Biochemical characterization of sialoprotein "anti-agglutinin" purified from boar epididymal and seminal plasma.
Sialoprotein "anti-agglutinin," previously shown to inhibit sperm head-to-head agglutination, is found in both boar epididymal and seminal plasma. The present report characterizes anti-agglutinin by mass spectrometry, by N-terminal amino acid sequence analysis, and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting techniques to assess phosphate content of the molecule. Anti-agglutinin had the SDS-PAGE mobility of approximately 25 kDa. By electrospray ionization-mass spectrometry, however, mass spectra of anti-agglutinin were characterized by two major peaks (19,379-19,382 Da and 19,395-19,397 Da) and several minor peaks. Mass spectrometry of tryptic peptide fragments of deglycosylated anti-agglutinin and amino acid sequence analysis revealed that the protein has a unique peptide-mass fingerprinting of fragments (12,668 Da, 5,209 Da, 1,226 Da, and 1,168 Da) and a novel N-terminal amino acid sequence (KTDDY AISGA KEEEF YDYME ELYAV), respectively. Additionally Western blot techniques, using commercially available monoclonal antibodies, were used to detect presence of phosphothreonine and phosphoserine substituents, but two different monoclonal antibodies did not detect phosphotyrosine. Moreover, treatment with two different alkaline phosphotases converted the molecule, as assessed by SDS-PAGE and detection by silver stain, from the parent form of about 25 kDa to forms of approximately 19 kDa (similar to that assigned by mass spectrometry) and/or 15 kDa. Original antiserum generated toward, and reacting with native anti-agglutinin, reacted only with 19 kDa form. These results are consistent with the conclusion that the native anti-agglutinin may be a novel protein that is phosphorylated at serine and/or threonine residues.
Harayama
H
H
Graduate School of Science and Technology, Kobe University, Kobe, Japan. harayama@ans.kobe-u.ac.jp
Liao
P C
PC
Gage
D A
DA
Miyake
M
M
Kato
S
S
Hammerstedt
R H
RH
D000373:Agglutinins / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D012661:Semen / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D013552:Swine; D013997:Time Factors
D000373:Agglutinins; D012795:Sialoglycoproteins; D000469:Alkaline Phosphatase