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Sperm Antigen 6 Is the Murine Homologue of the Chlamydomonas reinhardtii Central Apparatus Protein Encoded by the PF16 Locus¹

^a Center for Research on Reproduction and Women's Health, ^b Center for Neurobiology and Behavior, and ^c Molecular Genetics in Psychology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A cDNA encoding sperm antigen 6 (Spag6), the murine homologue of the Chlamydomonas reinhardtii PF16 protein—a component of the flagella central apparatus—was isolated from a mouse testis cDNA library. The cDNA sequence predicted a 55.3-kDa polypeptide containing 8 contiguous armadillo repeats with 65% amino acid sequence identity and 81% similarity to the Chlamydomonas PF1 protein. An antipeptide antibody generated against a C-terminal sequence recognized a 55-kDa protein in sperm extracts and localized Spag6 to the principal piece of permeabilized mouse sperm tails. When expressed in COS-1 cells, Spag6 colocalized with microtubules. The Spag6 gene was found to be highly expressed in testis and was mapped using the T31 radiation hybrid panel to mouse chromosome 16. Mutations in the Chlamydomonas PF16 gene cause flagellar paralysis. The presence of a highly conserved mammalian PF16 homologue (Spag6) raises the possibility that Spag6 plays an important role in sperm flagellar function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Despite the diversity of cell types that have cilia and flagella, the structural components of their axonemes are remarkably conserved across species, including nine microtubule doublets with the associated force-generating dynein arms and radial spokes surrounding the central apparatus [1, 2] (the first reference contains a description of central apparatus structure and a description of central apparatus proteins identified to date). The central apparatus is composed of two structurally and biochemically dimorphic singlet microtubules [1, 3]. Attached to the central pair of microtubules are projections that link the two microtubules. The unique waveforms produced by the beating of flagella are the result of coordinated regulation of the dynein arms, due in part to the existence of different dynein isoforms, alterations in their state of phosphorylation, and the function of other proteins in the axoneme that form the radial spokes and the central apparatus [1, 4–6]. The importance of the central apparatus is exemplified by mutants of the algae, Chlamydomonas reinhardtii, a well-studied model system of cilia and flagella function. Mutants that lack the central apparatus or some of its components have paralyzed flagella [7, 8].

Polypeptide mapping of the Chlamydomonas central apparatus suggests the presence of at least 23 different proteins in addition to tubulin [9]. Chemical and ultraviolet light-induced mutagenesis of Chlamydomonas led to the identification of six mutant phenotypes with central apparatus defects resulting in flagellar paralysis [10]. Four of the genetic loci encoding Chlamydomonas central apparatus proteins have been cloned [1]. The Chlamydomonas pf16 mutant has paralyzed flagella and an unstable C1 microtubule [7, 11]. The protein encoded by the PF16 gene contains 8 armadillo-type repeats and is localized along the length of the C1 tubule [11].

Although studies carried out on model systems such as Chlamydomonas strongly suggest that the central apparatus plays a regulatory role in controlling flagellar activity, relatively little is known about the mammalian flagellum central apparatus, and virtually nothing is known about its function. If studies on Chlamydomonas and other species are indicative of the function of the central apparatus in mammalian cilia and flagellum, then knowledge of the structure and the function of proteins making up this structure may lead to a better understanding of the control of mammalian cell motility. Here we report the cloning of the cDNA encoding a mouse homologue of the product of the Chlamydomonas PF16 locus, the localization of the murine gene to chromosome 16, and the immunolocalization of the protein product in the sperm flagellum.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Isolation and Characterization of cDNA Clones Homologous to the Product of ChlamydomonasPF16 Locus

An expression library from mouse mixed germ cells (containing primarily pachytene spermatocytes, round spermatids, and condensing spermatids) was screened. The oligo(dT)-primed cDNAs were cloned unidirectionally into the Uni-ZAP vector (Stratagene, La Jolla, CA) with the 5' end of the cDNA nearest the lacZ promoter. The ZAP Express library was plated in Xl-1 blue MRF' host cells at density of 50 000 per 150-mm dish and screened using standard procedures with a 254-base pair (bp) probe generated by polymerase chain reaction (PCR) from a mouse EST clone (number 918390) that we had previously noted had high homology to the recently identified human homologue of the product of the Chlamydomonas PF16 locus, SPAG6 [12]. Several positive plaques were selected for characterization. Plasmid was recovered from the phage clones using procedures recommended by Stratagene, and the inserts were subjected to DNA sequence analysis using Taq DyeDeoxy Terminator Cycle Sequencing and reagents and a model 373A Automated Sequencer (all from Applied Biosystems, Foster City, CA). Sequence ambiguities were resolved by sequencing both strands. The amino acid sequence of the encoded protein was deduced using the MacVector (IBI, New Haven, CT) molecular biology programs. Homology searches of GenBank and other sequence databases were performed using the BLAST program of the National Center for Biotechnology Information [13].

