John H, Preissner K T, Forssmann W G, Ständker L
Lower Saxony Institute for Peptide Research (IPF), Hannover, Germany.
Biochemistry. 1999 Aug 10;38(32):10217-24. doi: 10.1021/bi990787+.
Circulating elongated forms of the angiogenesis inhibitor and potential anti-cancer drug endostatin were isolated from human blood filtrate. Immunoreactive endostatin was identified by a polyclonal rabbit antiserum raised against an N-terminal epitope of the polypeptide and purified by consecutive chromatographic steps and immunoblotting. N- and C-terminal sequence analyses of the isolated molecules revealed different forms of endostatin starting with V(117)HLRPAR. lacking the last and final three residues of the noncollagenous domain 1 (NC-1) of collagen XVIII, respectively. These polypetides are found to be O-glycosylated at T(125) (residue 9) with a glycan structure of the mucin type consisting of galactose N-acetylgalactosamine and N-acetylneuraminic acid residues. Carbohydrate analyses were performed via the semiquantitative HPLC-electrospray ionization mass spectrometry (ESMS) technique after exoglycosidase hydrolysis. Circulating endostatins are present as sialoglycoprotein (22 000 and 21 841 Da +/- 0.02%) and asialoglycoprotein structures (21 710 and 21 549 Da +/- 0.02%), while the two completely deglycosylated forms are obtained only after enzymatic incubation. The described glycosylated endostatins may represent intermediates in the proteolytic pathway of the NC-1 domain of collagen XVIII resulting in bioactive endostatins. Furthermore, immunoreactive endostatin-related C-terminal fragments of human collagen XV are found in the hemofiltrate. These polypeptides exhibit the N-terminal sequences P(66)HLLPPP. and Y(81)EKPALH. of the collagen XV NC-1 domain. ESMS and immunoblotting analyses reveal three glycosylated polypeptides with a molecular mass ranging from 16 to 21 kDa. Due to the high degree of homology between collagen XV and collagen XVIII as well as their analoqous proteolytic processing, functional similarities of collagen XVIII- and XV-related fragments should be revealed in future experiments.
从人血液滤液中分离出血管生成抑制剂及潜在抗癌药物内皮抑素的循环延长形式。通过针对该多肽N端表位产生的兔多克隆抗血清鉴定免疫反应性内皮抑素，并通过连续的色谱步骤和免疫印迹进行纯化。对分离出的分子进行N端和C端序列分析，发现了不同形式的内皮抑素，分别以V(117)HLRPAR开头，缺少胶原蛋白XVIII非胶原结构域1（NC-1）的最后三个残基。发现这些多肽在T(125)（第9位残基）处发生O-糖基化，糖基结构为粘蛋白型，由半乳糖、N-乙酰半乳糖胺和N-乙酰神经氨酸残基组成。在外切糖苷酶水解后，通过半定量高效液相色谱-电喷雾电离质谱（ESMS）技术进行碳水化合物分析。循环内皮抑素以唾液酸糖蛋白（22 000和21 841 Da±0.02%）和去唾液酸糖蛋白结构（21 710和21 549 Da±0.02%）的形式存在，而两种完全去糖基化的形式仅在酶孵育后获得。所述糖基化内皮抑素可能代表胶原蛋白XVIII的NC-1结构域蛋白水解途径中的中间体，从而产生生物活性内皮抑素。此外，在血液滤过液中发现了人胶原蛋白XV的免疫反应性内皮抑素相关C端片段。这些多肽具有胶原蛋白XV NC-1结构域的N端序列P(66)HLLPPP和Y(81)EKPALH。ESMS和免疫印迹分析显示三种糖基化多肽，分子量范围为16至21 kDa。由于胶原蛋白XV和胶原蛋白XVIII之间的高度同源性以及它们类似的蛋白水解过程，未来的实验应揭示胶原蛋白XVIII和XV相关片段的功能相似性。
