Kubo H et al. (1999), Molecular basis for oviductin-mediated processi...

XB-ART-12554

Dev Genet 1999 Jan 01;252:123-9. doi: 10.1002/(SICI)1520-6408(1999)25:2<123::AID-DVG6>3.0.CO;2-3.

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Molecular basis for oviductin-mediated processing from gp43 to gp41, the predominant glycoproteins of Xenopus egg envelopes.

Kubo H , Matsushita M , Kotani M , Kawasaki H , Saido TC , Kawashima S , Katagiri C , Suzuki A .

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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.

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Species referenced: Xenopus laevis
Genes referenced: furin gsr prss1 tbx2 zp3 zp3

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	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.