Northern Blot Analysis

Northern blots containing total RNA (20 µg/lane) extracted from mouse brain, heart, kidney, liver, lung, spleen, and testes were probed with the same 254-bp fragment that was used in the screening of the library and then an actin cDNA. Blots were incubated with the 254-bp-labeled fragment generated by the random primer method (1 x 10⁶ cpm/ml of hybridization buffer consisting of 50% formamide, 5% Denhardt's solution, 0.1% SDS, 10% dextran sulfate, and 0.2 mg/ml sheared salmon sperm DNA) at 42°C overnight. Blots were washed in double-strength saline-sodium phosphate-EDTA buffer (SSPE), 0.1% SDS, and then in 0.1-strength SSPE and 0.1% SDS, both for 15 min at 40°C and 55°C, and then air dried and exposed to film overnight at -70°C with an intensifying screen.

Western Blotting

Equal amounts of protein (24 µg/lane) from epididymal sperm were heated at 95°C for 4 min in sample buffer, loaded onto 10% SDS-PAGE gels, and electrophoretically separated for 3 h at constant current (30 mA). Protein molecular markers were used to calibrate the gel. Separated proteins were electrotransferred overnight onto nitrocellulose membranes. Membranes were blocked in Tris-buffered saline solution (TBS) containing 5% nonfat dry milk and then incubated with an antipeptide antibody generated against amino acid residues 438–452 (KVLPHDSKARRLFVT) in the C-terminus of murine Spag6, a sequence that is conserved in the human protein [12]. After washing in TBS, the blots were incubated with an anti-rabbit immunoglobulin conjugated to horseradish peroxidase for 24 h at 4°C (1:1000 dilution). After washing in TBS, Spag6 protein was detected with the ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to film.

Cell Culture and Transient Transfection

COS-1 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum at 37°C. At 60% confluence, cells were washed twice with serum-free medium before addition of 1 µg of the mouse cDNA encoding Spag6 in pSV-SPORT-1 or 1 µg of the empty vector and 10 µl of Lipofectamine (Gibco-BRL, Gaithersburg, MD). After 5-h incubation, 1 ml of 20% fetal calf serum was added. After 48 h of culture, cells were fixed in 100% methanol containing 0.1 mM EGTA at -20°C for 8 min, or transfected cells were scraped into the lysis buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1 mM PMSF, and 1 µg/ml aprotinin) for Western blot analysis.

In order to determine the subcellular localization of mouse Spag6, transfected COS-1 cells were collected into a buffer consisting of 100 mM 2-[N-morpholino]ethanesulfonic acid, 1 mM EGTA, 0.5 mM MgSO₄, pH 7.4, at 4°C for 20 min; they were then sonicated for 5 min and centrifuged at 30 000 x g for 30 min. The resulting pellet and the supernatant were analyzed for Spag6 or -tubulin by Western blotting.

Preparation of Sperm for Immunolocalization of Spag6

Sperm were obtained from mouse epididymis and centrifuged at 3000 x g, washed twice in PBS, resuspended in PBS, and layered onto polylysine-coated slides. The preparations were fixed in 4% paraformaldehyde, permeabilized with methanol, and then treated with primary antibody and fluorescein-conjugated second antibody.

Immunofluorescence Microscopy

After permeabilization, COS-1 and sperm cells were blocked in PBS containing 10% goat serum (1 h at 37°C) and incubated with primary antibody (rabbit anti-Spag6 or mouse anti-ß-tubulin) overnight at 4°C. Secondary antibodies were fluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG (1:500). An Olympus (Tokyo, Japan) IX-70 epifluorescence microscope and digitally cooled camera and the Meta Morph Imaging System software (Universal Imaging Corp., West Chester, PA) were used for imaging.

