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Spin-trapping agent alpha-phenyl N-Tert-butylnitrone as inhibitor of trypsin and enhancer of the oxidative degradation of the enzyme by heparin
No full textThe oxidative mechanism of heparin interferes with radical production by glucose and reduces the degree of glycooxidative modifications on human serum albumin
No full textImmune complexes of glucose-regulated protein94 (Grp94) with IgG, present in the plasma of type 1 diabetic subjects, show strong angiogenic-transforming capacity on HUVECs
No full textInduction of angiogenesis by the heat shock protein Glucose-regulated protein94 (Grp94) mediated by a cytokine-like mechanism.
No full textIdentification and purification from the plasma of Type 1 diabetic subjects of a proteolytically active Grp94Evidence that Grp94 is entirely responsible for plasma proteolytic activity.
Full text linkAIMS/HYPOTHESIS:
The overall increase in proteolytic activity in diabetes is known to be associated with the development and progression of vascular complications. Our aim was to investigate in detail the molecular nature of this activity in the plasma of Type 1 diabetic subjects.
METHODS:
Plasma of both diabetic and control subjects was subjected to various purification procedures (ion exchange and affinity chromatography, HPLC, immunoprecipitation, electrophoresis, immunoblot and mass analyses) to identify the proteins of interest. Biological activities were measured on specific substrates.
RESULTS:
In diabetic but not normal plasma we identified the presence of two heat shock proteins, Grp94 (Glucose-regulated protein94) and HSP70. The higher-than-normal proteolytic activity of Grp94 was: (i) directed against casein, but not against endogenous plasma proteins; (ii) fully and specifically inhibited only by anti-Grp94 polyclonal antibodies; and (iii) coupled with low-level ATPase activity. In addition, ATP binding to Grp94 was able to modulate proteolytic activity. We found that Grp94 in plasma circulates only as high molecular mass homo- and hetero-complexes, the latter mostly formed with IgG to which Grp94 is also linked by tenacious binding. Proteolytically-active Grp94 was purified by immunoprecipitation, which co-immunoprecipitated alpha(1)antitrypsin.
CONCLUSION/INTERPRETATION:
Our results show the unexpected extracellular location and characteristic biological function of Grp94 even at a late stage of disease. These findings have physiopathological relevance for predicting activation of both autoimmune and inflammatory processes potentially associated with vascular complications
Comment on: Antibodies to the endoplasmic reticulum-resident chaperones calnexin, Bip and Grp94 in patients with rheumatoid arthritis and systemic lupus erythematosus
No full textProteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects
Full text linkThe 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
Characterization of immune complexes of idiotypic catalytic and anti-idiotypic inhibitory antibodies in plasma of type 1 diabetic subjects
No full textAn increase in proteolytic activity is an early common feature of diabetes, and is associated with the development of vascular complications. We performed an extensive proteomic investigation on plasma of type 1 diabetic subjects to discover why some of them apparently lacked any measurable proteolytic activity. Activity was found enclosed in immune complexes in which Fab/(Fab)(2) displayed a serine-like catalytic activity. Disaggregation of complexes by means of Protein G affinity chromatography led to the separation of free subunits of Fab, showing a specific amidolytic activity, from Fab that displayed activity on casein and remained closely complexed with whole IgG. On both types of Fab the serine catalytic site appeared to be the same, being located in close vicinity to the antigen-binding site. The distinct substrate specificity was due to the different conformation adopted by the catalytic site depending on the structure of Fab/(Fab)(2), whether in complexes or as free subunits. Catalytic Fab/(Fab)(2) originated from idiotypic antibodies developed against Grp94, identified as the primary antigen covalently complexed with Fab. Whole IgG present in immune complexes were instead mostly formed with anti-idiotypic antibodies developed against the adduct of Fab/(Fab)(2) with Grp94, and were responsible for blocking any catalytic activity. In dot-blot experiments with native Grp94, we confirmed that in any diabetic plasma circulated anti-Grp94, idiotypic, and anti-idiotypic antibodies
Immune complexes of Glucose-regulated protein94 (Grp94) with IgG are present in the plasma of Type 1 diabetic subjects and show strong angiogenic-transforming capacity on HUVECs (oral communication)
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