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

Abstract

-Pregnancy-associated glycoproteins (PAG), structurally related to aspartic proteinases, are expressed in the outer epithelial cell layer (chorion/trophectoderm) of the ungulate placenta. The aim of the present study was to isolate as many PAG molecules as possible from placentae collected between day 60 and day 100 of gestation and to characterize their amino-terminal amino-acid sequences. Three heterologous radioimmunoassays were used to monitor PAG immunoreactivity throughout the isolation procedures. Sequential use of DEAE-cellulose, gel filtration, and CM ceramic chromatographies led to the isolation of several fractions rich in PAG immunoreactivity. The fractions with a large amount of proteins were also purified by chromatofocusing. The analysis of immunoreactive fractions by SDS-PAGE, Western blotting and two-dimensional electrophoresis followed by amino-terminal microsequencing on PVDF membranes allowed to identify eight different ovPAG with apparent molecular masses ranging from 55 to 66 kDa and isoelectric points from 4.0 to 6.8. The N-terminal sequences were determined and their comparison to those previously identified revealed that four of them are identical to those encoded by previously known cDNA, while the additional four sequences appear to be novel since they have not yet been described. ovine / pregnancy-associated glycoprotein / placenta / multiple forms / N-terminal microsequencing

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].…”

Variations in plasma matrix metalloproteinases and placental pregnancy - associated glycoproteins during gestation in rabbits

“…During pregnancy, ovine and bovine PAGs exhibit spatially and temporally distinct expression patterns (Green et al, 2000), and their concentration decreases rapidly after lambing, reaching basal levels (Ranilla et al, 1994). In the same species, there are several PAGs which differed in amino acid sequence and apparent molecular mass and each PAG presents several isoforms with different isoelectric points (El Amiri et al, 2004).…”

“…These glycoproteins are produced by trophoblasts and are expressed by chorial epithelium of the placenta (Xie et al, 1991). They have been extensively studied in domestic and wild ruminants Beckers et al, 1998;de Sousa Melo et al, 2003, El Amiri et al, 2004, but have not yet been studied in rabbits.…”