Mapping of the Spag6 Gene with a Radiation Hybrid Panel

The mouse radiation hybrid panel T31 was constructed by fusing irradiated mouse embryo primary cells (129 amino acids) with hamster cells [14]. The mouse cells were irradiated with 3000 rad and fused with A23, a thymidine kinase-deficient (TK-) hamster cell line. A selected set of 100 hybrid lines, with the average retention rate of 20–25% and estimate of 98 kilobases (kb)/CR3000, provides potential resolution of 378 kb. In contrast to genetic mapping, which requires polymorphic markers between the inbred strains used to generate the cross, PCR-based radiation hybrid mapping requires only that markers be present in mouse and absent in hamster DNA, or alternatively, that the amplicons detected in these DNAs be of different size. DNA from the 100 radiation hybrid clones from the mouse-hamster T31 Radiation Hybrid panel was purchased from Research Genetics (Huntsville, AL). Samples were arrayed in a 96-well 0.5-ml polypropylene deep-well plate (Marsh Biomedical, Rochester, NY) and diluted threefold. Twenty-five nanograms of DNA was transferred with a multichannel pipette to a second reaction plate.

Gene primers were designed using sequence data from the 3' end of the Spag6 cDNA. The primer sequences were as follows: left primer 5'-TTCCTCTTCCCTGAAGGGTC-3'; right primer 5'-ATCGGAAAACATGACTTGGG-3'. PCR reactions consisted of 10-µl total volume containing 25 ng genomic DNA, 0.23 µM primers, 0.23 mM of each dNTP, 0.25 U Taq polymerase, and single-strength PCR buffer containing 1.5 mM MgCl₂. Thermal cycling included a 5-min denaturization step at 94°C; 35 cycles of 30 sec at 94°C, 30 sec at 55°C, 60 sec at 72°C; and a final extension step for 10 min at 72°C. Analysis of PCR products was performed by electrophoresis on 3% NuSieve (FMC BioProducts, Rockland, ME) agarose gels followed by ethidium bromide staining.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Characterization of the Spag6 cDNA and Its Deduced Amino Acid Sequence

The initial screen of the mouse testis expression library yielded multiple plaques that were positive in a second round of screening. The sequence of one of the isolated cDNAs (2.0 kb) encompassed a partial amino acid sequence homologous to the product of the Chlamydomonas PF16 locus (64% amino acid identity, 80% similarity) including the presence of 8 contiguous armadillo repeats [1, 15, 16]. Rescreening of the testes library using the 5' end of this clone as a probe yielded a 2.5-kb cDNA insert containing a 507-amino acid open reading frame (Fig. 1) coding for a 55.3-kDa protein. The deduced amino acid sequence of this clone showed 65% amino acid identity and 81% similarity to the Chlamydomonas PF16 protein and 96% amino acid identity and 98% similarity to SPAG6, the human homologue of the Chlamydomonas PF16 protein [12] (Fig. 2). Chlamydomonas PF16 has a C-terminal extension of 50 amino acid residues compared to Spag6, which also lacks the two Asn residues that form the C-terminus of the human homologue.

View larger version (50K): [in this window] [in a new window]   FIG. 1. Nucleotide sequence of the mouse Spag6 clone and deduced amino acid sequence

View larger version (114K): [in this window] [in a new window]   FIG. 2. Alignment of the mouse Spag6, human SPAG6, and Chlamydomonas PF16 amino acid sequences. Sequence identities are boxed and similar residues are shaded. The 8 contiguous armadillo repeats are indicated by underlining

A motif analysis of the mouse Spag6 amino acid sequence revealed three consensus sequences for potential protein kinase C-mediated phosphorylation (amino acid residues 2–4, 304–306, and 400–402) and three potential casein kinase II phosphorylation sites (amino acid residues 91–94, 204–207, and 392–395). There were no other motifs of interest besides the 8 armadillo repeats.

Tissue-Specific Expression of Spag6

Northern hybridization analysis demonstrated Spag6 transcripts of 1.7 and 2.8 kb in mouse testes but no detectable mRNA in spleen, thymus, prostate, ovary, small intestine, colon, and peripheral blood leukocytes (Fig. 3).

View larger version (69K): [in this window] [in a new window]   FIG. 3. Northern blot analysis of Spag6 expression. Northern blot of mRNA extracted from the indicated tissues (A). The blot was reprobed with an actin cDNA (B)

Western Blot Analysis of Spag6

Western blotting using the anti-Spag6 antibody demonstrated the presence of a 55-kDa protein in extracts of epididymal sperm consistent with the predicted molecular mass of Spag6 (Fig. 4).

