Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma.
Ecto-5'-nucleotidase (ecto-5'-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5'-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln(547) and/or Phe(548). Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn(403). Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5'-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.
Fini
Carlo
C
Dipartimento di Medicina Interna, Università di Perugia, via del Giochetto 6, Italy. cfini@unipg.it
Talamo
Fabio
F
Cherri
Silvia
S
Coli
Marcello
M
Floridi
Ardesio
A
Ferrara
Lino
L
Scaloni
Andrea
A
D015720:5'-Nucleotidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D008297:Male; D008958:Models, Molecular; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D012661:Semen / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D009844:Oligosaccharides; D010446:Peptide Fragments; D015720:5'-Nucleotidase; D014357:Trypsin