Type-2 diabetes and innate immunity : new connections revealed by multi-dimensional fractionation of blood plasma prior to proteomic analysis
Laffoon, Scott Bradley.
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We compared levels of protein isoforms in human blood plasma from patients with newly diagnosed and untreated type-2 diabetes (T2DM) with non-diabetic controls in samples obtained from US NIH. We immunodepleted fourteen of the most abundant proteins from pooled plasma samples and separated the depleted samples into six fractions by reverse-phase liquid chromatography at 80°C. Proteins from these fractions were labeled with new high quantum yield, hydrophilic and spectrally resolved fluorescent detection dyes developed at MSU and resolved on large-format (24cm x 20cm) two-dimensional gels. By fluorescence analysis of 2D gels, using >1.4 fold change and p<0.05 acceptance criteria, we have identified five T2DM associated proteins and isoforms, including: two isoforms of zinc-alpha glycoprotein (ZAG), one isoform of serum amyloid A-1 (SAA-1) preprotein, one isoform of cysteine-rich secreted protein-3 (CRISP-3), one isoform of haptoglobin, and an A1-apolipoprotein fragment. Complement factor H related-5 (CFHR-5) is the likely identification of a sixth protein found significantly down in T2DM. Changes in the plasma levels of CRISP-3 and CFHR-5 strengthen the hypothesis that T2DM is a disease involving innate immunity. Three of these proteins are known to specifically bind to the transport protein, human serum albumin (HSA). Also, CRISP-3 is a specific and high-affinity ligand of alpha 1 beta glycoprotein, which is an HSA binder. To investigate HSA binding properties, we quantitatively measured the binding of a dye probe by HSA at neutral pH. These measurements revealed that HAS binding of the probe correlates with several metabolic parameters of central importance to the diagnosis of T2DM, including fasting plasma glucose (FPG). Therefore, this assay may reveal altered properties of HSA that could be developed for the clinical assessment of individuals' metabolic status. We sought modifications of HSA or altered cargo of HSA that may cause the difference in binding. 1D gels of plasma proteins reacted with maleimide dye showed no changed levels of the oxidation state of HSA's lone thiol, Cys-34. However, 1D blots of plasma proteins reacted with the oxidative carbonyl probe, hydrazide-biotin conjugate, and probed with luminol reactive HRP-neutravidin showed a surprising anti-correlation of HSA oxidation with hemoglobin A1c, an indicator of glycemic control.