View larger version (37K): [in this window] [in a new window]   FIG. 4. Western blot analysis of Spag6 in mouse sperm extracts. Western blot of sperm protein extracts probed with an antibody generated against amino acid residues 438–452 in the C-terminus of the Spag6 protein. Arrow shows an immunoreactive protein band migrating near 55.3 kDa compatible with the predicted size of Spag6

Localization of Spag6 in Mouse Sperm and Transfected COS-1 Cells

Spag6 immunoreactivity was localized to the principal piece of the mouse sperm flagellum by indirect immunofluorescence studies in permeabilized mouse cauda epididymal sperm (Fig. 5). A weaker signal was observed in the sperm tail midpiece and head. Neutralization of the antiserum with peptide ablated the signal. Nonpermeabilized sperm did not stain, indicating that the Spag6 protein was localized beneath the membrane of the tail.

View larger version (76K): [in this window] [in a new window]   FIG. 5. Localization of Spag6 in mouse sperm flagella. A) Phase contrast and (B) immunofluorescence of methanol-permeabilized sperm incubated with the antibody against Spag6. C) Nonpermeabilized mouse sperm incubated with the same antibody (left, phase contrast; right, immunofluoresence). D) Mouse sperm reacted with antibody neutralized with the 438–452 peptide (left, phase contrast; right, immunofluoresence)

Transfection of COS-1 cells with the Spag6 cDNA in the pSV-SPORT-1 vector led to the expression of a 55-kDa protein detected with the anti-Spag6 peptide antibody, whereas this protein was not detected in cells transfected with empty vector (Fig. 6). The Spag6 was localized to structures that stained for tubulin in transfected COS-1 cells, suggesting that it interacts with microtubules (Fig. 7). Moreover, Spag6 was detectable only in the 30 000 x g pellet of homogenates of the transfected COS-1 cells, but tubulin was detected in both the supernatant and pellet. Collectively, the immunocytochemical studies and the Western blot analyses of cell fractions indicated that all or most of the expressed Spag6 protein is associated with the polymerized tubulin.

View larger version (23K): [in this window] [in a new window]   FIG. 6. Western blot analysis of Spag6 in extracts of transfected COS-1 cells. COS-1 cells were transfected with mouse Spag6 cDNA (pSPORT-Spag6) or empty pSV-SPORT-1 vector. A) Total extract of COS-1 cells showing expression of Spag6 in cells transfected with Spag6 cDNA, but no expression in cells transfected with the empty plasmid vector. B) Presence of Spag6 in the 30 000 x g pellet, but not the supernatant, of COS-1 cells transfected with Spag6 cDNA. C) Presence of tubulin in the 30 000 x g pellet and supernatant of the same COS-1 cells transfected with Spag6 cDNA

View larger version (27K): [in this window] [in a new window]   FIG. 7. Colocalization of Spag6 and tubulin in transfected COS-1 cells. Top) COS-1 cells transfected with the Spag6 cDNA reacted with antibody against the Spag6 peptide. Middle) COS-1 cells transfected with Spag6 cDNA reacted with anti-ß-tubulin. Bottom) Merged image showing colocalization of Spag6 and tubulin seen in yellow. Bars = 10 µm

Mapping of the Spag6 Gene

Radiation hybrid data were submitted to the MIT Auto-RHMAPPER web site (http://www.genome.wi.mit.edu/cgi-bin/mouse_rh/rhmap-auto/rhmapper.cgi). Auto-RHMAPPER places loci relative to the existing MIT radiation hybrid framework map, which contains over 2400 loci mapped across the mouse genome [15]. Spag6 was linked most closely with the chromosome 16 marker, D16Mit34, at a LOD score of 13.7 (Fig. 8).

