Actin and annexins I and II are among the main endothelial plasmalemma-associated proteins forming early glucose adducts in experimental diabetes

An immunochemical and biochemical study was performed to reveal which of th e endothelial plasma membrane proteins become glycated during the early pha ses of diabetes. The blood front of the lung microvascular endothelial plas malemma was purified by the cationic colloidal silica method from normal an d diabetic (streptozotocin-induced) rats and comparatively analyzed by two- dimensional electrophoresis. No major qualitative differences in the genera l spectrum of endothelial plasmalemmal proteins were recorded between normo glycemic and hyperglycemic animals. By probing with antiglucitollysine anti bodies, we found that at 1 month after the onset of diabetes, several endot helial membrane polypeptides contained glucose covalently linked to their l ysyl residues. Ten days of insulin treatment restored euglycemia in the dia betic animals and completely abolished the membrane nonenzymatic glycosylat ion. All the glycated polypeptides of the endothelial plasma membrane belon g to the peripheral type and are associated with its cytoplasmic face (cell cortex). They were solubilized by buffers of high pH and were not detected in the lung cytosolic fraction (100,000 g). By microsequencing, the major proteins labeled by the anti-glucitollysine have been identified as being a ctin, annexin I, annexin II, the p34 subunit of the Arp2/3 complex, and the Ras suppressor protein-1. Conversely, the intrinsic endothelial membrane p roteins do not seem to be affected by hyperglycemia. This defines the inter nal face of the:endothelial plasma membrane, particularly the cortical :cyt oskeleton, as a preferential target for nonenzymatic glycosylation in diabe tes, with possible consequences on the fluidity of the endothelial plasmale mma and impairment of the endothelial mechanotransducing ability.