Kubo H et al. (2000), Egg envelope glycoprotein gp37 as a Xenopus hom...

XB-ART-10388

Dev Growth Differ 2000 Aug 01;424:419-27. doi: 10.1046/j.1440-169x.2000.00526.x.

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Egg envelope glycoprotein gp37 as a Xenopus homolog of mammalian ZP1, based on cDNA cloning.

Kubo H , Kawano T , Tsubuki S , Kotani M , Kawasaki H , Kawashima S .

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The egg envelope is a kind of extracellular matrix, which surrounds growing oocytes, ovulated eggs and early embryos. Among the glycoprotein components of the Xenopus laevis egg envelope, gp43/gp41 and gp69/64 have already been shown to be frog homologs of the mammalian zona pellucida components ZP3 and ZP2, respectively. To determine the structure of another major component of egg envelope, gp37, the peptides isolated from the lysyl endopeptidase digests of gp37 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 a postovulated 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 gp37 cDNA comprises 1674 bp and contains one open reading frame encoding a polypeptide with 543 amino acids. The predicted amino acid sequence of the gp37 cDNA has a close similarity to that of mammalian ZP1. Northern blot and in situ hybridization studies indicated that the transcript (1.8 kb) is exclusively expressed in the oocytes, particularly in the previtellogenic young oocytes, just like the expression pattern of gp43 mRNA, suggesting a coordinate transcription of the gp43 and gp37 genes in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: furin zp2 zp3 zp3 zp3.2 zp4

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	Fig. 1. Nucleotide sequence of gp37 cDNA and its deduced amino acid sequence. The cDNA comprises 1674 bp, containing one open reading frame that encodes a polypeptide with 543 amino acids. The sequence in the thicker box indicates the poly(A)+ signal (AATAAA) overlapping the stop codon TGA (asterisk). Solid underlines indicate sequences of the peptides 17p (Ala151âCys163) and 16p (Asp320âTyr332) from the digestion of gp37 with lysyl endopeptidase. Amino acids adjacent to the N-terminus of these peptides are Lys150 and Arg319, indicating that the peptides were indeed released by lysyl endopeptidase digestion. The N-terminal sequence analysis of gp37 from vitelline envelope (VE) and coelomic envelope (CE) gave an identical sequence of 15 amino acids (Asp130âAsp144) as indicated in the box with thinner lines. The deduced sequence includes a ZP domain (Gln183âMet462) and a transmembrane domain (Gly510âIle529), as indicated by dotted underline and italicized letters in the thinner box, respectively. Broken underlines show putative N-glycosylation sites. Single and double arrowheads indicate putative digestion sites with a signal peptidase and a furin-like processing protease, respectively. The encircled Thr457 indicates the putative Cterminal amino acid deduced from the generation process of the C-terminus of gp43. The sequence data will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the accession number AB037700.
	Fig. 2. Multiple alignment of the amino acid sequences of gp37 and mammalian ZP1 homologs. huZPB indicates human ZP1. piZP3a and ra55K denote pig ZP3a and rabbit rc55, respectively, both of which are ZP1 homologs. Solid boxes show amino acids common to the ZP1 homologs. Cysteine residues are hatched, and 20 of 21 of the residues present in gp37 occupy a conserved position in the mammalian ZP1. The line from Gln183 to Met462 indicates the ZP domain of gp37.
	Fig. 3. Northern blot analysis of gp37. All the organs except testis were from Xenopus females. Ovary was from a female 2 days after ovulation. Total RNA (10 Î¼g) and poly(A)+ RNA (1 Î¼g) were electrophoresed on a formaldehyde-denaturing agarose gel and blotted onto a nylon membrane. They were hybridized with a digoxigenin (DIG)-labeled antisense riboprobe (nucleotides 200â€“570). The signal was detected with an alkaline phosphatase-conjugated anti-DIG antibody and a chemiluminescent substrate. The transcript was present exclusively in the ovary with a length of 1.8 kb. The lower panel shows 18S and 28S rRNA.
	Fig. 4. Distribution of gp37 transcripts in the ovary by in situ hybridization. Paraffin sections of Xenopus ovary were hybridized with digoxigenin (DIG)-labeled antisense (a) or sense (b) riboprobes (nucleotides 200â€“570). The specifically hybridized probe was visualized with an alkaline phosphatase-coupled anti-DIG antibody. The signal was only detected in the cytoplasm of previtellogenic young oocytes, not in the somatic cells. A black pigment layer is visible on the peripheral zone of the vitellogenic oocytes. (b) Bar, 300 Î¼m.