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

Indirect immunofluorescence of mouse caput and caudal sperm shows distinctl y different distributions of Spam1 protein, which is associated with struct ural and functional differences of the molecule. Spam1 is uniformly distrib uted over the surface of the head of caput sperm while in caudal sperm, lig ht and confocal microscopy demonstrate that it is local ized to the anterio r and posterior regions. The hyaluronidase activity of Spam1 in acrosome-in tact caput sperm was significantly lower (4.3-fold; P < 0.0001) than that o f caudal sperm. The increase in enzymatic activity in caudal sperm is accom panied by a reduction in the molecular weight (MW): in extracts from caput sperm there was a major band at similar to 74 kDa and a minor band at simil ar to 67 kDa; while for the cauda there was a major band at similar to 67 k Da and minor bands at similar to 70 and similar to 56 kDa. Additionally, th e 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 suggest s the presence of different surface characteristics of the molecule from th e two epididymal regions. Computer analysis of the protein structure from S pam1 cDNA sequence reveals four putative N-linked glycosylation sites, and enzymatic deglycosylation suggests that all sites are functional. After end oglycosidase activity of extracts from caput and caudal sperm, both show a major band with a MW of similar to 56 kDa, the size of the membrane-anchore d polypeptide backbone. Based on the difference in size and intensity of th e 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. Mol. Reprod. Dev. 52:196-206, 1999. (C) 1999 Wiley-Liss, Inc.