title: Vacuolar system-associated protein-60: a protein characterized from bovine granulosa and luteal cells that is associated with intracellular vesicles and related to human 80K-H and murine beta-glucosidase II.
abstract: It has been suggested that proteins of molecular size 56-58 kDa play an important role in bovine ovarian follicular development and oocyte maturation. A polyclonal antibody was raised against a 56- to 58-kDa protein band purified from bovine granulosa cells and was used to screen granulosa or luteal cell cDNA expression libraries. This work resulted in the identification of a cDNA encoding for a protein of 60.1 kDa with a signal peptide of 13 residues. The bovine 60.1-kDa protein shared an overall 86.7% and 81.8% identity with, respectively, the human 80K-H protein and the mouse putative beta subunit of glucosidase II (beta-GII), and was named vacuolar system-associated protein-60 (VASAP-60). Marked differences in sequence identity were noted in a putative molecular adapter domain containing a tandem D and E amino acid stretch flanked by proline-rich sequences presenting the minimal PXXP SH3 motif. VASAP-60 was shown to be unglycosylated using endoglycosidase H treatment and was found mainly in a cellular membrane fraction of bovine corpus luteum. VASAP-60 was localized in a rat hepatic Golgi/endosome fraction and in wheat germ agglutinin (WGA) affinity chromatographic eluates, thereby suggesting the presence of interactions with membrane glycoproteins. A polyclonal antibody was raised against the putative adapter domain of the recombinant VASAP-60; this was shown to recognize a major 88-kDa and two minor 58-kDa and 50-kDa proteins, suggesting that the major 88-kDa protein band represents the complete VASAP-60 protein whereas the 58-kDa and the 50-kDa bands represent its proteolytic fragments. Northern blot analysis demonstrated the presence of a single 2.3-kilobase transcript in all the bovine tissues analyzed with variation in the steady state level between tissues. Immunohistochemical observations showed that VASAP-60 was widely distributed in bovine tissues and was localized in pericytoplasmic and perinuclear membranes. In epithelial cells, the staining presented a basolateral or apical polarity associated with intracellular vacuoles. In conclusion, we have characterized a novel acidic membrane protein, associated with organelles of the vacuolar system, that is widely and histospecifically expressed in bovine tissues. VASAP-60 represents either the bovine ortholog or a new family member of the previously characterized human 80K-H and murine beta-GII proteins. Our results suggest that VASAP-60 presents characteristics of a molecular adaptor protein with functions in membrane-trafficking events.
authors.affiliation: Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6.
authors.forename: S
authors.initials: S
authors.lastname: Brûlé
authors.forename: F
authors.initials: F
authors.lastname: Rabahi
authors.forename: R
authors.initials: R
authors.lastname: Faure
authors.forename: J F
authors.initials: JF
authors.lastname: Beckers
authors.forename: D W
authors.initials: DW
authors.lastname: Silversides
authors.forename: J G
authors.initials: JG
authors.lastname: Lussier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002417:Cattle; D003338:Corpus Luteum / Q000378:metabolism*; D018076:DNA, Complementary; D005260:Female; D005786:Gene Expression Regulation; D005959:Glucosidases; D006031:Glycosylation; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000378:metabolism; D001617:beta-Glucosidase / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; C512949:Prkcsh protein, mouse; C512948:Prkcsh protein, rat; D011994:Recombinant Proteins; C406303:VASAP-60 protein, Bos taurus; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D005959:Glucosidases; C070368:PRKCSH protein, human; D001617:beta-Glucosidase