N-glycodiversity of the Pregnancy-Associated Glycoprotein family (PAG) produced in vitro by trophoblast and trophectoderm explants during implantation, placentation and advanced pregnancy in the pig

Abstract

The PAG family is encoded by distinct genes expressed in extra-embryonic chorionic membranes (TR--trophoblast, TRD--trophectoderm) of various pregnant mammals. The objective of our study was to determine N-glycodiversity of porcine PAG protein family (pPAG) produced in vitro by TR or TRD explants of gilts (n=26) throughout pregnancy (16-77 dpc). Explants were cultured for over 1200 h (TR, 16 dpc) or for 8 h (TRD, 17-77 dpc). Released proteins were isolated from media by separating ultra-filtration (>10 kDa). A deglycosylation (removal of N-linked carbohydrate side chains) of proteins was performed by glycopeptidase F, and compared to non-deglycosylated forms by PAGE-Western blotting with anti-pPAG sera and additionally to polypeptide pPAG precursors, coded by ORF of their cloned cDNAs. We demonstrated gestation-stage dependent diversity of deglycosylated/glycosylated forms of the pPAG proteins produced in vitro in the pig. TR explants harvested on 16 dpc during long term culture released 43 kDa pPAG proteins. These proteins were deglycosylated to approximately 36.9 and approximately 39.6 kDa (16 dpc). Tissue harvested on 17 dpc in vitro secreted 65-68 kDa pPAG proteins which were reduced to three forms, 50.6, 58.7 and 63.5 kDa. In addition, approximately 73.3 kDa major pPAG proteins (77 dpc) were reduced to at least three forms: approximately 39.6, approximately 36.9 and approximately 33.4 kDa. Such N-deglycosylation was not detected on days 25-61. N-deglycosylation of native pPAG proteins clearly corresponded to three N-glycosylation sites of asparagines (N-x-S/T) found in ORF of the pPAG2-like precursors, identified by their in silico translated cDNAs. Thus, the pregnancy-stage dependent N-glycodiversity of the pPAG protein family, containing an average 9.66% of N-linked oligosaccharides, may play some role(s) in porcine conceptus attachment, successful implantation and during advanced pregnancy.