View larger version (14K): [in this window] [in a new window]   FIG. 8. Map of chromosome 16 showing the existing framework. Spag6 localization on the radiation hybrid map of mouse chromosome 16. Spag6 was placed between D16Mit34 and D16Mit101 and was linked closely to D16Mit34 (LOD = 13.7). CentiRay distances are shown between markers. For mouse chromosome 16, cR = 112.3 kb [15]

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We describe here the cloning and nucleotide sequence of a cDNA encoding a mouse protein that has high homology with SPAG6, a human sperm antigen recently described in this laboratory [12], and with the product of the Chlamydomonas PF16 locus. These proteins are members of the armadillo repeat family, named for a repetitive 42-amino acid motif originally identified in the Drosophila segment polarity gene product, armadillo [16, 17] The armadillo repeats are thought to mediate protein-protein interactions, suggesting that the PF16 homologues interact with other molecules to perform structural or regulatory functions [1, 11]. The nature of the protein(s) with which Spag6 interacts is presently unknown. The yeast two-hybrid system could be a useful approach for identification of interacting partners as demonstrated by the cloning of Spag4, a protein that binds to the outer dense-fiber protein Odf1 [18]. Suppressor mutations in the ß-dynein heavy chain restore flagellar motility in the Chlamydomonas pf16 mutant [19], raising the possibility that proteins that interact with Spag6 interact directly or indirectly with the outer dynein arms. The presence of consensus sites for phosphorylation that are conserved in both the mouse and human Spag6 sequences deserves future attention given the recognized importance of protein phosphorylation in flagellar motility [20, 21].

The product of the Chlamydomonas PF16 locus is localized to the C1 microtubule of the central pair of the flagellum [1, 11]. The molecular basis for the asymmetric localization of PF16 in the central pair of Chlamydomonas flagella has not been ascertained. However, the two central pair microtubules contain different tubulin isoforms that may determine the binding of PF16 or proteins linking PF16 to the C1 microtubule [22]. The immunocytochemical detection of Spag6 protein primarily in the principal piece of permeabilized sperm, and the inability to detect a signal when sperm were not permeabilized, are consistent with an axonemal localization. However, studies at the ultrastructural level will be required to determine whether the mouse protein resides in the central apparatus and to determine whether it is associated with a specific microtubule of the central pair. Transfection of COS-1 cells, which do not normally contain Spag6, with the Spag6 cDNA resulted in colocalization of the expressed protein with microtubules. Subcellular fractionation of the transfected COS-1 cells indicated that little if any of the expressed protein was in the cytoplasm, with the only detectable protein being recovered in the cell pellet containing polymerized tubulin. It remains to be determined whether Spag6 binds directly to polymerized tubulin or whether it is linked to microtubules through an intermediary protein(s).

The high level of Spag6 expression in the testis and its apparent absence in other tissues are consistent with a role for Spag6 in sperm flagellar structure or function. It should be noted, however, that the Spag6 gene may be expressed at low levels in tissues containing ciliated cells (i.e., trachea and oviduct). The abundance of Spag6 mRNA in the total RNA preparations we analyzed may not have been sufficient to be detected in the Northern blotting procedure.

The gene encoding SPAG6, the human PF16 homologue, has been mapped to chromosome 10p11.2-p12 [12]. The mapping of the murine homologue to mouse chromosome 16 was not initially anticipated from the known syntenic relationships between human chromosome 10p and murine chromosomes (Chr 2 and 18). However, it should be noted that relatively few genes on human 10p have been mapped in the mouse genome. The localization of Spag6 to chromosome 16 extends the catalogue of genes on human chromosome 10p to a murine chromosome not previously known to contain 10p-encoded genes.

The phenotype of the Chlamydomonas pf16 mutant includes flagellar paralysis and an unstable C1 microtubule [9]. These observations suggest a structural role for the PF16 protein and possibly a regulatory role in controlling the flagellar beat. The availability of molecular probes for the murine PF16 homologue, Spag6, creates opportunities to study the role of the mammalian protein in flagellogenesis as well as flagellar function using gene-targeting technology.

	ACKNOWLEDGMENTS

 
We thank Ms. Judith Wood for assistance in preparation of this manuscript.

	FOOTNOTES

 
First decision: 21 September 1999.

¹ Supported in part by D43-TW-00671 from the Fogarty Center (J.F.S.), HD37416 (J.F.S.), a grant from the Rockefeller Foundation (J.F.S.), and a Scholarship from the Organization of American States (PRA-56721) (R.S.). The cDNA sequence reported here has been deposited in GenBank (Accession No. AF173866).

² Correspondence: Jerome F. Strauss III, 1354 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104. FAX: 215-573-5408; jfs3{at}mail.med.upenn.edu

³ R.S. is a visiting investigator from the Department of Histology and Embryology, Faculty of Medicine, Montevideo, Uruguay.

Accepted: October 5, 1999.

Received: August 11, 1999.

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