Systematic characterization of sperm-specific membrane proteins by targeted proteomics
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Because it is imperative to develop a comprehensive understanding of mammalian fertilization, we systematically characterized the alloantigen proteome of sperm membranes in a single species to identify sperm-specific membrane proteins (SSMPs) and lipid raft proteins (SSLRPs) likely to have unique functions in gamete interactions. The decreased fertility of immunized gilts with high immune response to proteins in membrane- or lipid raft-enriched particulate fractions of pig spermatozoa suggests that one or more of these SSMPs and SSLRPs function in fertilization, and are therefore potential targets for fertility control. Mass spectrometric analysis identified the immunodominant alloantigens of pig sperm membranes as the ADAMs fertilin α, fertilin ß, and cyritestin. Less abundant alloantigens included previously unknown SSMPs ATP synthase F1 subunit, myo-inositol monophosphatase-1 and zymogen granule membrane glycoprotein-2. Immunodominant SSLRPs included SAMP14 and the epididymal sperm protein E12. In all, fourteen SSMPs and three SSLRPs were positively identified. De novo peptide sequences from SSMP “M6” and SSLRP “R4” displayed no homology to known sequences. We then determined the amino acid sequence, tissue expression and localization of SSMP “M6” or SSMP20 by cDNA cloning, northern blotting, immunofluorescence and immunoelectron microscopy. A SSMP20 composite cDNA was amplified from pig testis by 3’RACE with nested degenerate oligonucleotide primers and 5’RACE with specific antisense primers. Northern blotting confirmed that this 1.1 kb composite cDNA, encoding a 126 amino acid polypeptide with two predicted transmembrane segments, was full-length and testis-specific. BLASTp searches with the deduced SSMP20 sequence identified a single mouse cDNA and a single hypothetical gene product from macaque. Comparative genomic sequence analysis identified six SSMP20 orthologs not previously or incorrectly annotated in syntenic regions of human, cow, macaque, horse, rat and mouse genomes. The N-terminal alanine-proline-rich region of SSPM20 containing putative SH3 binding motifs showed high divergence among the six species compared, and was in fact absent from the rat and mouse sequences. SSMP20 localized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and in the acrosome of ejaculated spermatozoa. We conclude that SSMP20 is a rapidly evolving sperm-specific integral membrane protein in the sperm apical head with biochemical properties suggesting a function in early events of fertilization.