Purification and microsequencing of the intra-acrosomal protein SP-10. Evidence that SP-10 heterogeneity results from endoproteolytic processes.
The human sperm antigen SP-10 has been shown to be a testis-specific, intra-acrosomal protein that is associated with the membranes and matrix of the acrosomal vesicle. Sperm extracts, analyzed on Western blots with a monoclonal antibody to SP-10, have shown heterogeneity of SP-10 peptides ranging from 17.5-34 kDa. Although the entire SP-10 amino acid sequence of 265 amino acids (28.3 kDa) has been deduced from sequencing SP-10 cDNAs, the nature of multiple SP-10 peptide bands is incompletely understood. In this study, we developed a three-step purification method for SP-10 peptides using monoclonal antibody affinity chromatography, reverse-phase HPLC, and preparative gel electrophoresis. Eight SP-10 peptides separated by this protocol and sequenced using Edman degradation showed amino termini that corresponded to regions on the deduced SP-10 amino acid sequence. Peptides with progressively lower apparent mass aligned further toward the carboxy terminus. On the basis of putative cleavage sites on the SP-10 sequence, endoproteases that act at five different peptide bonds are predicted to cleave SP-10: these hydrolyze following arginine (a trypsin-like protease, possibly acrosin), and following serine, proline, glycine, and glutamic acid (previously undescribed intra-acrosomal protease specificities). The present studies 1) provide a purification method for SP-10 peptides; 2) confirm that the SP-10 cDNAs previously sequenced encode authentic SP-10; and 3) yield indirect evidence that endoproteases act to contribute to SP-10 heterogeneity.
Herr
J C
JC
Department of Anatomy and Cell Biology, University of Virginia, Charlottesville 22908.
Klotz
K
K
Shannon
J
J
Wright
R M
RM
Flickinger
C J
CJ
D000177:Acrosome; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D005971:Glutamates / Q000032:analysis; D005998:Glycine / Q000032:analysis; D012739:Gonadal Steroid Hormones / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D011392:Proline / Q000032:analysis; D012694:Serine / Q000032:analysis
C512729:ACRV1 protein, human; D000911:Antibodies, Monoclonal; D000941:Antigens; D005971:Glutamates; D012739:Gonadal Steroid Hormones; D008565:Membrane Proteins; D012694:Serine; D004247:DNA; D011392:Proline; D010450:Endopeptidases; D005998:Glycine