Kubo H et al. (1997), A major glycoprotein of Xenopus egg vitelline e...

XB-ART-16142

Dev Growth Differ 1997 Aug 01;394:405-17. doi: 10.1046/j.1440-169x.1997.t01-3-00001.x.

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A major glycoprotein of Xenopus egg vitelline envelope, gp41, is a frog homolog of mammalian ZP3.

Kubo H , Kawano T , Tsubuki S , Kawashima S , Katagiri C , Suzuki A .

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A predominant glycoprotein in the vitelline envelope (VE) of the anuran Xenopus laevis is gp41, known to be proteolytically converted from gp43 of the coelomic egg envelope concomitant with the acquisition of egg fertilizability. To characterize the protein core of gp41, purified gp41 from VE was digested with lysyl endopeptidase, and peptides isolated from the digests were sequenced for amino acids to design degenerate primers for polymerase chain reaction. By reverse transcription-polymerase chain reaction with a poly(A)+ RNA from the ovary of an ovulated female Xenopus, a specifically amplified band was obtained and sequenced. The upstream and downstream sequences of the sequenced region were completed by 5'- and 3'-rapid amplification of cDNA ends, respectively. The cDNA, referred to as gp43 cDNA, comprises 1423 base pairs and contains one open reading frame with a sequence for 460 amino acids. The predicted amino acid sequence of gp43 cDNA has a close similarity with that of mammalian ZP3. Northern blot and in situ hybridization studies indicated that gp43 mRNA is expressed in oocytes, particularly in the previtellogenic oocytes. A comparison of the N-terminal sequences of gp41 and gp43 strongly suggested that gp41 is generated at least by processing of the N-terminal portion of gp43 with oviductin.

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Species referenced: Xenopus laevis
Genes referenced: zp3 zp3 zp3.2

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	Fig.1. t\ucleotide sequence of gp43 eDNA and its deduced amino acid sequence. The eDNA is composed of 1423b.p. containing one open read1ng frame that encodes a polypeptide with 460 amino acids. The sequence in the thicker lined box indicates the poly(Aj+ signal (AATAAA) overlapping with the stop codon (TAA: indicated as an asterisk). Underlines indicate sequences ot the peptides (27p, 21 p, 13p, 9p, and 3p in this order from theN-terminus) obtained by lysyl endopeptidase-digestion of gp41. All the amino acids N-terminally adjacent to the peptides are Lys or Arg, indicating that the peptides were released by digestion of gp41 with lysyl endopeptidase. Broken underlines show tentative N-glycosylation sites. The N-terminal sequence analysis of gp41 gave an amino acid sequence identical with 15 amino acids indicated in the box with thinner lines. An arrowhead indicates a tentative digestion site with a signal peptidase. Thesequence data will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number D86568.
	Fig. 2. Multiple alignment of the amino acid sequences of mammalian ZP3 and non-mammalian ZP3 homologs. L-SF41 is a fish homolog of mammalian ZP3, and huZP3 and moZP3 indicate human and mouse ZP3, respectively. Solid boxes show amino acids (88 amino acids) common to four sequences. Cysteine residues are hatched, and 12 out of 13 of these residues present in gp43 occupy the positions conserved in mammalian ZP3.
	Fig. 3. K-D (Kyle-Doolittle) plot analysis of human ZP3 (upper) and Xenopus gp43 (lower), showing that the position and shape of both peaks are extremely similar to one another.
	Fig. 4. Northern blot analysis of gp43. All the organs except testis were from Xenopus females. Ovary (hCG) was from a female previously ovulated with hCG. Total RNA (10 j.Jg each) and poly(Aj+ RNA (11Jg) were electrophoresed on a formaldehydedenaturing agarose gel and blotted on a nylon membrane. They were hybridized with a radio-labeled probe prepared with a 1 1 k.b.p. DNA fragment (250-1361 nt) as a template. The signal was detected exclusively in the ovary with a length of 1.6 kb. A longer exposure gave a faint signal of the same length in the oviposited eggs (UF).
	Fig. 5. Dist'ibution of gp43 mRNA in the ovary by in situ hybridization. Paraffin sections of Xenopus ovary were hybridized with DIGlabeled antisense (a) or sense (b) riboprobe of approximately 0.45 kb in length. The specifically hybridized probe was visualized with an alkaline phosphatase-coupled anti-DIG antibody. The signal was only detected in the cytoplasm of young oocytes (stained dark blue) but not in the somatic cells. The rests. except the cortical pigment layer, were observed as faint and brownish. I-IV indicate Dumont stage numbers. Bar in (b) represents 300 ~m.
	Fig. 6. Hypothetical steps for generation of gp41 by N-terminal processing. The protein natively synthesized with a length of 460 amino acids is digested at Ala20 with a signal peptidase, giving rise to Leu21 as the N-terminus. The signal peptidase-digested protein is supposed to be further digested at an unspecified site followed by N-terminal blocking, which constitutes CE as gp43. When coelomic eggs bearing gp43 pass through the PR, oviductin hydrolyses at GSR61 of gp43, resulting in gp41 with Gln62 as the N-terminus.