Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
The Occurrence of Nicked Human Chorionic Gonadotropin (hCG) by a Thermolytic Endoprotease
鍵本 明男榊原 隆三福島 直美池田 宣子石黒 正恒
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1995 年 18 巻 6 号 p. 810-817

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A nicked form of human chorionic gonadotropin (nicked hCG), in which only one peptide bond between residues 47 and 48 (-Gly-Val-) of β-subunit is cleaved, has been found in the urine and blood of pregnant women. In this study, we investigated the occurrence of nicked hCG and the localization of the nicking enzyme for hCG. First, to determine what type of protease nicks hCG, an in vitro proteolytic study using various proteases was performed. Amino-terminal amino acid sequence analysis of the β-subunit purified from protease-treated hCG indicated that thermolysin actively nicks hCG. Secondly, to determine which tissues are related to the formation of nicked hCG, the distribution of radioactivity in various tissues after i. v. administration of radiolabeled hCG to female rats was examined. The radioactivity accumulated predominantly in the kidney (17%), liver (9.3%) and ovary (0.9%) after 30 min of injection. Analysis of molecular species of β-radiolabeled hCG in various tissues and body fluids, using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography, indicated that a nicked hCG-like molecule was found in the kidney, predominantly, as well as in the serum and urine. To examine the role of the kidney in producing nicked hCG, hCG was incubated with rat kidney particulate fraction (KPF). Immunoblot analysis of KPF-treated hCG indicated that KPF produced a nicked hCG-like molecule. Furthermore, the possibility that placental trophoblast cells produce nicked hCG was also examined using the choriocarcinoma cell BeWo. It was revealed that 1 to 5% of the hCG secreted from BeWo cells to the medium was assessed to be the nicked form by immunoblot analysis of the medium. Taken together, these results suggested that the nicking of hCG may be caused by thermolytic protease in trophoblast cells before or concomitantly on secretion, as well as in the kidney cortex after secretion. In the kidney proximal tubular cells, neutral endopeptidase EC 3. 4. 24. 11 (endopeptidase 24. 11) is localized, and its substrate specificity is very similar to that of thermolysin. Thus, the possibility is raised that endopeptidase 24. 11 might be a nicking enzyme for hCG. However, we have been unable to demonstrate that purified endopeptidase 24. 11 forms nicked hCG from hCG, indicating that the hCG-nicking enzyme may be an endoprotease unidentified so far.

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