heparitin-sulfate and trifluoromethanesulfonic-acid

Proteoglycans interact with soluble proteins such as growth factors and thereby regulate extracellular signals. During B lymphocyte maturation, secretion of proteoglycans may be functionally related to the different requirements of the respective maturation stage. In order to address this question we compared structures of proteoglycans released by three B lymphocyte lines which correspond to different maturation stages. Plasma-cell type U266 cells secreted the largest proteoglycans (150 kDa), followed by mature B cells JOK-1 (130 kDa) and pre-B cells Nalm 6 (90 kDa). On average, secreted proteoglycans carried four glycosaminoglycan chains with molecular masses ranging each from 32 kDa (U266) to 23 kDa (Nalm 6). All three cell lines secreted more than 90% of their proteoglycans possessing chondroitin sulfate chains having chondroitin-4-sulfate (delta Di-4S) as the prevalent disaccharide unit. In these proteochondroitin sulfates, unsulfated chondroitin (delta Di-0S) was present in smaller quantities and chondroitin-6-sulfate (delta Di-6S)-containing proteoglycan was released only by Nalm 6 and U266 cells. Cell line Nalm 6 exclusively produced proteochondroitin sulfate, whereas in culture medium of JOK-1 and U266 a small amount of proteoheparan sulfate was found also. In all three cell lines, treatment with chondroitinase ABC released a protein of 30 kDa and chemical deglycosylation resulted in a core protein of 21 kDa. In addition to pure proteochondroitin sulfate, a small portion of proteoheparan sulfate with a protein moiety of 30 kDa was detected after heparitinase treatment in supernatants of JOK-1 and U266. Thus, our results indicate that released proteoglycans may undergo modulations in their glycosaminoglycan moieties during B-cell differentiation. This may have functional consequences at the level of growth factor regulation.

Topics: B-Lymphocytes; Cell Line; Centrifugation, Density Gradient; Chondroitin Lyases; Chondroitin Sulfates; Chromatography, Agarose; Chromatography, Gel; Culture Media; Electrophoresis, Polyacrylamide Gel; Glycosylation; Heparitin Sulfate; Humans; Mesylates; Molecular Weight; Polysaccharide-Lyases; Proteoglycans

In the very old, there is an increased prevalence of immunologically mediated vascular disease. Vascular heparan sulfate proteoglycan (vHSPG) is a molecule which plays an important structural and functional role in the vasculature. Autoimmunity to vHSPG may play a role in the pathogenesis of vascular disease. The presence of autoantibodies to vHSPG was investigated in sera from aged nursing home patients who have a high prevalence of vascular disease. The results showed an association of the presence of autoantibodies to vHSPG in the sera from patients in this aged population with vascular disease, particularly cerebrovascular disease, renal disease and diabetes. In addition, autoantibodies to vHSPG protein core were associated with cerebrovascular disease in this frail elderly population. These studies support the hypothesis that autoimmunity may play a role in vascular disease in the aged.

Topics: Aged; Aged, 80 and over; Autoantibodies; Autoimmunity; Blood Vessels; Enzyme-Linked Immunosorbent Assay; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Male; Mesylates; Proteoglycans; Vascular Diseases

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

Topics: Animals; Centrifugation, Density Gradient; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Female; Glycosylation; Guanidine; Guanidines; Heparan Sulfate Proteoglycans; Heparin Lyase; Heparitin Sulfate; Liver; Male; Mesylates; Molecular Weight; Peptide Mapping; Polysaccharide-Lyases; Rats; Rats, Inbred Strains; Serine Endopeptidases

1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had an apparent molecular mass of 18 kDa. Results of heparitinase digestion and HNO2-treatment indicated a clustering of sulfate groups in the distal part of the HS side chains. 5. Peptide mapping after trypsin, clostripain or V8 protease digestion of radiolabeled human and equine heparan sulfate proteoglycans (HSPG) preparations with three different separation techniques showed large differences. 6. Polyclonal antisera raised against the HSPGs reacted against the core proteins. Both HSPG preparations and their antisera showed ca 40% cross-reactivity. About 50% of monoclonal antisera elicited against one HSPG preparation showed reaction with both HSPG preparations. 7. Polyclonal antisera stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies of kidney sections from horse, man and various mammalian species. 8. Biochemical and immunological data indicate that HSPGs from equine and human GBM have a comparable structure, but the core proteins differ considerably.

Topics: Amino Acids; Animals; Basement Membrane; Carbohydrates; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Chromatography, Ion Exchange; Epitopes; Fluorescent Antibody Technique; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Horses; Humans; Immune Sera; Immunoblotting; Kidney Glomerulus; Mesylates; Molecular Weight; Peptide Mapping; Polysaccharide-Lyases

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.

Topics: Amino Acids; Antibodies, Monoclonal; Basement Membrane; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Chromatography, Ion Exchange; Fluorescent Antibody Technique; Glycosaminoglycans; Guanidine; Guanidines; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Hydrolysis; Kidney Glomerulus; Kidney Tubules; Mesylates; Polysaccharide-Lyases; Proteoglycans