PARENT SESSION
Pregnancy and Parturition

SIMULTANEOUS SECRETION OF MULTIPLE PREGNANCY ASSOCIATED GLYCOPROTEINS IN CATTLE.

Patel, Osman¹, Takahashi, Toru¹, Ishiwata, Hiroko¹, Hashizume, Kazuyoshi¹, ¹

ABSTRACT- A multitude of genes have been identified in the ruminants that regulate the expression of pregnancy-associated glycoprotein (PAG) across gestation. Interestingly, recent studies suggest that the expression of the same may be stage specific rather than uniform throughout gestation. Therefore, the objective of our study was to characterize the different forms of bPAG secreted at specific stages of gestation. Briefly, 40 grams of placentome (210 days pregnancy) were manually minced and suspended in DMEM/F12 and incubated for 24 hours at 37 C. The conditioned medium was harvested and subjected to DEAE-ion exchange chromatography, Pepstatin-A affinity chromatography and preparative SDS-PAGE. The presence of bPAG in chromatographed fraction revealed a 62-64 kDa molecule following immunoblotting (anti-bPAG-1 peptide 39-58). The purified protein was enzymatically deglycosylated. The enzymes cleaved all N and O-linked oligosacharides plus all Ser/Thr linked Gal and all Sialic acid substituted Gal. The deglycosylated PAG was then subjected to one-dimensional (ODE) SDS-PAGE, silver staining (SS) and immunoblotting and later to two-dimensional electrophoresis (TDE) and subsequent immunoblotting. It revealed 5 isoforms of varying molecular weights (35-60 kDa) in ODE and SS. Similarly, TDE revealed 5 isoforms of varying pI (5.0-7.4) and molecular weights (35-60kDa) of the deglycosylated peptide. Notwithstanding this, a recombinant bPAG (56 kDa) derived from bPAG-1 gene transfected HEK 293 cells and undergoing the same purification protocol, enzymatic deglycosylation and identical electrophoresis showed a minor shift in the molecular weight on ODE and SS, however, depicted 4-5 isoforms of varying pI (6.5-7.4) and molecular weights (35-45kDa) on TDE. These results suggest that the placenta produces multiple forms of PAG at specific stages of gestation and that a single bPAG-1 gene has the potential of expressing an array of proteins following post-translational modification.

KEY WORDS: bovine, pregnancy, pregnancy associated glycoprotein, recombinant protein