Katagiri C et al. (1997), Molecular cloning of Xenopus hatching enzyme an...

XB-ART-16948

Int J Dev Biol 1997 Feb 01;411:19-25.

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Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells.

Katagiri C , Maeda R , Yamashika C , Mita K , Sargent TD , Yasumasu S .

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UVS.2 has been known as a cloned cDNA expressed selectively in the hatching gland cells of Xenopus laevis. To determine the molecular identity and function of UVS.2-encoded proteins, antibodies were raised against a bacterially-expressed fusion protein comprising glutathione-S-transferase (GST) and UVS.2. Anti-GST-UVS.2 antibodies inhibited the vitelline envelope digesting activity of the medium (hatching medium) in which dejellied prehatching embryos were cultured. On Western blotting, hatching medium contained 60 kDa and 40 kDa molecules reactive with these antibodies. Whole-mount immunostaining showed a specific localization of UVS.2 protein in the hatching gland cells which appeared first at stage 20, increased in number and intensity to stage 31 then decreased gradually thereafter. Immunoelectron microscopy revealed that UVS.2 protein is localized exclusively in the secretory granules in the hatching gland cells. A cDNA library from the dorsoanterior portion of stage 25 embryos was screened with UVS.2, and a 1.8 kb insert thus cloned contained additional 619bp and 204bp at the 5' and 3' ends of UVS.2, respectively. This clone, designated XHE, contained an open reading frame encoding 514 amino acids including both signal and propeptide sequences. The predicted mature enzyme comprising 425 amino acids consists of about 200 amino acid-long metalloprotease sequence of astacin family at the N-terminus, followed by two repeats of CUB domain each 110 amino acid-length. We conclude that UVS.2 represents an approximately 3/4 C-terminal portion of the hatching enzyme.

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Species referenced: Xenopus laevis
Genes referenced: astl2c astl3a.1 serpinb1
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	Fig. 1. Western blot analysis of hatching media using anti-GST-UVS.2 antiserum (lanes 1,3) or the same antiserum pre absorbed with cuIture medium of posterior embryonic halves (lane 2), Lanes 1 and 2 were derived from the same hatching medium preparation. Numbers to the left refer to the molecular weIght (kDa) of markers.
	Fig. 2, Whole-mount immunostaining of embryos with anti-GST.UVS.2 antiserum and rhodamine 8-conjugated anti-rabbit IgG. showing distribution of reactive cells at st. 31 fA.BI. st. 20 ICI. st. 25 (DI. st. 35/36 lEI. and st. 43 (FI.IBI Higher magnificatIon of (AI. Arrowheads IndIcate the frontal slce of embryos. c, cement gland. A, x 12,' B-F. x320
	~Fig.3. Number of anti-GST-UVS.2 reactive cells per embryo during pre- and posthatching stages.
	Fig. 4. Immunoelectron microscopic localization of UVS.2 in ultrathin section of stage 31 embryo. showing hatching gland cells (HGCJ and common epidermal cell (CECJ, and colloidal gold particles distributed e.\clusively in secretory granules (arrows) In apical cytoplasm of HGG but not in those in GEG (arrowheads). m, mitochondrion: n. nucleus; y, yolk platelet. Bar, 3,unl.
	Fig. 5. Nucleotide sequence and predicted amino acid sequence of XHE. The numbers to the feft and right refer to those of nucleotides and amino acids, respectively. Arrow and arrowhead indicate the putative signal sequence cleavage site and the putative N-terminus of mature enzyme, respectively. An active site consensus sequence of HExxHxxGFxHE motif of astacin family is indicated by shaded boxes. Underline indicates the poly(A) signal sequence.
	Fig. 6. Domain structure of XHE. Structure of mature enzyme including Astacus protease domain (AS) and CUB domain (C; is represented by shaded boxes. S. signal sequence; P, prosequence. Numbers at the top refer to the starring amino acid residues of the domain structures.