Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.
The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.
Barbato
O
O
Department of Biopathological Veterinary Science, Faculty of Veterinary Medicine, University of Perugia 06126, Italy. barbato@unipg.it
Sousa
N M
NM
Klisch
K
K
Clerget
E
E
Debenedetti
A
A
Barile
V L
VL
Malfatti
A
A
Beckers
J F
JF
D000362:Agar; D000595:Amino Acid Sequence; D000818:Animals; D002020:Buffaloes / Q000502:physiology*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D005260:Female; D047109:Fetal Development / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry; D010920:Placenta / Q000737:chemistry*; D037121:Plant Lectins; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011270:Pregnancy, Animal / Q000502:physiology*
D010446:Peptide Fragments; D037121:Plant Lectins; D011257:Pregnancy Proteins; C042242:Vicia lectins; D000362:Agar