Identification of TEX101 functional interactome through proteomic measurement of human spermatozoa homozygous for the missense variant rs35033974

SUMMARY TEX101 is a testis-specific cell-surface protein expressed exclusively in the male germ cells 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 healthy fertile men and sub-fertile patients for a common and potentially deleterious missense variant rs35033974 of TEX101 , and identified 52 heterozygous and 4 homozygous patients. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with near-complete degradation (>97%) of the corresponding G99V TEX101 form, and suggested that spermatozoa of homozygous patients 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 8 cell-surface and 9 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 fusion (including LY6K and ADAM29) were confirmed by targeted proteomics and western blotting assays. Since recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a single pathogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found 5-fold lower ( P =0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction. Non-standard abbreviations TEX101 Testis-expressed sequence 101 protein LY6K Lymphocyte antigen 6 complex locus K ADAM29 A disintegrin and metalloproteinase domain-containing protein 29 DPEP3 Dipeptidase 3 BH-adjusted t-test Benjamini-Hochberg-adjusted t-test FDR False discovery rate FWHM Full width at half maximum GPI Glycosylphosphatidylinositol LC-MS/MS liquid chromatography - tandem mass spectrometry LFQ Label-free quantification MS Mass spectrometry mAb Monoclonal antibody MWU Mann Whitney Unpaired t-test PRM Parallel reaction monitoring ROC AUC Receiver operating characteristic area under the curve SCX strong cation exchange chromatography SP seminal plasma SNV Single nucleotide variation SRM Selected reaction monitoring WT wild-type