Five monoclonal antibodies (MAb), 7D4, 4C3, 6C3, 4D3, and 3C5, were produced in mice immunized with high buoyant density embryonic chick bone marrow proteoglycans (PGs) as antigen. All of these MAb recognized epitopes in native chick bone marrow and cartilage PGs which could be selectively removed by chondroitinase ABC and chondroitinase AC II, indicating that their epitopes were present in chondroitin sulfate glycosaminoglycans (GAGs). These MAb recognized epitopes present in purified cartilage PGs obtamed from a wide variety of different vertebrate species. However, none ofthe new MAb detected epitopes in Swarm rat chondrosarcoma PG. On the basis of these results, we propose that these MAb recognize novel epitopes located in chondroitin sulfate/dermatan sulfate glycosaminoglycan (CS/DS GAG) chains, representing at least four and possibly five different structures. Immunocytochemical studies have shown that the epitopes identified by these new MAb
820BIOCHEMICAL SOCIETY TRANSACTIONS tion staining, and indicated that in all probability BMCSPG is absent from the mature glomerular capillary loop. Our data suggest, therefore, that BMCSPG does not have an obligatory structural role in terms of basement membrane assembly, as has been proposed for the large HSPG. Neither does BMCSPG appear to have a role in regulating glomerular permselectivity.We are currently studying kidney development to monitor the appearance and distribution of BMCSPG. Early data support our findings in rat skin [ 121, which showed that this basement membrane component, unlike many others, is strongly developmentally regulated. BMCSPG staining is absent from newly formed basement membranes surrounding early post-induction structures, but appears later at the time of vascular invasion. It may also be that the proteoglycan is transiently expressed in the glomerular basement membrane around the time of its maturation. Its presence, therefore, coincides with the completion of morphogenetic events, and indicates that BMCSPG has a role in basement membrane stability. Such a hypothesis is supported by a study of patients with dystrophic forms of epidermolysis bullosa, where basement membrane chondroitin sulphate was shown to be absent or present to a much reduced extent [ 131. In contrast, antibodies to other basement membrane components, including HSPG [ 131 stained the skin basement membranes of dystrophic epidermolysis bullosa patients normally. Characteristically, these patients have skin basement membrane fragility with cleavage from the underlying dermis. It therefore appears that a specific defect in BMCSPG expression correlates with severe basement membrane abnormality.Finally, the lack of BMCSPG in the normal adult rat glomerular capillary loop raised the question of potential abnormality in diabetes, where mesangial expansion and thickening of the capillary loop and other basement membranes is a corollary of the disease process. Indeed our preliminary data with a streptotozocin model of diabetes in the rat indicate that BMCSPG is abnormally expressed in the kidney glomerulus of rats which have been diabetic for 2 months. It seems, therefore, that although a minor basement membrane component, BMCSPG may be important in development and some diseases where basement membrane integrity or stability are compromised.Chondroitin sulphate (CS) proteoglycans are major components of cartilage and lesser constituents of many other connective tissues. Current literature indicates that the major biomechanical functions of the polyanionic CS glycosaminoglycan chains of the cartilage proteoglycans are to contribute towards maintenance of a Donnan osmotic pressure within the cartilage, to participate in excluded volume effects and to contribute to the viscoelastic properties of the tissue (see [ 1-31 for reviews). Biochemical analysis of articular cartilage proteoglycans indicates that expression of different isomeric forms of CS occurs during normal growth and development, and in pathology [4-61. The pu...
Vitamin C deficiency causes morphologic changes in the endothelial and smooth muscle compartments of guinea pig blood vessels. Endothelial cells synthesize the basement membrane components, type IV collagen and laminin, and smooth muscle cells synthesize elastin in blood vessels. Therefore, we examined the possibility that vitamin C deficiency affects the expression of these proteins. Decreased expression of types I and II collagens in other tissues of vitamin C-deficient guinea pigs is associated with weight loss and the consequent induction of insulin-like growth factor binding proteins; thus we also used food deprivation to induce weight loss. Female guinea pigs received a vitamin C-free diet, supplemented orally with ascorbate. Vitamin C-deficient guinea pigs received the same diet but no ascorbate, and the food-deprived group received no food, but were supplemented with vitamin C. Concentrations of mRNAs for basement membrane components and elastin in blood vessels were measured by Northern blotting; overall basement membrane metabolism was assessed by measuring immunoreactive laminin and type IV 7S collagen in serum. Laminin mRNA in blood vessels and serum laminin concentrations were unaffected by vitamin C deficiency. Concentrations of type IV collagen and elastin mRNAs in blood vessels were not significantly affected in moderately scorbutic guinea pigs (0-7% weight loss), but with increased weight loss, type IV collagen mRNA was 57% (P < 0.05) and elastin mRNA was 3% (P < 0. 01) of normal values. In food-deprived guinea pigs, type IV collagen mRNA was 51% (P < 0.05) and elastin mRNA was 35% (P < 0.05) of normal. Serum type IV 7S collagen concentrations were 25% of normal in scorbutic guinea pigs with extensive weight loss. The lower expression of type IV collagen and elastin mRNAs in blood vessels may contribute to defects observed in blood vessels during scurvy.
Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.
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