Identification of TEX101-associated Proteins Through Proteomic Measurement of Human Spermatozoa Homozygous for the Missense Variant rs35033974[S]

TEX101 protein is a validated biomarker of male infertility and a potential germ cell-surface chaperone. Near-complete degradation of variant G99V TEX101 protein was discovered in men homozygous and heterozygous for the missense variant rs35033974. Differential proteomic profiling revealed TEX101-associated proteins down-regulated in rs35033974hh spermatozoa, including LY6K protein. [Display omitted] Highlights •Missense variant rs35033974 resulted in significantly reduced levels of human TEX101 protein in seminal plasma and spermatozoa.•Differential proteomics revealed TEX101-associated testis-specific proteins, including LY6K, which were down-regulated in rs35033974hh spermatozoa.•Deep proteome of human spermatozoa, including some “missing” proteins, was identified. TEX101 is a germ-cell-specific protein and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm–oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied in vitro due to the lack of human germ cell lines. Here, we genotyped 386 men for a common missense variant rs35033974 of TEX101 and identified 52 heterozygous and 4 homozygous men. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with the near-complete degradation (>97%) of the corresponding G99V TEX101 form and suggested that spermatozoa of homozygous men could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified eight cell-surface and nine secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm–oocyte interaction (including LY6K and ADAM29) were confirmed by targeted proteomics and Western blotting assays. Because recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a monogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found fivefold lower (p = 0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men