Isolation and characterization of eight pregnancy-associated glycoproteins present at high levels in the ovine placenta between day 60 and day 100 of gestation.
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.
Physiology of Reproduction, Faculty of Veterinary Medicine, University of Liege, 4000, Belgium.
Bouchra
B
El Amiri
Benoit
B
Remy
Noelita Melo
NM
De Sousa
Jean-François
JF
Beckers
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D005591:Chemical Fractionation; D002845:Chromatography / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000737:chemistry; D005260:Female; D005865:Gestational Age; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000737:chemistry*; D011247:Pregnancy; D017385:Sequence Homology; D012756:Sheep
D006023:Glycoproteins