title: Isolation of pregnancy-associated glycoproteins from placenta of the American bison (Bison bison) at first half of pregnancy.
abstract: This paper describes the successful purification and characterisation of pregnancy-associated glycoproteins (PAG) extracted from placenta (3-4 months) of American bisons (Amb). Chorionic AmbPAG proteins were purified from foetal cotyledonary tissues (CT) and liquid cotyledonary-carrying proteins (LCP) leaking from damaged cells. Our protocols successfully indicated the usefulness of AmbPAG protein identification, especially from LCP fraction. The AmbPAGs were extracted, precipitated and eluted during DEAE cellulose chromatography. The richest protein fractions were further chromatographed on VVA (Vicia villosa agglutinin affinity column), then characterised by mono- and bi-dimensional electrophoresis, Western blot and N-terminal amino acid (aa) sequence. After being transferred to PVDF membranes, three selected VVA-purified AmbPAG isoforms differing in molecular masses and isoelectric points (Ip 4-4.6) were selected for sequencing. One identified N-terminal 25aa sequence of AmbPAG72kDa CT form was identified as completely new (RGSNLTSLPLQNVIDLFYVGNITIG). Two other AmbPAG proteins purified from different sources (74kDa CT and 76kDa LCP forms; RGSNLTIHPLRNIRDIFYVGNITIG) were identical or corresponded to N-terminus of various bovine PAGs (boPAG). The two AmbPAGs (74kDa CT and 76kDa LCP) revealed identical micro-sequence to boPAG7; and were similar mainly to bovine PAG4, -6, -15 and -17 precursors that were identified by full-length sequencing derived from cDNA cloning. The novel sequence of the AmbPAG (72kDa CT) was related to some boPAG and various other ruminant PAG precursors (caprine and ovine). All three identified AmbPAG sequences were also relatively similar to mature forms of purified native boPAG(56-75kDa) proteins. This is the first report indicating aa sequences of native AmbPAG proteins purified from placenta (CT and LCP) of bison species. The N-terminal sequences of the AmbPAGs have been deposited in the EMBL-EBI database (UniProtKB; Accession Nos.: P84916, P84917 and P84918).
authors.affiliation: Department of Animal Physiology, Faculty of Biology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn-Kortowo, Oczapowskiego 1A/222, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Kiewisz
authors.forename: Noelita Melo de
authors.initials: NM
authors.lastname: Sousa
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Beckers
authors.forename: Hilde
authors.initials: H
authors.lastname: Vervaecke
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Panasiewicz
authors.forename: Bozena
authors.initials: B
authors.lastname: Szafranska
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D016164:Bison / Q000502:physiology; D002846:Chromatography, Affinity; D005260:Female; D005865:Gestational Age; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry*; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011270:Pregnancy, Animal; D017386:Sequence Homology, Amino Acid
chemical_list: D006023:Glycoproteins; D011257:Pregnancy Proteins