ABSTRACT
Glycation of basement membrane collagen IV has been implicated as a major pathogenetic process leading to diabetic microvascular complications. To evaluate the relevance of carbohydrate-induced modifications on collagen IV in diabetic nephropathy, we isolated the cross-linking domains 7S and NC1 from the glomerular basement membrane (GBM) of patients with diabetes mellitus. Modifications characteristic for glycated proteins were identified when the domains from diabetic kidney were compared with the same domains from human placenta as an unmodified control. In both domains a marked formation of inter-and intramolecular cross links could be demonstrated by SDS-PAGE. Furthermore circular dichroism studies showed a decrease in helicity of the 7S domain from human diabetic kidneys of 13%, indicating denaturation already at room temperature. Thermal transition profiles, showing a shift of the denaturation temperature towards a lower temperature, with loss of a distinct second melting point, confirmed this observation. Our data provide further evidence for a possible role of protein-modification by glycoxidative reactions in the onset of diabetic nephropathy in vivo.
Subject(s)
Collagen/chemistry , Diabetic Nephropathies/metabolism , Kidney/metabolism , Basement Membrane/metabolism , Blotting, Western , Circular Dichroism , Collagen/metabolism , Diabetes Mellitus, Type 2/metabolism , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Humans , In Vitro Techniques , Kidney Glomerulus/metabolism , Molecular Weight , Placenta/metabolism , Protein ConformationABSTRACT
Non-enzymic interactions of carbohydrates and proteins are a major feature of cumulative modification in basement membranes in the course of diabetic microvascular complications. To evaluate the significance of both glycation and glycoxidation reactions for subsequent alterations of biochemical properties, we examined the effects of in vitro glycation on distinct collagen IV domains under different experimental conditions. The 7 S domain and the major triple-helical domain from human placental collagen IV were incubated for various time intervals up to 14 days at 37 degrees C in the presence of different concentrations of either glucose or ribose under oxidative and antioxidative conditions. Carbohydrate-induced non-enzymic modification in two collagen IV domains was revealed by increased cross-linking and fluorescence. In addition, these non-enzymic modifications apparently have a major impact on molecular conformation and thermal stability of collagen IV, which in turn might influence both cell-matrix interactions and matrix assembly.
Subject(s)
Collagen/chemistry , Collagen/metabolism , Glucose/metabolism , Protein Conformation , Ribose/metabolism , Basement Membrane/chemistry , Circular Dichroism , Collagen/isolation & purification , Female , Glycosylation , Humans , Molecular Weight , Placenta/metabolism , Pregnancy , Protein Denaturation , Spectrometry, FluorescenceABSTRACT
The expression of vigilin was followed during chick embryonal development by in situ hybridization. Vigilin mRNA is abundantly expressed in tissues of mesenchymal and ectomesenchymal origin. The mesenchymal primordial cells of cartilage and bone did not show any significant expression of vigilin. As tissue differentiation proceeded, vigilin mRNA levels increased in hyaline cartilage and in both endochondral as well as intramembranous bone. The results suggest that the expression of vigilin mRNA in cartilage- and bone-forming cells, chondrocytes and osteoblasts, is dependent on the stage of development and cellular differentiation, although not a unique process of bone formation. Most striking is the correlation of the maximum vigilin mRNA expression in osteoblasts and hypertrophic chondrocytes to periods when cell-specific genes were highly transcribed and substantially translated, e.g., synthesis of procollagen and formation of extracellular matrix in bone and cartilage.
Subject(s)
Bone and Bones/metabolism , Carrier Proteins , Cartilage/metabolism , Proteins/metabolism , RNA-Binding Proteins , Animals , Bone and Bones/embryology , Cartilage/embryology , Cell Differentiation , Chick Embryo , Gene Expression , In Situ Hybridization , Osteoblasts/cytology , Osteoblasts/metabolism , Procollagen/genetics , Procollagen/metabolism , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
A fusion protein composed of about two vigilin domains and beta-galactosidase was used to raise polyclonal antibodies which were affinity-purified and employed for immunoblotting and immunohistochemistry. A protein of an apparent molecular mass of 155 kDa could be stained in extracts of a variety of cells from different species and organs. Immunohistological studies on single cells showed that vigilin is accumulated in the cytoplasm. During in vitro maintenance of primary cell cultures, as well as of a growth-factor-dependent cell line, vigilin expression decreases and ceases in senescent cells. In contrast, vigilin is constitutively expressed in all other transformed cell lines of various origin studies so far. Vigilin expression can be induced in peripheral blood lymphocytes by mitogen stimulation. These observations suggest an involvement of vigilin in processes of cell activation. Immunoblot experiments demonstrating the presence of vigilin in a broad range of eukaryotes, indicate a high degree of evolutionary conservation.
