Determination of different gonadotropin synthesis mechanisms in trophoblast and choriocarcinoma cells

人绒毛膜促性腺激素（hCG）是一种糖蛋白激素，由非共价键合的α和β亚基组成。两个亚基都含有两个Asn（N）连接的糖链，此外，β亚基含有四个Ser（O）连接的糖链。已经表明，绒毛膜癌产生的hCG的α-和β-亚基都诱导了糖基化的改变。本研究旨在探讨正常滋养层细胞和绒毛膜癌细胞系（BeWo）合成、分泌激素及未结合亚基结构差异的本质。用免疫结合法分析了正常滋养层细胞中hCG亚单位的胞内形式。结果表明，hCG亚单位积累在滋养层细胞中作为主要的中间体（未成熟的形式）含有高甘露糖糖链。另一方面，未成熟β亚基中未发现丝氨酸连接的糖链。分别纯化未成熟的hCG亚基，并测定其理化性质。各未成熟亚基的N-末端氨基酸序列与各自的成熟形式相同。将纯化的未成熟亚基相互重组并重建未成熟hCG。在滋养层细胞中发现的未成熟hCG以及用纯化的未成熟亚基重建的hCG的表观分子量大于预期。提示未成熟hCG以寡聚体形式存在于滋养层细胞中.比较绒毛膜癌BeWo细胞和正常滋养层细胞hCG及其亚单位的结构特征。发现BeWo细胞hCG亚基的蛋白质和糖链结构均不同于滋养层细胞。

Human chorionic gonadotropin (hCG) is a glycoprotein hormone consisting of noncovalently bonded subunits, alpha and beta. Both subunits contain two Asn(N)-linked sugar chains, in addition, the beta-subunit contains four Ser(O)-linked sugar chains. It has been suggested that altered glycosylation is induced in both alpha- and beta-subunits of hCG produced by choriocarcinoma. The objective of this study was to determine the nature of the structural differences of hormones synthesized in the cells and secreted as well as the uncombined subunits in normal trophoblasts and a choriocarcinoma cell line (BeWo).The results obtained from this study are as follows:1. Intracellular forms of hCG subunits in normal trophoblast cells were analyzed by immunobinding method. It was revealed that hCG subunits accumulate in trophoblast cells as predominant intermediates (immature forms) containing high-mannose sugar chains. On the other hand, Ser-linked sugar chain was not found in the immature beta-subunit.2. The immature hCG subunits were purified, separately, and their physicochemical properties were determined. N-terminal amino acid sequence of each immature subunit was identical to respective mature form. The purified immature subunits were recombined each other and reconstituted an immature hCG. The apparent molecular weights of the immature hCG found in trophoblast cells as well as hCG reconstituted with the purified immature subunits were larger than those expected. It was suggested that the immature hCG is present in trophoblast cells as an oligomer.3. The structural characteristics of hCG and its subunits in choriocarcinoma, BeWo cells, were compared to those of normal trophoblast cells. It was found that both protein and sugar chain structures of hCG subunits of BeWo cells were differed from those of trophoblast cells.