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 hat ching gland cells of Xenopus laevis. To determine the molecular identi ty and function of UVS.2-encoded proteins, antibodies were raised agai nst a bacterially-expressed fusion protein comprising glutathione-S-tr ansferase (GST) and UVS.2. Anti-GST-UVS.2 antibodies inhibited the vit elline 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 localiz ation of UVS.2 protein in the hatching gland cells which appeared firs t at stage 20, increased in number and intensity to stage 31 then decr eased gradually thereafter. Immunoelectron microscopy revealed that UV S.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 cl oned contained additional 619bp and 204bp at the 5' and 3' ends of UVS .2, respectively. This clone, designated XHE, contained an open readin g frame encoding 514 amino acids including both signal and propeptide sequences. The predicted mature enzyme comprising 425 amino acids cons ists of about 200 amino acid-long metalloprotease sequence of astacin family at the hi-terminus, followed by two repeats of CUB domain each 110 amino acid-length. We conclude that UVS.2 represents an approximat ely 3/4 C-terminal portion of the hatching enzyme.