An X-linked Gene Encodes a Major Human Sperm Fibrous Sheath Protein, hAKAP82
1998; Elsevier BV; Volume: 273; Issue: 48 Linguagem: Inglês
10.1074/jbc.273.48.32135
ISSN1083-351X
AutoresRegina M. Turner, Linda R. Johnson, Lisa Haig‐Ladewig, George L. Gerton, Stuart B. Moss,
Tópico(s)Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
ResumoMammalian sperm motility is regulated by a cascade of cAMP-dependent protein phosphorylation events mediated by protein kinase A. A-kinaseanchor proteins (AKAPs) direct protein kinase A activity by tethering the enzyme near its physiological substrates. We have characterized a major human sperm fibrous sheath AKAP, hAKAP82, and its precursor, pro-hAKAP82, the homologues of the mouse fibrous sheath proteins mAKAP82 and pro-mAKAP82. The cDNA sequence of pro-hAKAP82 was highly homologous to the mouse sequence, and the functional domains of the pro-hAKAP82 protein, the protein kinase A binding, and the pro-hAKAP82/hAKAP82 cleavage sites were identical to those of the mouse protein. The genomic organization of mousepro-AKAP82 was determined. Alternative splicing occurred in both the mouse and human pro-AKAP82 genes that resulted in at least two distinct transcripts and possibly two different proteins. Compared with pro-mAKAP82, considerably less pro-hAKAP82 was processed to hAKAP82 in human sperm. Although pro-mAKAP82 localizes only to the proximal portion of the principal piece of the flagellum, pro-hAKAP82 localized to the entire length of the principal piece. The pro-hAKAP82 gene mapped to human chromosome Xp11.2, indicating that defects in this gene are maternally inherited. These studies suggest several roles for hAKAP82 in sperm motility, including the regulation of signal transduction pathways. Mammalian sperm motility is regulated by a cascade of cAMP-dependent protein phosphorylation events mediated by protein kinase A. A-kinaseanchor proteins (AKAPs) direct protein kinase A activity by tethering the enzyme near its physiological substrates. We have characterized a major human sperm fibrous sheath AKAP, hAKAP82, and its precursor, pro-hAKAP82, the homologues of the mouse fibrous sheath proteins mAKAP82 and pro-mAKAP82. The cDNA sequence of pro-hAKAP82 was highly homologous to the mouse sequence, and the functional domains of the pro-hAKAP82 protein, the protein kinase A binding, and the pro-hAKAP82/hAKAP82 cleavage sites were identical to those of the mouse protein. The genomic organization of mousepro-AKAP82 was determined. Alternative splicing occurred in both the mouse and human pro-AKAP82 genes that resulted in at least two distinct transcripts and possibly two different proteins. Compared with pro-mAKAP82, considerably less pro-hAKAP82 was processed to hAKAP82 in human sperm. Although pro-mAKAP82 localizes only to the proximal portion of the principal piece of the flagellum, pro-hAKAP82 localized to the entire length of the principal piece. The pro-hAKAP82 gene mapped to human chromosome Xp11.2, indicating that defects in this gene are maternally inherited. These studies suggest several roles for hAKAP82 in sperm motility, including the regulation of signal transduction pathways. fibrous sheath protein kinase A A-kinase anchor protein regulatory subunit of PK-A mouse AKAP82 precursor of mAKAP82 human AKAP82 precursor of hAKAP82 pro domain of pro-hAKAP82 bacterial artificial chromosome antibody against the pro domain of pro-hAKAP82 expressed sequence tag sequence representing the 5′-untranslated region of the alternative spliced variant of pro-hAKAP82 fluorescence in situ hybridization base pair(s) polymerase chain reaction phosphate-buffered saline. Eukaryotic flagellar assembly and the regulation of flagellar motility are complex processes. For example, in the blue-green algaChlamydomonas reinhartii, more than 25 loci have been characterized that affect flagellar assembly, and over 52 loci have been identified that result in altered motility (1Bell C. Trends Biochem. Sci. 1994; 19: 427-429Abstract Full Text PDF PubMed Scopus (1) Google Scholar). In contrast toChlamydomonas, only a few of the molecular defects underlying naturally occurring cases of human flagellar immotility have been characterized (2Afzelius B.A. Eliasson R. J. Ultrastruct. Res. 1979; 69: 43-52Crossref PubMed Scopus (112) Google Scholar, 3Eliasson R. Mossberg B. Camner P. Afzelius B. N. Eng. J. Med. 1977; 297: 1-6Crossref PubMed Scopus (259) Google Scholar, 4Narayan D. Krishnan S.N. Upender M. Ravikumar T.S. Mahoney M.J. Dolan T.F. Teebi A.S. Haddad G.G. J. Med. Genet. 