Isolation and partial characterization of three pregnancy‐associated glycoproteins from the ewe placenta

Abstract

Pregnancy-associated glycoproteins (PAGs) are synthesized in the outer epithelial layer of the placenta in artiodactyls. In this work, three novel ovine PAGs were isolated from late-pregnancy fetal cotyledons and characterized biochemically. The isolation procedure included acid and ammonium sulfate precipitations and anion and cation exchange chromatographies. The isolated PAGs have different NH(2)-terminal amino acid sequences (RGSXLTILPLRNMRDIVY, ISRVSXLTIHPLRNIMDML, and RGSNLTIHPLRNIRD) and apparent molecular masses (55, 57, and 59 kDa). Each shows several isoforms with different pI values. The three proteins share high sequence identity with each other and with other ovine, bovine, and caprine PAGs. They have not been described previously. The ovPAG-59 sequence differs from the previously identified ovPAG-4 sequence (determined by DNA cloning and sequencing) at only one position among the 15 N-terminal residues. The newly characterized ovPAGs and the procedure used to isolate them will be helpful in producing new antisera for investigating PAG secretion in pregnant ewes.

Isolation and characterization of eight pregnancy-associated glycoproteins present at high levels in the ovine placenta between day 60 and day 100 of gestation

“…These molecules were named according to their molecular masses: ovPAG 55 , ovPAG 60 , ovPAG 61 , and ovPAG 65 . We very recently, isolated three novel molecules (ovPAG-55, ovPAG-57, and ovPAG-59) with different amino-acid sequences from ovine fetal cotyledons (on or after day 100) [10]. These proteins show similarity to all PAG identified earlier, especially with regards to the cleavage point (Arg or Ile), the consensus sequences for the carbohydrate sulfaction site (ProLeu-Arg), and N-glycosylation site.…”

“…The stage of pregnancy was assessed by measuring the crown-rump length of the foetuses [15]. As in previous works, the placentae were collected after day 100 [9,10]; in this study the period was focused on days 60 to 100. Total protein (TP) concentrations of all fractions were determined by the Lowry method [16].…”

“…The cotyledons were cut into small pieces and washed with saline to remove blood. The tissues were homogenized in potassium phosphate buffer (0.10 M, pH 7.6) containing a cocktail of proteinase inhibitors as described by El Amiri et al [10]. The extract was subjected to acid precipitation (pH 4.5) and then to ammonium sulfate fractionation.…”

Evaluation of a bovine pregnancy-associated glycoprotein enzyme-linked immunosorbent assay kit for serological diagnosis of pregnancy in sheep

“…Bovine and ovine cDNA encoding PAG-1 have 86% similarity in nucleotide sequences and results in encoded proteins which differ in only 2 amino acids (XIE et al, 1991). There are numerous isoforms of PAGs produced from ovine placenta (e.g., ovPAG-55, 57, 59, 60, 61 and 65), which were identified at random stages of pregnancy and used to develop PAGs-based assays with placental antigens from ewes (XIE et al, 1997;EL AMIRI et al, 2003;EL AMIRI et al, 2004). These studies involving sheep PAGs were based on radioimmunoassay (RIA), a radioactive technique (DE SOUSA et al, 2003;KAREN et al, 2003).…”

Biodiversity of multiple Pregnancy-Associated Glycoprotein (PAG) family: gene cloning and chorionic protein purification in domestic and wild eutherians (Placentalia) – a review

“…In sheep, oPAG65 protein revealed a similar micro-sequence to the sequences of two distinct precursors, oPAG3 and oPAG7 [9,11]. Moreover, the N-terminus of some lately identified native oPAG proteins (55−60 kDa) revealed high homology to the sequences of the polypeptide oPAG1, 4 and 6 precursors [76][77][78].…”