TROPHOBLAST-SPECIFIC PROCESSING AND PHOSPHORYLATION OF PREGNANCY-ASSOCIATED GLYCOPROTEIN-1 IN DAY 15 TO 25 SHEEP PLACENTA

Bovine and ovine pregnancy-associated glycoproteins-1 (PAG-1) are prod ucts of binucleate trophoblast cells and belong to the aspartic protei nase gene family. Estimates of their relative molecular masses have va ried considerably, from 47 to 90 kDa, even though the mature polypepti de has been inferred to be no more than 330 amino acids in length and that the glycosylated recombinant form synthesized in Chinese hamster ovary(CHO) or COS-1 cells had an apparent mass of 46 kDa. To establish the relationships among the various molecular forms, metabolic labeli ng, immunoprecipitation, and electrophoretic analysis were used to fol low the biosynthesis of ovine PAG-1 (ovPAG-1) in placental explants. I n time-course studies, ovPAG-1 could first be detected within 10 min a s a 70-kDa form within the tissue. With time, forms of intermediate (5 3-61 kDa) and low (47 kDa) molecular mass began to accumulate. The lat ter predominated in medium after 6 h labeling. Pulse chase studies est ablished that the 70-kDa forms were the precursors of the smaller spec ies. Inhibition of glycosylation with tunicamycin or treatment with N- glycosidase F confirmed that ovPAG-1 contained N-linked oligosaccharid e chains, but that this carbohydrate accounted for only a relatively s mall fraction (8-10 kDa) of the apparent mass, Consecutive treatment w ith neuraminidase and O-glycanase also reduced the apparent molecular mass of the precursor by approximately 11 kDa. OvPAG-1 incorporated P- 32 from [P-32]orthophosphate into phosphoserine and phosphothreonine, but there was no incorporation of S-35 from [S-35]sulfate. The basis o f the differences in molecular mass between the precursor and the fina l products remains to be elucidated, but the differences seem likely t o be due to some unusual form of posttranslational modification introd uced in the binucleate cell. The results of the study appear to explai n the disparate size values that have been reported for these placenta -derived proteins.