Characterization of native and recombinant bovine pregnancy-associated glycoproteins.
Pregnancy-associated glycoproteins (PAGs) are products of the ruminant placenta that belong to the aspartic proteinase family. Extensive glycosylation may account for the size and heterogeneity of their molecules. To assess this we investigated the effect of glycosidase and tunicamycin treatments on native (n) and mammalian-cell generated recombinant (r) bovine PAGs. Native PAG came from explant culture conditioned medium (150 days pregnancy) while rPAG was obtained by transfection of HEK 293 cells with the bPAG-1 gene employing the PRcRSV expression vector. The undigested nPAG gave a homogenous band at 67 kDa after one-dimensional SDS-PAGE, silver staining and Western blotting, but rPAG gave dual bands at 54 and 52 kDa. PNGase F digestion of nPAG gave five bands ranging from 60 to 37 kDa and digestion of rPAG gave three bands ranging from 54 to 37 kDa. On two-dimensional electrophoresis, the undigested pI ranges of n- and rPAGs were 4.7-5.6 and 7.3-8.8, respectively. The digested isoforms of n- and rPAGs had pI ranges from 5.1 to 8.5 and 7.9-8.5, respectively. Tunicamycin treatment had no effect on the mobility of nPAG but it had a pronounced time-dependant effect on the mobility of rPAG. Our findings indicate that both n- and rPAGs have principally N-linked oligosacharides.
Department of Developmental Biology, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki 305-8602, Japan.
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Patel
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Takahashi
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Imai
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Hashizume
D000818:Animals; D015153:Blotting, Western / Q000379:methods / Q000662:veterinary; D002417:Cattle; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods / Q000662:veterinary; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods / Q000662:veterinary; D005260:Female; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D007526:Isoelectric Point; D007668:Kidney / Q000166:cytology / Q000187:drug effects; D008970:Molecular Weight; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D016622:Silver Staining / Q000379:methods / Q000662:veterinary; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D006023:Glycoproteins; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D014415:Tunicamycin