Identification of functional TEX101 interactome through proteomic profiling of human spermatozoa homozygous for a missense variant rs35033974
Schiza, C., Korbakis, D., Jarvi, K., Diamandis, E. P., & Drabovich, A. P. (2018). Identification of TEX101-associated proteins through proteomic measurement of human spermatozoa homozygous for the missense variant rs35033974. Molecular & Cellular Proteomics. http://doi.org/10.1074/mcp.RA118.001170
- Organism: Homo sapiens
- Instrument: Q ExactiveTM Plus Hybrid Quadrupole-Orbitrap and TSQ QuantivaTM triple quadrupole mass spectrometer
Human TEX101 is a testis-specific cell membrane protein expressed exclusively in male germ cells and a previously validated biomarker of male infertility. TEX101 was suggested to function as a cell surface chaperone involved in maturation of numerous proteins involved in sperm migration and sperm-egg interaction. However, the precise functional role of human TEX101 is not known and can hardly be studied in vitro due to the lack of human male germ cell lines. Here, we genotyped 386 healthy fertile men and sub-fertile patients for a predicted deleterious missense variant rs35033974 of TEX101, and identified 52 heterozygous and 4 homozygous men. We then discovered by targeted proteomics that the variant allele rs35033974 resulted in a near-complete degradation (>97%) of the corresponding G99V TEX101 form, and suggested that spermatozoa of homozygous men could serve as a knock-down model to study TEX101 function in human. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified 9 membrane-bound and 9 secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific membrane proteins involved in sperm migration and sperm-oocyte fusion (including LY6K and ADAM29) were confirmed by targeted proteomics and western blotting. Elucidation of TEX101 functional role will advance biology of human reproduction and may facilitate development of better diagnostics of unexplained male infertility.
Spermatozoa pellets were washed twice with PBS, lysed with 0.1% RapiGest SF (Waters, Milford, MA) in 50 mM ammonium bicarbonate and sonicated three times for 30 s. Cell lysates were then centrifuged at 15,000 g for 15 min at 4°C. Total protein in each spermatozoa or SP sample was measured by bicinchoninic acid assay. Ten µg of total protein per patient sample were prepared in 50 mM ammonium bicarbonate. Purified recombinant human TEX101 protein and proteins from spermatozoa and SP were denatured and disulfide bonds were reduced by 0.05% RapiGest SF and 5 mM dithiothreitol (DTT) at 65°C for 30 min. Proteins were then alkylated with 10 mM iodoacetamide in the dark for 40 min at RT. Protein digestion was performed overnight at 37°C in the presence of sequencing grade Glu-C enzyme (Promega) (1:20, Glu-C: total protein) and 5% acetonitrile (ACN) for enhanced Glu-C activity. Digestion in the presence of ammonium bicarbonate (pH 7.8) provided enzyme specificity and cleavage after glutamine. RapiGest SF cleavage and Glu-C inactivation were achieved with 1% trifluoroacetic acid (TFA). Two heavy isotope-labeled TEX101 peptides, AITIVQHSSPPGLIV*TSYSNYCE and AITIVQHSSPPVLIV*TSYSNYCE (L-Valine 13C5, 15N), for the wild-type and the G99V form, were pooled and diluted to a final concentration of 100 fmol/µL and 250 fmol/µL, respectively. Two µL of the heavy peptide pool were spiked into each sample after digestion. Digests were desalted and peptides were extracted by C18 OMIX tips (Varian Inc., Lake Forest, CA). Peptides were eluted in 3 µL of 70% acetonitrile and 0.1% formic acid. Peptides were analyzed by an EASY-nLC 1000 nanoLC coupled to Q ExactiveTM Plus Hybrid Quadrupole-OrbitrapTM Mass Spectrometer (Thermo Fischer Scientific).
Semen samples (N=386) were collected with the informed consent from patients with the approval of the institutional review boards of Mount Sinai (approval #08-117-E) and University Health Network (#09-0830-AE). Samples were obtained from healthy fertile men before vasectomy, and individuals diagnosed with unexplained infertility and oligospermia. Clinical parameters were summarized in Table 1. Unexplained infertility group included men who were not able to father a pregnancy after one year of regular unprotected intercourse, despite the higher than 15 million/mL sperm concentration. After liquefaction, semen samples were centrifuged three times at 13,000 g for 15 min at room temperature. Spermatozoa and SP were separated and stored at -80°C. Samples were analyzed retrospectively.
For the differential proteomic analysis, sperm cell samples were obtained from four men homozygous for TEX101 c.296G>T variant, diagnosed with oligospermia (N=2) and unexplained infertility (N=2), median age of 29.5 years, sperm concentration 2 – 30 million/mL, and TEX101 concentration in SP 3.5 – 47 ng/mL. Fertile control men (N=4), homozygous for wild-type TEX101 genotype, age matched, with normal sperm concentration (>15 million/mL), and TEX101 concentration in SP 8,000 – 12,500 ng/mL, were selected from the pre-vasectomy group. Before cell lysis, sperm pellets were washed three times with PBS.
Eight individual sperm cell samples (N=4 TEX101 wild-type and N=4 TEX101 c.296G>T homozygous) were lysed with 0.1% RapiGest SF in 50 mM ammonium bicarbonate. Cell lysates were centrifuged at 15,000 g for 15 min at 4°C to remove debris, and total protein concentration was measured using BCA assay. Proteins (225 µg total protein per sample) were denatured, and reduced (5 mM DTT), alkylated (10 mM iodoacetamide), and digested with trypsin (Sigma-Aldrich) overnight at 37°C.
Created on 3/22/18 8:24 AM