Secretion of egg envelope protein ZPC after C‐terminal proteolytic processing in quail granulosa cells

Abstract

In avian species, an egg envelope homologous to the mammalian zona pellucida is called the perivitelline membrane. We have previously reported that one of its components, a glycoprotein homologous to mammalian ZPC, is synthesized in the granulosa cells of the quail ovary. In the present study, we investigated the proteolytic cleavage of the newly synthesized ZPC and the secretion of ZPC from the granulosa cells. Western blot analysis of the cell lysates demonstrated that the 43-kDa protein is the precursor of mature ZPC (proZPC), and is converted to the 35-kDa protein before secretion. The accumulation of proZPC in the presence of brefeldin A, and conversion of proZPC to ZPC in the presence of monensin, indicate the possibility that the proteolytic processing of ZPC occurs in the Golgi apparatus. An analysis of amino-acid sequence identified that the C terminus of mature ZPC protein is Phe360, and the N-terminal amino-acid sequence of the proZPC-derived fragment was determined as Asp363. These results suggest that newly synthesized ZPC is cleaved at the consensus furin cleavage site, and the resulting two basic residues at the C terminus are subsequently trimmed off to generate mature ZPC prior to secretion.

A duplicated motif controls assembly of zona pellucida domain proteins

“…4). This finding is consistent with the fact that ZP protein homologues from fish to mammals are all cleaved between the ZP domain and EHP before incorporation into the ZP͞VE (5)(6)(7)(8)(9)(10)(11). Mammalian ZP protein constructs that lack a TMD are not C-terminally processed, such that the EHP is retained after secretion (Fig.…”

“…Cleavage of the C-terminal portion of ZP domain protein precursors is required for secretion and assembly into extracellular structures (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). To determine whether truncation of ZP3 before its TMD affected proteolytic processing, a truncated construct with a Cterminal FLAG-tag was produced (ZP3-373-FLAG).…”

“…During secretion, but before incorporation into the ZP, a furin-like enzyme excises the propeptide from ZP precursors by cleavage at the CFCS (5)(6)(7)(8). C-terminal processing of precursors also occurs for ZP homologues from fish to birds (9)(10)(11), as well as for other ZP domain proteins (12)(13)(14). Recombinant ZP proteins synthesized from cDNAs mutated at the CFCS are not secreted and accumulate in the endoplasmic reticulum of transfected cells (15)(16)(17).…”

Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica)

“…The granulosa layer from the largest preovulatory follicles was isolated as a sheet of granulosa cells sandwiched between the pvm and basal laminae, as described previously (Gilbert et al 1977). The pvm was isolated according to a procedure described by Sasanami et al (2002). The isolated pvm was used for in vitro assay for sperm-egg interaction.…”

“…The signals were detected with HRP-conjugated antimouse IgG (Cappel) as a secondary antibody. The immunoblot for ZP1 and ZP3 was performed as described previously (Sasanami et al 2002(Sasanami et al , 2006. For ligand blot analysis, a PVDF strip electrotransferred with pvm lysate or purified ZP1 was incubated with [ 3 H]-labeled ZP3 as described previously (Ohtsuki et al 2004).…”