Discovery of a Human Testis-specific Protein Complex TEX101-DPEP3 and Selection of Its Disrupting Antibodies

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.

Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein with Orthogonal Immunoprecipitation-Mass Spectrometry Assays

“…We have previously demonstrated that some high-affinity monoclonal antibodies could disrupt protein-protein interactions. 39 Likewise, our present data suggested that the N-term monoclonal antibody could interfere with binding of some ERG-interacting proteins, and that some ERG-interacting proteins (especially very large ~2,000 kDa BAF complexes and 150-260 kDa transcriptional regulators) were binding to the 251 aa region between the N-term epitope and ETS DNA-binding domain. These large proteins were probably displaced by the N-term epitope antibody and the full ERG antibody, but not by the C-term antibody.…”

“…44 Immunoprecipitation substantially reduces sample complexity and facilitates quantification of multiple peptides per protein, thus enabling in-depth analysis of post-translational modifications and resolution of splicing isoforms. In our previous studies, IP-SRM assays have been utilized to quantify low-abundance kallikrein-related peptidases, 45 resolve protein isoforms, 46 screen for antibody clones, 47 discover the TEX101-DPEP3 protein complex, 39 and detect a low-abundance missense variant of TEX101 protein. 48 In this study, we focused on development of IP-MS and IP-SRM assays for the low-abundance TMPRSS2-ERG fusion protein, which triggers malignant transformation of prostate epithelial cells in nearly 50% of prostate cancer cases.…”

“…Similar case on antibody-mediated disruption of protein-protein interactions we have previously observed for the TEX101-DPEP3 complex. 39 This unexpected finding led us to the hypothesis that the ERG interactome could be disrupted by some short synthetic peptides representing the known 9FY epitope, thus paving the road to development of targeted anti-ERG therapies.…”

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

“…TEX101 was previously shown to be essential for the production of fertilization-competent spermatozoa through maturation of ADAM 3–6 proteins in mice (9, 10, 56–58). We have also recently completed a co-immunoprecipitation-mass spectrometry study on TEX101 and identified its physical interactome, including DPEP3 protein (59). Our motivation for the present study was to apply an orthogonal proteomic approach to discover TEX101-associated proteins, some of which could be proteins with weak and transient interactions and thus missed by co-immunoprecipitation.…”

Crystal structure of TEX101, a glycoprotein essential for male fertility, reveals the presence of tandemly arranged Ly6/uPAR domains

“…ADAM3 is then processed into its mature form in the epididymis. In addition, the testis-specific cell-surface dipeptidase 3 (DPEP3) has been recently identified as a TEX101-interacting partner with peptidase activity that could potentially be modulated by TEX101 function, and facilitate the cleavage of ADAM pro-domains [16]. However, the detailed molecular mechanism through which TEX101 regulates ADAM3 function in the process of spermatogenesis is largely unknown.…”