Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells.
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.
Katagiri
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C
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan. katagiri@bio.hokudai.ac.jp
Maeda
R
R
Yamashika
C
C
Mita
K
K
Sargent
T D
TD
Yasumasu
S
S
D000367:Age Factors; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D007158:Immunologic Techniques; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D014018:Tissue Distribution; D014817:Vitelline Membrane / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics*
D018076:DNA, Complementary; D008666:Metalloendopeptidases; C024344:envelysin