Characterization of buffalo native pregnancy-associated glycoprotein: mass spectrometry-based glycan composition analysis, sugar-binding characteristics and proteolytic activity assay
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Pregnancy-associated glycoproteins constitute a large family of extensively glycosylated secretory proteins which are specifically expressed in outer epithelial cell layer (chorion/trophectoderm) of placenta. The present study reports purification and characterization of buffalo pregnancy-associated glycoprotein (BuPAG) from foetal cotyledons during early pregnancy (< 3 months). Native BuPAG was purified by anion-exchange and Vicia villosa affinity chromatography. Three distinct bands were observed in SDS-PAGE and Western blot with higher molecular weight range of ~ 55–70 kDa in comparison to the calculated one (~ 40 kDa) which may be attributed to the presence of glycosylation in BuPAG. Glycan analysis of purified BuPAG by high-resolution mass spectrometry (ESI-qTof) revealed the presence of nine N-glycan structures and three O-glycan structures. Interaction analysis of various sugars with purified BuPAG using fluorescence quenching assay showed decrease in fluorescence intensity without any shift in emission maxima. BuPAG exhibited proteolytic activity within pH range of 2.0–5.5 with highest activity at pH 3.0. In the presence of inhibitor pepstatin A, proteolytic activity reduced by 84%. The glycan structures and sugar-binding characteristics of BuPAG are identified for the first time in this study. The activity of BuPAGs as active peptidases indicates their possible role in various molecular pathways required for sustenance of pregnancy.
KeywordsPlacenta Cotyledons Glycan Aspartic proteinases Glycosylation Trophoblast
The authors sincerely thank National Fund for Basic, Strategic & Frontier Application Research in Agriculture (NFBSFARA) and Indian council of Agricultural research for their funding support. Masoud Lotfan is thankful to Indian Council of Cultural Relations, Government of India for providing scholarship to carry out his Ph. D. work.
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Conflict of interest
The authors declare that they have no competing interests.
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