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a Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
b Unité de recherche en pédiatrie, Centre hospitalier universitaire, Université Laval, Ste-Foy, Québec, Canada G1V 4G2
c Faculté de médecine vétérinaire, Université de Liège, Liège, Belgium
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 ß subunit of glucosidase II (ß-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 ß-GII proteins. Our results suggest that VASAP-60 presents characteristics of a molecular adaptor protein with functions in membrane-trafficking events.
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the "Fonds de la recherche scientifique médicale" (FRSM) of Belgium, the "Fonds pour la Formation de Chercheurs et l'Aide à la Recherche" (FCAR) of Quebec. F.R. was supported by the "Agence Francophone des Universités pour l'Enseignement Supérieur et la Recherche" (AUPELF-UREF).
2 Correspondence: Jacques G. Lussier, Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, P.O. Box 5000, St-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; lussij{at}medvet.umontreal.ca
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