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Acquisition of fertilizability in Xenopus coelomic eggs is correlated with the conversion from coelomic to vitelline envelope during passage of the eggs through the pars recta portion of oviduct. The conversion includes processing of a major envelope constituent gp43 of coelomic envelopes to gp41 of vitelline envelopes by a trypsin-type protease, oviductin, which is secreted from the pars recta. Our recent sequencing analyses [Kubo et al., (1997): Dev Growth Diff 39:405-411] strongly suggested that the N-terminal portion of gp41 is exposed as a result of oviductin digestion. In this study, a monoclonal antibody specific to the predicted N-terminus of gp41 was raised by immunizing mice with a synthetic N-terminal hexapeptide (QLPVSP) coupled to keyhole limpet hemocyanin. The antibody specifically reacted to gp41, but not to gp43, indicating that Gln62 is exposed as the N-terminal amino acid of gp41 by oviductin-mediated cleavage of gp43 at Arg61 in GSR61. The C-terminal sequencing of gp43 and gp41 indicated that Arg373 in GSR373 as the C-terminus of gp41 is generated by cleavage of three amino acid (WNQ) residues from the C-terminus of gp43. The resulting polypeptide moiety of gp41 has a molecular mass of 33900 Da with 312 amino acid residues. We propose that oviductin possessing the substrate specificity of GSR simultaneously digests gp43 at Arg residues in GSR61 and GSR373 to generate the N- and C-terminus of gp41, respectively.
Fig. 1. Specificity of monoclonal antibody against the N-terminus of
gp41 in Xenopus vitelline envelope (VE). Coelomic envelope (CE) and
VE were separated by SDS-PAGE with a gradient gel of 4–20%
acrylamide, and electroblotted onto a PVDF membrane. The blots
were probed with anti-N41 (a-N41; culture supernatant), followed by
incubation with peroxidase-labeled goat anti-mouse Ig and chemiluminescent
detection. For Coomassie Brilliant Blue staining (CBB) and
anti-N41 detection, 8 µg each and 1 µg each of CE and VE were
electrophoresed, respectively. For specific inhibition of antibody binding
to VE gp41, 5-fold-diluted culture supernatant was incubated for
2 h with the same volume of PBS containing 80 µM N-terminal
hexapeptide [QLPVSP(1)] or PBS only [QLPVSP(2)] before probing
the blots of VE.
Fig. 2. Putative C-terminal processing steps for generation of gp43
and gp41 of Xenopus egg envelopes. The membrane-anchored form of
gp43 is first digested at Arg378 (double underline) with a Xenopus
furin homologue in the trans-Golgi network. For effective digestion by
furin, the sequence has to fulfill the following rules: (i) Arg at position
21 (double underline) is essential; (ii) at least two of the three basic
residues are required at position 22, 24, and 26 (underline); (iii) at
position 11 (dotted underline), a hydrophobic aliphatic residue is not
suitable. Because Arg373 (underline), Lys377 (underline), Arg378
(double underline), and Glu379 (dotted underline) occupy the positions
26, 22, 21, and 11, respectively, the sequence is digestible at Arg378
by a furin-like convertase. Next, the C-terminal basic residues (Arg378
and Lys377) are removed successively by carboxypeptidase H-like
enzymes in the secretory granules, resulting in the exposure of Gln376
as the C-terminus of gp43. In the oviductal pars recta, the C-terminal
region of gp43 is digested by oviductin at Arg373 in GSR373, resulting
in the generation of Arg373 as the C-terminus of gp41. CPH indicates
carboxypeptidase H. C41 and C43 denote the C-terminus of gp41 and
gp43, respectively
Fig. 3. Hypothetical steps for generation of gp41 of Xenopus VE.
The protein natively synthesized with 460 amino acid residues is
digested at Ala20 with a signal peptidase. The signal peptidasedigested
protein is further digested at an unidentified site, followed by
N-blocking. In the C-terminal portion, the protein is digested at
Arg378 with a furin-like convertase in the trans-Golgi network,
followed by removal of basic residues (Lys377-Arg378) with a carboxypeptidase
H-like enzymes in the secretory granules, resulting in
generation of the C-terminus (Gln376) of gp43 constituting CE (see
also Fig. 2). During passage of coelomic eggs bearing gp43 through the
oviductal pars recta, oviductin hydrolyses at Arg61 and Arg373 in
GSR61 and GSR373, generating gp41 with Gln62 and Arg373 as the
N- and C-terminus, respectively. CPH indicates carboxypeptidase H.
TM denotes transmembrane domain.
