Uncovering Novel Protein Partners of Inducible Nitric Oxide Synthase in Human Testis

Abstract

Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.

Keywords: co-immunoprecipitation; male infertility; nitric oxide synthase; protein–protein interaction; yeast two-hybrid assay.

Figure 1

Three domains of human inducible nitric oxide synthase with corresponding amino acid residues are shown above (not according to scale). CaM-BD = calmodulin-binding domain.

Figure 2

Western blot of iNOS bait in yeast-transformants using anti-cMyc antibody. Lane 1: pre-stained protein marker; Lane 2: negative control—untransformed yeast lysate; Lane 3: positive control—yeast transformed with pGBKT7-53; Lanes 4 and 6: other iNOS protein fragments faintly displayed (not used in this study); Lane 5: iNOS peptide fragments from 181 to 335aa used in this study. cMyc is a fused tag.

Figure 3

ProLabel assay after co-immunoprecipitation. Panel (A) shows the PL assay with positive control p53 and its interacting partner T antigen. Panel (B) shows the PL assay with candidate proteins, Spaca7 and RbBP4. The negative control included AcGFP1-Lam plus ProLabel-SV40 T.

Figure 4

ProLabel assay with full-length iNOS as bait. K2 is the iNOS peptide fragment as used before compared to the full-length iNOS, as shown by WiNOS. Here, the incubation time was increased to 180 min. The Spaca7 and RbBP4 show stronger signals.

Figure 5

Co-localization of SPACA7 and iNOS in HEK293 cells transfected with pProLabel-SPACA7 and pAcGFP-WiNOS. The nucleus appears blue under the DAPI filter due to the DAPI staining of the nuclear DNA (A). The iNOS appears green due to the GFP under the FITC filter (B). The SPACA7 appears red under the TRITC filter because of using red-fluorescent secondary antibodies specific to the primary SPACA7 antibody (C). Merged image of (B,C) (D). (Magnification 40×).

Figure 6

Co-localization of RbBP4 and iNOS in HEK293 cells transfected with pProLabel-RbBP4 and pAcGFP-WiNOS. The nucleus appears blue under the DAPI filter due to the DAPI staining of the nuclear DNA (A). The iNOS appears green due to the GFP under the FITC filter (B). The RbBP4 appears red under the TRITC filter because of using red-fluorescent secondary antibodies specific to the primary SPACA7 antibody (C). Merged image of (B,C) (D). (Magnification 40×).

Figure 7

The SPACA7-WiNOS stable cell line under a fluorescent microscope (FITC Filter, 40×).

Figure 8

Sequencing of the Spaca7 cDNA revealed a spliced variant. Original spliced mRNA of human SPACA7 (A); spliced variant SPACA7 that we found (B).