Abstract
The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications.
Highlights
Previous studies indicated that plasma of Type 1 (T1) diabetic subjects contains elevated concentrations of the heat shock protein (HSP) glucose-regulated protein94 (Grp94) [1, 2]
No immune reaction for Grp94 was shown in control plasma, whereas an intense positivity was detected in any diabetic plasma, with some interindividual difference (Figure 1)
Previous observations suggested that these complexes could be immune in nature, based on the finding that anti-Grp94 Abs were present in diabetic plasma in response to extracellular exposure of Grp94 [8]
Summary
Previous studies indicated that plasma of T1 diabetic subjects contains elevated concentrations of the heat shock protein (HSP) glucose-regulated protein (Grp94) [1, 2]. In plasma of T1 diabetic patients Grp does not circulate as free protein but always binds to IgG, forming stable complexes that have been interpreted as immune in nature [1, 2] This was supported by the finding that anti-Grp antibodies are present in diabetic plasma in response to the immune stimulation driven by extracellular Grp94 [2]. Other observations indicated that Grp94-IgG complexes in diabetic plasma can induce an intense activation of inflammatory cell signaling pathways with angiogenic-like transformation of vascular cells [8]. This has led to the proposal that Grp94-IgG complexes might be a marker of increased risk of vascular complications in T1 diabetes [2]. The nonimmune nature of Grp94IgG complexes could shed new light on the pathogenetic mechanisms associated with diabetes and its complications
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