to meet the needs of what is being studied. For instance, in the studies below, a quaternity ammonium compound (QAC) has been attached to a glycodendrimer to determine the antimicrobial activity of a multivalently presented QAC in studies of minimum inhibitory concentration (MIC), biofilm prevention, and bacterial resistance. Results include comparable MICs to those of established antibiotics, prevention of biofilm formation but not disruption of an established biofilm, and establishment of multivalency as a strategy to counteract bacterial resistance. Another heterogeneously functionalized dendrimer was synthesized to study drug release characteristics of a prodrug attached to a cleavable substrate. In these studies, the upregulation of several proteins during cancer progression was taken advantage of including; MMP-2, -7, -9, and galectin-3. Glycodendrimers are tools used to study protein carbohydrate interactions. Study of galectins and their corresponding Beta-galactosides have illuminated their role in several essential biological processes. Multivalency plays a crucial role in many protein-carbohydrate interactions. Galectins are known to interact multivalently with various ligands. Although the role of galectins in this process is not yet fully understood, galectins have been proposed to serve as protective proteins during periods of high oxidative stress. We describe the synthesis of GalBeta1-4Fuc functionalized poly(amidoamine) (PAMAM) dendrimers in order to test C. elegans' response to high oxidative stress. In order to test the function of GalBeta1-4Fuc in vivo, C. elegans were treated with RNAi to knockdown lec-1 or lec-10, and then treated with glycodendrimer and exposed to oxidative stress. C. elegans that were pre-treated with the glycodendrimers were less susceptible to oxidative stress than untreated controls. The glycodendrimers mainly appeared within the digestive tract of the worms, and uptake into the vulva and proximal gonads could also be observed in some instances. This study indicates that multivalently presented GalBeta1-4Fuc can protect C. elegans from oxidative stress by binding to galectins.Item The synthesis of N-acetyllactosamine functionalized dendrimers, and the functionalization of silica surfaces using tunable dendrons and beta-cyclodextrins(Montana State University - Bozeman, College of Letters & Science, 2017) Ennist, Jessica Helen; Chairperson, Graduate Committee: Mary J. Cloninger; Mary J. Cloninger was a co-author of the article, 'The synthesis of N-acetyllactosamine functionalized dendrimers and their role in galectin-3 mediated cancer cellular aggregation studies' which is contained within this thesis.; Eric A. Gobrogge, Kristian H. Schlick, Robert A. Walker and Mary J. Cloninger were co-authors of the article, 'Cyclodextrin-functionalized chromatographic materials tailored for reversible adsorbtion' in the journal 'ACS applied materials and interfaces' which is contained within this thesis.Galectin-3 is beta-galactoside binding protein which is found in many healthy cells. In cancer, the galectin-3/tumor-associated Thomsen-Friedenreich antigen (TF antigen) interaction has been implicated in heterotypic and homotypic cellular adhesion and apoptotic signaling pathways. However, a stronger mechanistic understanding of the role of galectin-3 in these processes is needed. N-acetyllactosamine (LacNAc) is a non-native ligand for galectin-3 which binds with comparable affinity to the TF antigen and therefore an important ligand to study galectin-3 mediated processes. To study galectin-3 mediated homotypic cellular aggregation, four generations of polyamidoamine (PAMAM) dendrimers were functionalized with N-acetyllactosamine using a four-step chemoenzymatic route. The enzymatic step controlled the regiochemistry of the galactose addition to N-acetylglucosamine functionalized dendrimers using a recombinant beta-1,4-Galactosyltransferase-/UDP-4'-Gal Epimerase Fusion Protein (lgtB-galE). Homotypic cellular aggregation, which is promoted by the presence of galectin-3 as it binds to glycosides at the cell surface, was studied using HT-1080 fibrosarcoma, A549 lung, and DU-145 prostate cancer cell lines. In the presence of small LacNAc functionalized PAMAM dendrimers, galectin-3 induced cancer cellular aggregation was inhibited. However, the larger glycodendrimers induced homotypic cellular aggregation. Additionally, novel poly(aryl ether) dendronized silica surfaces designed for reversible adsorbtion of targeted analytes were synthesized, and characterization using X-ray Photoelectron Spectroscopy (XPS) was performed. Using a Cu(I) mediated cycloaddition 'click' reaction, beta-cyclodextrin was appended to dendronized surfaces via triazole formation and also to a non-dendronized surface for comparison purposes. First generation G(1) dendrons have more than 6 times greater capacity to adsorb targeted analytes than slides functionalized with monomeric beta-cyclodextrin and are 2 times greater than slides functionalized with larger generation dendrons. This study reported beta-cyclodextrin functionalized surfaces can undergo a triggered release of the adsorbent, but otherwise retained the targeted analyte through multiple aqueous washes. Therefore, a new generation of G(1) dendronized surfaces capable of reversible adsorption were developed by heterogeneously appending sulfonic acid/pyridine end-groups. Auger Electron Spectroscopy (AES) was