Shworak N W, Shirakawa M, Mulligan R C, Rosenberg R D
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1994 Aug 19;269(33):21204-14.
The specificity of the glycosaminoglycan (GAG) acceptor sites of ryudocan was examined by stably expressing epitope-tagged ryudocan cDNA constructs in mouse L cells, which normally produce this proteoglycan. Immunopurified ryudocan was glycanated with both heparan sulfate (HS) and chondroitin sulfate (CS). The attachment of GAGs to ryudocan was prevented by creating Ser-->Thr mutations in all possible combinations at positions 44, 65, and 67. The resulting ryudocan of exogenous origin was immunopurified and evaluated with regard to attached GAG chains. The data reveal that ryudocan possesses three functional GAG attachment sites, that the sites are always occupied with GAG chains, and that each site is capable of bearing either HS or CS. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of GAG lyase digests of intact ryudocan reveal the production of the following multiple isoforms: pure HS-ryudocan, various HS/CS-hybrids, and pure CS-ryudocan. The data suggest that the occupancy bias of each site for HS or CS is slight and that each site functions in a relatively independent fashion. The GAG lyase analysis of partially purified L cell proteoglycans shows two pure CS-homoglycans with core proteins of M(r) = 130,000 and 52,000, respectively. A similar analysis of immunopurified L cell glypican demonstrates that this species only exists as a pure HS-homoglycan. The production of pure homoglycans by this clonal cell line strongly suggests that the functional promiscuity of GAG attachment sites of ryudocan must be encoded in the core protein structure. This property of ryudocan is not peculiar to L cells, as ryudocan synthesized by early passage human endothelial cells also bears both HS and CS. The production of multiple isoforms of ryudocan may serve to expand the functional versatility of this cell surface component and allow it to participate in many different biologic processes.
通过在通常产生这种蛋白聚糖的小鼠L细胞中稳定表达带有表位标签的ryudocan cDNA构建体，研究了ryudocan的糖胺聚糖（GAG）受体位点的特异性。免疫纯化的ryudocan与硫酸乙酰肝素（HS）和硫酸软骨素（CS）都发生了糖基化。通过在第44、65和67位以所有可能组合产生丝氨酸到苏氨酸的突变，阻止了GAG与ryudocan的结合。对所得的外源性ryudocan进行免疫纯化，并对附着的GAG链进行评估。数据显示ryudocan具有三个功能性GAG附着位点，这些位点总是被GAG链占据，并且每个位点都能够携带HS或CS。完整ryudocan的GAG裂解酶消化产物的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱显示产生了以下多种异构体：纯HS-ryudocan、各种HS/CS杂种以及纯CS-ryudocan。数据表明每个位点对HS或CS的占据偏向较小，并且每个位点以相对独立的方式发挥作用。对部分纯化的L细胞蛋白聚糖的GAG裂解酶分析显示了两种分别具有M(r)=130,000和52,000核心蛋白的纯CS同聚糖。对免疫纯化的L细胞磷脂酰肌醇蛋白聚糖的类似分析表明，该物种仅以纯HS同聚糖的形式存在。这种克隆细胞系产生纯同聚糖强烈表明，ryudocan的GAG附着位点的功能混杂性必定编码在核心蛋白结构中。ryudocan的这种特性并非L细胞所特有，因为早期传代的人内皮细胞合成的ryudocan也同时携带HS和CS。ryudocan多种异构体的产生可能有助于扩展这种细胞表面成分的功能多样性，并使其能够参与许多不同的生物学过程。
