Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy.
Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.
Klisch
Karl
K
Microscopical Anatomy, Medical School Hannover, Hannover, Germany. klisch.karl@mh-hannover.de
De Sousa
Noelita Melo
NM
Beckers
Jean-François
JF
Leiser
Rudolf
R
Pich
Andreas
A
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D005260:Female; D015726:Giant Cells / Q000378:metabolism*; D013058:Mass Spectrometry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014327:Trophoblasts / Q000378:metabolism*
D011257:Pregnancy Proteins; D016282:Aspartic Acid Endopeptidases; C094766:pregnancy-associated glycoprotein 1; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase