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

Abstract

Indirect immunofluorescence of mouse caput and caudal sperm shows distinctly different distributions of Spam1 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 Spam1 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 ~74 kDa and a minor band at ~67 kDa; while for the cauda there was a major band at ~67 kDa and minor bands at ~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-Spam1 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 ~56 kDa, the size of the membrane-anchored polypeptide backbone. Based on the difference in size and intensity of the Spam1 bands and hyaluronidase activities from caput and caudal sperm, the data suggest that the activation of Spam1 during epididymal maturation is regulated by deglycosylation.