Carboxy-terminal proteolytic processing at a consensus furin cleavage site is a prerequisite event for quail ZPC secretion.
In avian species, a glycoprotein homologous to mammalian ZPC is synthesized in the granulosa cells of developing follicles. We have previously reported that the newly synthesized ZPC (proZPC) in granulosa cells is cleaved at a consensus furin cleavage site to generate mature ZPC prior to secretion. In the present study, we examined the effect of the proteolytic cleavage of proZPC on ZPC secretion by using a specific inhibitor of furin endoprotease and site-directed mutagenesis of the furin cleavage site. Western blot analysis demonstrated that the furin inhibitor efficiently blocked both the proteolytic cleavage of proZPC and the subsequent ZPC secretion. A site-directed mutant that possessed a mutated sequence for furin cleavage was not secreted from the cells. The immunocytochemical observations indicated that proZPC produced in the presence of a furin inhibitor or those produced by the site-directed mutant of the furin cleavage site had accumulated in the endoplasmic reticulum. These results indicate that proZPC is proteolytically cleaved at the consensus furin cleavage site with furin-like protease, and the failure of this cleavage results in its accumulation in the endoplasmic reticulum. Therefore, the C-terminal proteolytic processing of proZPC at the consensus furin cleavage site is a prerequisite event for quail ZPC secretion.
Sasanami
Tomohiro
T
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Japan.
Toriyama
Masaru
M
Mori
Makoto
M
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D003370:Coturnix / Q000378:metabolism*; D006224:Cricetinae; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D045683:Furin / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006107:Granulosa Cells / Q000378:metabolism; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; D045683:Furin