Characterization and biological properties of chemically deglycosylated human chorionic gonadotropin. Role of carbohydrate moieties in adenylate cyclase activation.
Characterization and biological properties of chemically deglycosylated human chorionic gonadotropin. Role of carbohydrate moieties in adenylate cyclase activation.
复制标题
化学去糖基化人绒毛膜促性腺激素的表征和生物学特性。
DOI：
10.1016/s0021-9258(19)45401-x
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
K. Catt
中科院分区：
文献类型：
--
作者：
H. C. Chen;Y. Shimohigashi;M. Dufau;K. Catt
纯化的人绒毛膜促性腺激素 (hCG) 的化学去糖基化是通过在苯甲醚存在下在 0℃ 下用无水 HF 处理 1 小时来进行的。产品在 Sephadex G-100 上的凝胶色谱显示与完整 hCG 的 Kav= 0.21 相比，Kav= 0.28 的单峰。HF 处理的亚基解离率小于 5%。 合并峰（表示为 HF-hCG）的十二烷基硫酸钠凝胶电泳显示两条带，其分子大小比天然 a 和 P 亚基小 30%。没有检测到氨基酸组成的变化，但碳水化合物分析表明，HF 处理去除了 76-87% 的唾液酸、半乳糖、甘露糖和岩藻糖，52% N-乙酰基-D-葡萄糖胺和14%的N-乙酰基-D-半乳糖胺。这些发现表明，HF 裂解主要发生在天冬酰胺连接部分的甘露糖残基和丝氨酸连接部分的半乳糖残基处。去糖基化的 hCG 不与刀豆球蛋白 A 结合，并且对亚硫酸品红呈惰性 高碘酸盐氧化后的试剂。当分析与 hCG 和 P 亚基抗血清的结合时，HF-hCG 与抗 hCG 系统中的 hCG 交叉反应相同，但在抗 hCGP 和抗 hCGP-COOH-肽系统中效力较低。在大鼠睾丸黄体生成素受体测定中，HF-hCG 的结合活性比完整的 HF-hCG 高 2.5 倍 人绒毛膜促性腺激素。相比之下，HF-hCG 在刺激间质细胞中睾酮和 cAMP 产生以及间质膜中腺苷酸环化酶激活后的体外生物活性比 hCG 低至少 100 倍。在大鼠子宫重量测定中，2.5 pg/只大鼠未检测到 HF-hCG 的体内生物活性，而 12.5 ng hCG 子宫重量增加了一倍。 1 ng/ml hCG 对 cAMP 产生的激活被 0.1 pg/ml HF-hCG 完全拮抗。类似地，1 pg/ml hCG 对间质膜中腺苷酸环化酶的激活被 2 pg/ml HF-hCG 抑制至基础水平。有证据表明，HF-hCG 在大鼠体内被迅速清除 腹腔注射后30分钟测得的HF-hCG血清最大含量不到注射3小时后hCG达到的10%。这些结果表明 hCG 的碳水化合物部分在激素与促黄体生成素受体结合后激活间质细胞反应中的重要性，并证明了去糖基化的拮抗剂特性 hCG 对大鼠睾丸中腺苷酸环化酶的影响。
Chemical deglycosylation of purified human chorionic gonadotropin (hCG) was performed by treatment with anhydrous HF in the presence of anisole at 0 “C for 1 h. Gel chromatography of the product on Sephadex G-100 revealed a single peak with Kav= 0.28, as compared to the Kav= 0.21 of intact hCG. Subunit dissociation by the HF treatment was less than 5%. Sodium dodecyl sulfate-gel electrophoresis of the pooled peak, denoted as HF-hCG, showed two bands with molecular sizes 30% lower than the native a and P subunits. No change in amino acid composition was detected, but carbohydrate analysis showed that the HF treatment removed 76-87% of sialic acid, galactose, mannose, and fucose, 52% of N-acetyl-D-glucosamine, and 14% of N-acetyl-D-galactosamine. These findings indicate that HF cleavages occurred predominantly at the mannose residues of the asparagine-linked moiety and the galactose residues of the serine-linked moiety. Deglycosylated hCG did not bind to concanavalin A, and was inert to the fuchsin-sulfite reagent after periodate oxidation. When analyzed for binding to hCG and P-subunit antisera, HF-hCG cross-reacted equally with hCG in the anti-hCG system, but was less potent in anti-hCGP and anti-hCGP-COOH-peptide systems. In the rat testis luteinizing hormone receptor assay, HF-hCG displayed 2.5 times higher binding activity than intact hCG. In contrast, the in vitro biological activities of HF-hCG upon stimulation of testosterone and cAMP production in Leydig cells and the activation of adenylate cyclase in interstitial membranes were at least 100-fold less active than hCG. No in vivo biological activity of HF-hCG was detectable in the rat uterine weight assay at 2.5 pg/rat, whereas 12.5 ng of hCG doubled the uterine weight. Activation of cAMP production by 1 ng/ml of hCG was completely antagonized by 0.1 pg/ml of HF-hCG. Similarly, activation of adenylcyclase in interstitial membranes by 1 pg/ml of hCG was inhibited to the basal evel by 2 pg/ml of HF-hCG. HF-hCG was rapidly cleared in rats as evidenced by the fact that the maximal serum content of HF-hCG measured at 30 min after intraperitoneal injection was less than 10% of that reached by hCG after 3 h of injection. These results indicate the importance of the carbohydrate moieties of hCG in activation of Leydig cell responses following hormone binding to the luteinizing hormone receptors, and demonstrate the antagonist properties of deglycosylated hCG upon adenylate cyclase in the rat testis.