Radial spoke protein 44 (human meichroacidin) is an axonemal alloantigen of sperm and cilia

Abstract

To identify novel sperm alloantigens relevant to immune infertility, sera from infertile men containing antisperm antibodies (ASA) were employed on 2-D immunoblots of human sperm proteins. An immunoreactive protein spot (MW: 44 kDa, pI: 4.5) was microsequenced and the related cDNA was cloned yielding a 309 amino acid sequence corresponding to a gene currently annotated in Genbank as TSGA2 homolog (mouse) to signify ‘testis specific gene A2’. In Genbank the protein deduced from this gene is currently named human meichroacidin, an orthologue of meichroacidin previously identified in mouse spermatocytes. Human TSGA2 mapped to chromosome 21q22.3. Human meichroacidin (hMCA) contained a single potential tyrosine phosphorylation site and five casein kinase phosporylation motifs. The N-terminus contained a Membrane Occupation Recognition Nexus (MORN) motif found in the lipid kinase-phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family and junctophilins. However hMCA lacked the characteristic kinase homology domain of PIP5K. Northern blot analysis revealed 1.5 kb hMCA transcripts in testis and trachea with lower levels in thyroid and spinal cord. A semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated occurrence of the mRNA messages in all the ciliated tissues tested with highest levels of messages in testis and trachea. Western blot analysis showed the presence of hMCA protein in brain, thyroid and trachea at the identical mass, 44 kDa, as in human testis. However, this immunoreactive pattern differed from that of sperm in which a 38 kDa form was also evident suggesting that hMCA undergoes proteolytic processing. In human testis, hMCA localized to the tails of developing spermatids and did not localize to the nucleus of either spermatocytes or spermatids. EM immunocytochemistry localized hMCA within the radial spokes of the axonemal complex of the sperm flagellum, and immunofluorescence studies revealed h-meichroacidin in the cilia of epithelial cells in the trachea and ependyma. Bioinformatic identification of orthologues of meichroacidin in several lower organisms including ciliates and flagellates suggest the protein plays a role in flagellar motility across phyla. We propose the term radial spoke protein 44 as an accurate designation, preferable to human meichroacidin because it denotes the restricted localization of the protein to the radial spokes of the axonemes of both sperm and cilia. Further, since the human gene is expressed in brain, thyroid, trachea and lung in addition to testis, we suggest that the gene name be changed from TSGA2 [testis specific gene A2] to radial spoke protein 44 [RSP44].

Introduction

ASA are thought to impair fertility by inhibiting sperm motility (Haas, 1987), sperm penetration of the cervical mucus (Haas, 1986), capacitation (Caron and Saling, 1991), or the acrosome reaction (Lee et al., 1990); or they may invoke the complement cascade resulting in sperm lysis (D'Cruz et al., 1991, Bronson et al., 1982). Antisperm antibodies may also be formed as a result of cross-reactivity between sperm antigens and bacterial antigens (Witkin et al., 1995). A complete understanding of the mechanism behind immunologic infertility, as well as improved diagnosis and treatment, depends on knowledge of the identities of specific sperm antigens capable of eliciting the production of functionally relevant sperm antibodies. Utilizing vectorial labeling and 2-D electrophoresis by isoelectric focusing (IEF) our laboratory identified 6 auto- and iso-antigens which were not recognized by fertile sera and possibly relevant to antibody-mediated immunoinfertility (Shetty et al., 1999). Further studies led to the identification and characterization of several post-meiotically expressed sperm alloantigens residing in the acrosomal membranes (Hao et al., 2002, Shetty et al., 2003) and equatorial segment (Wolkowicz et al., 2003). In the current study an interesting alloantigen residing in the sperm tail is characterized.

The formation of the sperm tail is one of the final events in spermiogenesis, a process of marked morphological change during differentiation of haploid round spermatids into mature sperm. Mammalian spermatozoa and ciliated cells share a complex structure, the axoneme, which is responsible for motility of both cilia and flagella. The fine structural and molecular components of the axoneme are highly conserved through evolution from lower to higher eukaryotes (Pennarun et al., 2002). Axonemal ultrastructure reveals one central microtubular doublet and nine outer-doublet microtubules with attached inner and outer dynein arms, radial spokes and nexin links.

In addition to the axoneme and its associated proteins, the sperm flagellum also consists of a fibrous sheath (FS) and outer dense fibers (ODF), as well as proteins that interact with both FS and ODF, such as Oppo1 (Nakamura et al., 2002), Shippo 1(Egydio de Carvalho et al., 2002) and Spag 4 (Shao et al., 1999). However there are few reports on proteins associated with the sperm axonemal complex. Using a proteomic approach we report here the cloning and characterization of a human sperm axonemal complex protein, currently annotated in Genbank as h-meichroacidin. Mouse meichroacidin was originally reported to be a male meiotic chromosome associated acidic protein (meichroacidin), specific to pachytene spermatocytes through the round spermatid stages and not expressed in somatic cells (Tsuchida et al., 1998). The human orthologue of the same protein was later reported by Matsuoka et al. (2005), who demonstrated it to be a component of the sperm flagellum and specifically expressed in the testes. Our study of human meichroacidin indicates that this protein, in addition to be a component of the sperm tail, localizes to the radial spokes of the axoneme, as well as to cilia of epithelial cells suggesting a fundamental role in flagellar and ciliary action. Interestingly, the protein is a human alloantigen. Because this protein's distinct and specific localization in human spermatids and mature sperm differs from that reported for the mouse orthologue, we propose the name radial spoke protein 44 for this protein and gene (RSP44) in humans.