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

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Discovery of a Human Testis-Specific Protein Complex TEX101-DPEP3 and Selection of Its Disrupting Antibodies bioRxiv preprint doi: https://doi.org/10.1101/315713; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Discovery of a human testis-specific protein complex TEX101-DPEP3 and selection of its disrupting antibodies Christina Schiza1,2, Dimitrios Korbakis1,3, Efstratia Panteleli4, Keith Jarvi3,,5, Andrei P. Drabovich1,2,4, and Eleftherios P. Diamandis1-4* 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; 2Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada; 3Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada; 4Department of Clinical Biochemistry, University Health Network, Toronto, Canada; 5Department of Surgery, Division of Urology, Mount Sinai Hospital, Toronto, Canada *Correspondence should be addressed to: E. P. Diamandis, Ph.D., M.D., Mount Sinai Hospital, 60 Murray St [Box 32]; Flr 6 - Rm L6- 201-1, Toronto, ON, M5T 3L9, Canada. Tel: 416-586-8443; Fax: 416-619-5521; e-mail: [email protected] Non-standard abbreviations: TEX101, Testis-expressed sequence 101 protein; DPEP3, Dipeptidase 3; AC-MS, Affinity capture-mass spectrometry; co-IP-MS, Coimmunoprecipitation- mass spectrometry; GPI, Glycosylphosphatidylinositol; LFQ, Label-free quantification; mAb, Monoclonal antibody; NHS, N-hydroxysuccinimide; PRM, Parallel reaction monitoring; PTM, Posttranslational modification; SP, Seminal plasma; SRM, Selected reaction monitoring; FDR, false detection rate Key words: TEX101; testis-expressed sequence 101 protein; DPEP3; dipeptidase 3; affinity purification-mass spectrometry; interactome; protein-protein interactions; testis-specific proteins; spermatozoa; disrupting antibodies 1 bioRxiv preprint doi: https://doi.org/10.1101/315713; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. SUMMARY 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. 2 bioRxiv preprint doi: https://doi.org/10.1101/315713; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. INTRODUCTION Spermatogenesis is a highly organized process involving coordinated cell cycle progression, differentiation of spermatogonial stem cells and their transformation into mature spermatozoa. With no cell culture models of human germ cells available as yet, the molecular biology of spermatogenesis remains one of the least studied developmental processes in humans. Numerous animal studies emphasized the importance of protein-protein interactions (PPIs) in the production of fertile spermatozoa. In fact, the necessity to silence transcription and translation at the late stages of spermatogenesis resulted in the evolution of epididymis, in which spermatozoa are activated by epidydimis-secreted proteins through numerous proteolytic cascades and PPIs. Null mice models of selected testis-specific genes presented with male infertility phenotypes, presumably through disrupted PPIs and improper processing of proteins during spermatogenesis and sperm maturation (1-7). Early studies discovered the essential role of numerous cell surface proteins for sperm-oocyte interaction and fusion (8). Some of the most critical factors included metalloprotease-disintegrin ADAM2 (9), the cell adhesion tetraspanin CD9 (10) and the sperm-egg fusion protein IZUMO1 (11). The recent discovery of the cell surface recognition complex of IZUMO1 protein and the sperm-egg fusion protein JUNO provided detailed insights into gamete recognition and sperm-oocyte fusion (12, 13). Of 1,035 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. The identification of the exact roles of PPIs during maturation of male and female germ cells continues. Proteomics and mass spectrometry emerged as the techniques of choice to discover PPIs and to elucidate the molecular functions of proteins (15-17). Affinity purification or co-immunoprecipitation (co-IP) approaches followed by mass spectrometry identified numerous direct and indirect PPIs under native physiological conditions (18-20). Advances in sensitivity and throughput of mass spectrometry facilitated mapping of interactomes of bacteria (21), yeast (22, 23), insects (24) and human cells (25). High resolution mass spectrometry empowered by label-free quantification enabled identification of high-confidence PPIs after a single step of affinity purification (26). In this study, we focused on the testis-specific protein TEX101, which we previously discovered and validated as a biomarker of male infertility (27-31). TEX101 protein is exclusively expressed on the surface of testicular germ cells (32) and was suggested to be a cell-surface chaperone involved in trafficking and maturation of numerous cell surface proteins essential for fertilization in mice (7, 33). With four TEX101-regulated proteins (ADAM3-6) previously discovered in mice (7, 34), three correspond to pseudogenes in humans, while ADAM4 gene is not present in the human genome. While human TEX101 is an outstanding biomarker of male infertility (28), its functional 3 bioRxiv preprint doi: https://doi.org/10.1101/315713; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. role in human reproduction is not known. In this work, we established a quantitative co- IP-MS approach to discover the human TEX101 interactome. 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 non-hormonal male contraceptives. 4 bioRxiv preprint doi: https://doi.org/10.1101/315713; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. EXPERIMENTAL PROCEDURES Expression and purification of recombinant proteins TEX101 and DPEP3 Human recombinant proteins TEX101 and DPEP3 were produced in an Expi293F transient expression system according to manufacturer’s recommendations (Invitrogen). Briefly, DNA coding for the mature forms of TEX101 and DPEP3 (aa 26-222 and 36-463, respectively) were cloned into a pcDNA3.4 plasmid for mammalian protein expression (GeneArtTM Gene Synthesis, Invitrogen). Expi293F cells were grown in suspension and cell cultures containing secreted TEX101 and DPEP3 proteins were collected 72 and 96 hours post-transfection, respectively. Recombinant protein production was assessed by Western blot analysis with rabbit polyclonal antibodies, anti-TEX101 HPA041915 (Sigma-Aldrich) and anti-DPEP3 HPA058607 (Sigma- Aldrich). Purification of recombinant TEX101 and DPEP3 from culture supernatants was performed with an automated AKTA FPLC system on a pre-equilibrated 5-mL anion-exchange HiTrap Mono QTM Sepharose high performance column (GE Healthcare). Culture supernatants were diluted in 50 mM Tris (buffer A), pH 9.0 for TEX101, and pH 9.5 for DPEP3, and following binding and washing, proteins were eluted in 4-mL fractions with a linear gradient of 50 mM Tris, 1 M NaCl (buffer B), pH 9.0 (TEX101), and pH 9.5 (DPEP3). The concentration of TEX101 and DPEP3 in fractions was measured by TEX101 ELISA (35), and an SRM assay, respectively. Purity and molecular mass of recombinant proteins were determined by SDS-PAGE stained with Coomassie Blue. Gel bands were excised, subjected to in-gel digestion and analyzed by LC- MS/MS in a Q ExactiveTM Plus Hybrid Quadrupole-OrbitrapTM Mass Spectrometer
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