Biochemical maturation of Spam1 (PH-20) during epididymal transit of mouse sperm involves modifications of N-linked oligosaccharides

Indirect immunofluorescence of mouse caput and caudal sperm shows distinctly different distributions of Spaml protein, which is associated with structural and functional differences of the molecule. Spam1 is uniformly distributed over the surface of the head of caput sperm while in caudal sperm, light and confocal microscopy demonstrate that it is localized to the anterior and posterior regions. The hyaluronidase activity of Spaml in acrosome-intact caput sperm was significantly lower (4.3-fold; P < 0.0001) than that of caudal sperm. The increase in enzymatic activity in caudal sperm is accompanied by a reduction in the molecular weight (MW): in extracts from caput sperm there was a major band at approximately 74 kDa and a minor band at approximately 67 kDa; while for the cauda there was a major band at approximately 67 kDa and minor bands at approximately 70 and -56 kDa. Additionally, the bands from caput sperm were 4.9 to 7.7-fold less intense than those from caudal sperm. This decreased affinity for the polyclonal anti-Spaml suggests the presence of different surface characteristics of the molecule from the two epididymal regions. Computer analysis of the protein structure from Spam1 cDNA sequence reveals four putative N-linked glycosylation sites, and enzymatic deglycosylation suggests that all sites are functional. After endoglycosidase activity of extracts from caput and caudal sperm, both show a major band with a MW of approximately 56 kDa, the size of the membrane-anchored polypeptide backbone. Based on the difference in size and intensity of the Spaml bands and hyaluronidase activities from caput and caudal sperm, the data suggest that the activation of Spaml during epididymal maturation is regulated by deglycosylation.