Mass spectrometry study of ecto-5 '-nucleotidase from bull seminal plasma

The structure of ecto-5'-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry . MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for t he monomeric form, and it also showed a heterogeneous population of glycofo rms with the glycosidic moiety accounting for approximate to 6000 Da. MALDI -MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites o f N-glycosylation. GC-MS analysis provided information on the glycosidic st ructures linked to the four asparagines. Asn53, Asn311 and Asn333 were link ed to high-mannose saccharide chains, whereas the glycan chains linked to A sn403 contained a heterogeneous mixture of oligosaccharides, the high-manno se type structure being the most abundant and hybrid or complex type glycan s being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidy linositol anchor was obtained. MALDI spectral analysis indicated that the g lycosylphosphatidylinositol core contained EtN(P)Man(3)GlcNH(2)-myo-inosito l(P)-glycerol, principally modified by stearoyl and palmitoyl residues or b y stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylg lycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoy lglycerol. The combination of chemical and enzymatic digestions of the prot ein with the mass spectral analysis yielded a complete pattern of S-S bridg es. The protein does not contain free thiols and its eight cysteines are li nked by intramolecular disulfide bonds, the pairs being: Cys51-Cys57, Cys35 3-Cys358, Cys365-Cys387 and Cys476-Cys479. This work resolves details of th e structure of ecto-5'-nucleotidase, with particular regard to the localiza tion and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinosito l anchor.