The cattle tick antigen, Bm95, expressed in Pichia pastoris contains short chains of N- and O-glycans

Abstract Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein (SwissProt Accession No. P20736), which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N -glycans, from Man 5 GlcNAc 2 to Man 9 GlcNAc 2 of which, Man 8-9 GlcNAc 2 were the most abundant. O -Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn 122 ) is located between EGF-like domains 2 and 3, where the O -glycopeptides were found, two other N-glycosylated sites (Asn 329 and Asn 363 ) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr.