Recent studies on tissue specificity revealed ~900 genes with restricted expression in the testis germ cells. These genes encode proteins involved in spermatogenesis and may be essential for reproduction. A fraction of testis-specific genes, however, is functionally redundant and was generated by random duplications to eventually drive evolution of proteins with novel functions. We hypothesized that mapping of interactomes of testis germ cell-specific proteins may reveal their functional role in spermatogenesis and identify genes essential for human reproduction. Since male germ cells cannot not be cultured in vitro, we studied germ cell-specific proteins in human clinical samples, such as testicular tissues and spermatozoa.
We first investigated the interactome of TEX101, a germ cell-surface chaperone and a validated biomarker of male infertility . We identified by co-immunoprecipitation-mass spectrometry (co-IP-MS) the physical interactome of human TEX101. Germ cell-surface dipeptidase 3 emerged as a top hit, and TEX101-DPEP3 interaction was extensively validated .
We then designed a proteogenomic approach to identify germ cell-surface proteins which were degraded in the absence of TEX101 chaperone. Genotyping of 386 men revealed four men homozygous for rs35033974, a missense variant resulting in the near-complete degradation of the variant G99V TEX101 protein. Differential proteomics with label-free quantification measured 8,046 proteins in spermatozoa and identified germ cell-surface proteins down-regulated in patients homozygous for rs35033974. Significantly reduced levels of LY6K protein were confirmed by targeted proteomics and immunofluorescence. Since fathers homozygous for rs35033974 had biological children, TEX101 could be a nonessential and functionally redundant human gene . Our deep proteome profiling of the human spermatozoa also revealed 46 testis-specific proteins with no prior evidence at protein level.
Our current efforts are devoted to development of co-IP-MS and proteogenomic assays for numerous germ cell-surface proteins in order to map their interactomes, validate their essentiality, and investigate as male infertility biomarkers.