Abstract
TEX101 is a testis-specific protein expressed exclusively in male germ cells and is a validated biomarker of male infertility. Studies in mice suggest that TEX101 is a cell-surface chaperone which regulates, through protein-protein interactions, the maturation of proteins involved in spermatozoa transit and oocyte binding. Male TEX101-null mice are sterile. Here, we identified by co-immunoprecipitation-mass spectrometry the interactome of human TEX101 in testicular tissues and spermatozoa. The testis-specific cell-surface dipeptidase 3 (DPEP3) emerged as the top hit. We further validated the TEX101-DPEP3 complex by using hybrid immunoassays. Combinations of antibodies recognizing different epitopes of TEX101 and DPEP3 facilitated development of a simple immunoassay to screen for disruptors of TEX101-DPEP3 complex. As a proof-of-a-concept, we demonstrated that anti-TEX101 antibody T4 disrupted the native TEX101-DPEP3 complex. Disrupting antibodies may be used to study the human TEX101-DPEP3 complex, and to develop modulators for male fertility.
Highlights
TEX101 is a testis-specific protein expressed exclusively in male germ cells and is a validated biomarker of male infertility
Production of TEX101 Protein and Mouse Monoclonal Antibodies Recognizing Its Different Epitopes—The mature form of human TEX101 protein was expressed in Expi293F cells
The expression and purity of TEX101 protein were evaluated by Coomassie staining SDS-PAGE and Western blot analysis using an anti-TEX101 rabbit polyclonal antibody (Fig. 1A), and were confirmed by mass spectrometry
Summary
The interactome of human TEX101 protein in testicular tissues and spermatozoa was identified by applying a quantitative co-immunoprecipitation-MS approach. Validation of the human testis-specific protein complex TEX101-DPEP3. Disrupting antibodies may be used to study the human TEX101-DPEP3 complex, and to develop modulators for male fertility. Of 1035 highly testis-enriched proteins in the human proteome [14], nearly 160 proteins are membrane-bound and could be involved in spermatogenesis, remodelling of spermatozoa cell surface, sperm transit and sperm-oocyte interaction. We focused on the testis-specific protein TEX101, which we previously discovered and validated as a biomarker of male infertility [27,28,29,30,31]. Human Testis-specific Protein Complex TEX101-DPEP3 suggested to be a cell-surface chaperone involved in trafficking and maturation of numerous cell surface proteins essential for fertilization in mice [7, 33]. Taking into account the degradation of testis-specific ADAM proteins in TEX101-null mice and subsequent sterility of male mice [7], we hypothesized that disruptors of TEX101 PPIs could emerge as modulators of male fertility and nonhormonal male contraceptives
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