used to quantify the ratio of groups installed. Furthermore, G(1) dendronized surfaces were functionalized homogenously with sulfonic acid and pyridine for comparison and with chiral amino acids for chiral recognition studies.Item The synthesis and study of TF antigen functionalized dendrimers and dendrimer end group characterization and indium(III) promoted glycosylation(Montana State University - Bozeman, College of Letters & Science, 2017) Mattson, Amanda Lynn; Chairperson, Graduate Committee: Mary J. Cloninger; Anna K. Michel and Mary J. Cloninger were co-authors of the article, 'Using indium(III) as a promoter for glycosylation' in the journal 'Carbohydrate research' which is contained within this thesis.Polyamidoamine (PAMAM) dendrimers were functionalized with the Thomsen-Friedenreich (TF) antigen to study the multivalent effects on the galectin-3 mediated homotypic aggregation of A549 cells. TF antigen functionalized dendrimers of generations 2, 3, 4, and 6 were found to induce cellular aggregation. This is in contrast with previously observed results using lactose functionalized dendrimers, in which lactose functionalized generation 2 dendrimers were able to inhibit cellular aggregation. Additionally, TF antigen functionalized generation 6 dendrimers did not induce cellular aggregation as effectively as lactose functionalized generation 6 dendrimers. These preliminary results suggest that when compared to lactose functionalized dendrimers, the stronger galectin-3 binding affinity for TF antigen dendrimers may allow for more galectin-3 recruitment, creating aggregates with less freedom to rearrange into an optimized conformation. This suggests the reversibility of the binding event is important for effective protein interactions. The synthesis of TF antigen was achieved using indium triflate catalyzed glycosylation reactions. The development of indium(III) as a glycosylation promoter involved the analysis of indium bromide, indium chloride, and indium triflate for use in glycosylation reactions with a variety of alcohol acceptors. In(OTf) 3 mediated glycosylations of acetonide protected mannosides afforded exclusively alpha products in high yields. Acetylated mannosides gave moderate yields of exclusively alpha products using InBr 3 and InCl 3. Benzylated galactosides gave moderate yields of alpha, beta product mixtures using In(OTf) 3, with beta products formation being favored. Indium(III) was also used to synthesize alpha-1-2-dimannoside and alpha-galactose-1-2-mannoside in high yield. Additionally, spin labelled PAMAM dendrimers were preliminarily studied via electron paramagnetic resonance (EPR) to analyze the spatial arrangement of end groups. Dendrimers were functionalized with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) covalently tethered as a dimer. At low percent loading, a strong effect arising from the dimeric spin-labeled end groups was observed. EPR spectra of dendrimers bearing a higher loading of the dimeric spin-labeled end groups indicated that the end group arrangement approached a random distribution at approximately 40 to 50 percent loading. This suggests that covalently clustered pairs of end groups are significantly different from randomly distributed end groups on PAMAM dendrimers at low loading and become equivalent to randomly functionalized dendrimers around 50% functionalization.Item Effect of monomeric binding affinity on scaffold mediated protein aggregation(Montana State University - Bozeman, College of Letters & Science, 2015) Goodman, Candace Kay; Chairperson, Graduate Committee: Mary J. CloningerThe intermolecular interactions that occur in a system determine the degree and duration of the contact. They govern processes from signaling and recognition to aggregation and tumor formation. The ability to control and affect intermolecular processes requires an understanding of the assembly process and factors modulating the assembly, such as the strength of individual interactions (binding affinity) and the number of interactions between molecules (valency). Functionalized PAMAM dendrimers were used as nucleating scaffolds to study the significance of intermolecular interactions on aggregate assembly. Dendrimers functionalized with biotin, lactose and mannose units spontaneously aggregated when added to the appropriate protein binding partner (streptavidin, galectin-3, and Concanavalin A, respectively). Aggregates were characterized to provide insight regarding the effects of binding affinity, protein valency and concentration on the average diameter, regularity (polydispersity) and kinetics of aggregate formation. A number of tools were used in this investigation, including dynamic light scattering (DLS), fluorescence microscopy (FM) and fluorescence lifetime spectroscopy (FLS). FLS instrumentation was reconfigured to enable high thoughput formats. A discussion of the validation and re-design of the FLS instrumentation is included.Item The glycosyl moeity of sainfoin lectin(Montana State University - Bozeman, College of Letters & Science, 1978) Namen, Anthony EugeneItem Grasshopper lectin : cDNA sequence, amino acid sequence and computer-based homology model(Montana State University - Bozeman, College of Letters & Science, 1996) Radke, Jay RichardItem Grasshopper agglutinin : preparation and characterization by MALDI/TOF-MS(Montana State University - Bozeman, College of Letters & Science, 1996) Wenzlick, Donald Lee