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Identification of an Oolemmal Receptor for the Sperm Acrosomal Ligand, SLLP1.

2008; Oxford University Press; Volume: 78; Issue: Suppl_1 Linguagem: Inglês

10.1093/biolreprod/78.s1.69b

1529-7268

Arabinda Mandal, Monika Sachdev, Laura Digilio, Charles J. Flickinger, John C. Herr,

Proteins in Food Systems

The molecular mechanisms by which fertilization competent, acrosome reacted sperm bind to the oolemma prior to gamete fusion remains enigmatic. SLLP1 is a sperm intra-acrosomal, lysozyme like, non-bacteriolytic protein encoded by the gene Spaca3 which is conserved across mammals. Mouse SLLP1 (mSLLP1) is retained predominantly in the equatorial segment following the acrosome reaction. mSLLP1 binds to the microvillar domain of egg plasma membrane. Sperm binding to the oolemma as well as fertilization is blocked with recombinant SLLP1 suggesting the presence of oolemmal SLLP1 receptors. In order to identify receptor(s) of mSLLP1, an affinity panning experiment was performed on zona free mouse egg lysate with soluble mSLLP1 as bait employing surface plasmon resonance technique followed by tandem mass spectrometric microsequencing. Several proteins were identified as candidate SLLP1 receptors including an oocyte specific transmembrane protein, named SAS1R (sperm acrosomal SLLP1 receptor). SAS1R has a protease domain and is conserved among mammals. Full length SAS1R was cloned, expressed, and recombinant protein purified for characterization and functional studies including production of anti-SAS1R antibodies. SAS1R localized on the microvillar domain of mature live oocytes and was significantly lost after fertilization being virtually undetectable in blastocysts. Transfection of CHO-K1 cells with the full length SAS1R cDNA construct allowed the protein to be expressed on the surface of non-permeabilized cells, indicating the presence of an active transmembrane domain. Confocal studies revealed co-localization of mSLLP1 and SAS1R in unfertilized oocytes. To test the affinity between these two proteins in vitro, far-western analysis was performed with bacterially expressed purified recombinant proteins. Mouse SLLP1 showed stronger affinity to full length SAS1R than to its C-terminal fragment. Affinity between these two molecules in vivo was analyzed by the yeast two-hybrid system under stringent selection conditions. The N-terminus of SAS1R showed higher affinity than its C-terminus in binding to mSLLP1 and rescued the yeast cells on agar plate deficient in adenine and histidine confirming the interaction between these proteins. Furthermore, co-IP experiments using in vitro translated proteins from rabbit reticulocyte lysate containing radiolabelled methionine allowed immunoprecipitation of SAS1R by mSLLP1 and the converse, using anti-HA and/or anti-c-Myc tag monoclonal antibodies conjugated to agarose beads. The purified recombinant SAS1R was also found to be a protease active against the synthetic peptide as assayed by a FRET (fluorescence resonance energy transfer) based method. The present results suggest the identification of an oolemmal receptor, SAS1R, for the sperm acrosomal ligand, SLLP1. 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.