van den Heuvel L P, van den Born J, Veerkamp J H, van de Velden T J, Schenkels L, Monnens L A, Schröder C H, Berden J H
Department of Biochemistry, University of Nijmegen, The Netherlands.
Biochim Biophys Acta. 1990 Jun 11;1025(1):67-76. doi: 10.1016/0005-2736(90)90191-p.
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.
硫酸乙酰肝素蛋白聚糖（HSPG）用胍从人肾小管基底膜（TBM）中提取，经离子交换色谱和凝胶过滤纯化。该糖缀合物对乙酰肝素酶敏感，对软骨素酶ABC有抗性，表观分子量为200 - 400 kDa，由70%的蛋白质和30%的糖胺聚糖组成。其氨基酸组成的特点是甘氨酸、脯氨酸、丙氨酸和谷氨酸含量高。用三氟甲磺酸水解产生160 kDa和110 kDa的核心蛋白。碱性NaBH4处理后得到的硫酸乙酰肝素（HS）链分子量约为18 kDa。乙酰肝素酶消化和HNO2处理的结果表明HS侧链远端部分存在硫酸基团簇集。这些化学数据与先前在肾小球基底膜（GBM）HSPG上获得的数据相当（Van den Heuvel等人，（1989年）《生物化学杂志》264卷，457 - 465页）。胰蛋白酶、梭菌蛋白酶或V8蛋白酶消化TBM和GBM HSPG制剂后获得的肽谱显示出很大的相似性。针对两种HSPG制剂并针对核心蛋白产生的多克隆抗血清和一组单克隆抗体在ELISA和蛋白质印迹中显示出完全交叉反应性。在对人肾的间接免疫荧光研究中它们以强烈的线性方式染色所有基底膜。基于这些生化和免疫数据，我们得出结论，人GBM和TBM的HSPG是相同或至少密切相关的蛋白质。
