Molecular cloning of rat sperm galactosyl receptor, a C-type lectin with in vitro egg binding activity

Rat sperm galactosyl receptor is a member of the C-type animal lectin famil y showing preferential binding to N-acetylgalactosamine compared to galacto se. Binding is mediated by a Ca2+-dependent carbohydrate-recognition domain (CRD) identical to that of the minor variant of rat hepatic lectin recepto r 2/3 (RHL-2/3). The molecular organization of the genomic DNA, cDNA, and d erived amino acid sequence of rat testis galactosyl receptor have been dete rmined and in vitro fertilization studies were conducted to ascertain its r ole. We have determined that the rat testis galactosyl receptor gene genera tes two mRNA species: one species, designated liver-type, is identical to R HL-2/3; the other, designated testis-type, contains one unspliced intron (8 6 nt) which alters the reading frame and changes the amino acid sequence of the carboxyl terminus. As a result, the CRD (glutamine-proline-aspartic ac id/QPD) and flanked Ca2+-binding amino acid sequences were not present in t he testis-type protein. Northern and Southern blots demonstrated presence o f transcripts with unspliced intron in rat sperm but not liver. Similarly, antibody, raised against a synthetic 12-amino acid peptide (p12) encoded by the unspliced intron, recognized in immunoblots a 54 kDa receptor protein in protein extracts from testis but not from liver. Immunofluorescence and immunogold electron microscopy studies demonstrated that both protein speci es localized on the plasma membrane surface of the head and tail of rat spe rm. Furthermore, capacitated vat sperm preincubated with polyclonal antiser a to RHL-2/3 or to the CRD of the liver-type galactosyl receptor showed a s tatistically significant decrease in the in vitro fertilization rate. We co nclude that rat sperm galactosyl receptor may play a role in egg binding an d that an undetermined molecular mechanism operates to generate two protein s with identical intracellular amino terminal domain but only one of them d isplays a CRD and associated Ca2+-binding sites at the carboxyl terminal ex tracellular domain. Mol. Reprod. Dev. 56.401-411, 2000. (C) 2000 Wiley-Liss , Inc.