SAS1B Is an Egg Specific High Affinity Oolemmal Binding Partner for Sperm Specific Acrosomal SLLP1 During Fertilization.

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Molecules that interact on the oocyte membrane with the sperm's inner-acrosomal membrane during fertilization are poorly understood. Intra-acrosomal sperm lysozyme like protein, SLLP1, is retained predominantly in the equatorial segment following the acrosome reaction and shows binding to the microvillar domain of the oolemma. To identify SLLP1 interacting proteins on eggs, affinity panning with SLLP1 as bait was performed by surface plasmon resonance using mouse oocyte lysates. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants containing a zinc binding active site, a putative transmembrane domain, and possessing or lacking a signal peptide. Northern analysis revealed SAS1B transcripts only in ovary among 15 adult tissues. Immunofluorescence localized SAS1B to oocyte cytoplasm in secondary oocytes, Graafian follicles, and to the oolemma overlying the microvillar domain of M2 oocytes. After fertilization, SAS1B level decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native mouse SLLP1 co-localized with SAS1B in the microvillar domain in unfertilized M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western analysis, yeast two-hybrid studies and by co-immuno-precipitation analyses of recombinant full length and truncated SAS1B proteins. In addition, native SAS1B was immunoprecipitated from a mixture of mouse egg and sperm extracts using anti-SLLP1 antibody. SAS1B was found to have protease activity and showed inhibition of fertilization using recombinant SAS1B or SAS1B antibody. Study of affinity kinetics between SLLP1 and SAS1B revealed a high affinity interaction with KD of 0.32 nM. Together, the results suggest SAS1B is an oocyte specific, oolemmal metalloprotease binding partner for the sperm intra-acrosomal ligand SLLP1 during mammalian fertilization. This research was supported by the Kenneth A Scott Trust, a Keybank Trust, NIH R03 HD055129, and D43 TW/HD 00654 from the Fogarty International Center. (platform)