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 the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for approximately 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S-S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51-Cys57, Cys353-Cys358, Cys365-Cys387 and Cys476-Cys479. This work resolves details of the structure of ecto-5'-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor.
Fini
C
C
Dipartimento di Medicina Interna, Università di Perugia, Italy; INFM Sez. B Perugia, Perugia, Italy. cfini@unipg.it
Amoresano
A
A
Andolfo
A
A
D'auria
S
S
Floridi
A
A
Paolini
S
S
Pucci
P
P
D015720:5'-Nucleotidase / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D005227:Fatty Acids / Q000032:analysis; D008401:Gas Chromatography-Mass Spectrometry; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D012661:Semen / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005227:Fatty Acids; D017261:Glycosylphosphatidylinositols; D009844:Oligosaccharides; D015720:5'-Nucleotidase