Radial spoke protein 44 (human meichroacidin) is an axonemal alloantigen of sperm and cilia.
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].
Shetty
Jagathpala
J
Center for Research in Contraceptive and Reproductive Health, Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Klotz
Kenneth L
KL
Wolkowicz
Michael J
MJ
Flickinger
Charles J
CJ
Herr
John C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002923:Cilia / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007246:Infertility / Q000097:blood; D007519:Isoantigens / Q000276:immunology / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D013082:Sperm Tail / Q000378:metabolism* / Q000648:ultrastructure; D013116:Spinal Cord / Q000166:cytology
D000906:Antibodies; D004268:DNA-Binding Proteins; D007519:Isoantigens; D012333:RNA, Messenger; C484746:RSPH1 protein, human; D011994:Recombinant Proteins