1994; 31: 493-496Crossref PubMed Scopus (93) Google Scholar, 5Weil D. Blanchard S. Kaplan J. Guilford P. Gibson F. Walsh J. Mburu P. Varela A. Levilliers J. Weston M.D. Kelley P.M. Kimberling W.J. Wagenaar M. Levi-Acobas F. Larget-Piet D. Muunich A. Steel K.P. Brown S.D.M. Petit C. Nature. 1995; 374: 60-61Crossref PubMed Scopus (884) Google Scholar). It is likely that the molecular basis of human sperm motility is more complicated than that of Chlamydomonas because, in addition to the axoneme, mammalian sperm have several flagellar accessory structures including a fibrous sheath (FS).1 The FS is a structure found exclusively in the principal piece of the flagellum where it surrounds the outer dense fibers and axoneme. It is believed to play a structural role in sperm motility by restricting the plane of bending of the flagellum (6Lindemann C.B. Orlando A. Kanous K.S. J. Cell Sci. 1992; 102: 249-260PubMed Google Scholar). Although many of the target proteins have not been identified, it is well accepted that protein phosphorylation/dephosphorylation events are involved in the initiation and maintenance of mammalian sperm flagellar motility and that phosphorylation occurs via a cAMP-dependent pathway (7Tash J.S. Means A.R. Biol. Reprod. 1982; 26: 745-763Crossref PubMed Scopus (194) Google Scholar, 8Brokaw C.J. J. Cell. Biochem. 1987; 35: 175-184Crossref PubMed Scopus (130) Google Scholar, 9Tash J.S. Krinks M. Patel J. Means R.L. Klee C.B. Means A.R. J. Cell Biol. 1988; 106: 1626-1633Crossref Scopus (173) Google Scholar, 10Tash J.S. Bracho G.E. J. Androl. 1994; 15: 505-509PubMed Google Scholar). In mammalian sperm, the major downstream target of cAMP is protein kinase-A (PK-A), thus making it likely that this enzyme is involved in the regulation of sperm motility (11Visconti P. Johnson L. Oyaski M. Fornes M. Moss S. Gerton G. Kopf G. Dev. Biol. 1997; 192: 351-363Crossref PubMed Scopus (175) Google Scholar). Although typically soluble in somatic cells, PK-A also can be found tethered to subcellular organelles via binding of its regulatory (RII) subunit to A-kinaseanchor proteins (AKAPs) (12Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1907) Google Scholar, 13Rubin C.S. Biochim. Biophys. Acta. 1994; 1224: 407-479Google Scholar, 14Scott J.D. McCartney S. Mol. Endocrinol. 1994; 8: 5-11Crossref PubMed Scopus (152) Google Scholar). In response to cAMP binding to the RII subunit of PK-A, the catalytic subunit of the kinase is released and becomes free to catalyze phosphorylation of its substrates. By tethering PK-A close to certain substrates, AKAPs may play critical roles in determining the specificity of PK-A action (15Coghlan V.M. Bergeson S.E. Langeberg L. Nilaver G. Scott J.D. Mol. Cell. Biochem. 1993; 127/128: 309-319Crossref Scopus (55) Google Scholar, 16Rubin C.S. Biochim. Biophys. Acta. 1994; 1224: 467-479PubMed Google Scholar, 17Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (835) Google Scholar). We have cloned and characterized a cDNA encoding mouse AKAP82 (mAKAP82), the major protein of the sperm FS and a member of the AKAP family (11Visconti P. Johnson L. Oyaski M. Fornes M. Moss S. Gerton G. Kopf G. Dev. Biol. 1997; 192: 351-363Crossref PubMed Scopus (175) Google Scholar, 18Carrera A. Gerton G.L. Moss S.B. Dev. Biol. 1994; 165: 272-284Crossref PubMed Scopus (189) Google Scholar). Mouse AKAP82 is synthesized in the cell body of condensing spermatids as a M r 97,000 precursor (pro-mAKAP82, GenBank accession number U07423) (18Carrera A. Gerton G.L. Moss S.B. Dev. Biol. 1994; 165: 272-284Crossref PubMed Scopus (189) Google Scholar). This precursor polypeptide is transported down the flagellum to the principal piece where it is processed by the proteolytic cleavage of the amino-terminal 179 amino acids to produce mAKAP82 and the free 179 amino acid pro domain (19Johnson L. Foster J. Haig-Ladewig L. VanScoy H. Rubin C. Moss S. Gerton G. Dev. Biol. 1997; 192: 340-350Crossref PubMed Scopus (104) Google Scholar). Coincident with or following cleavage, mAKAP82 is assembled into the FS. Mouse AKAP82 could tether PK-A close to the axoneme and other components of the flagellum that are involved in sperm motility, thus regulating the action of PK-A by directing its activity to specific motility-related targets. With the characterization of mAKAP82 there is now experimental evidence for a functional role of the FS as a mediator of PK-A activity. Previous work from our laboratories has shown that the human homologue of mAKAP82, hAKAP82, localizes to the FS (20Carrera A. Moos J. Ning X. Gerton G. Tesarik J. Kopf G. Moss S. Dev. Biol. 1996; 180: 284-296Crossref PubMed Scopus (284) Google Scholar). Additionally, both hAKAP82 and its predicted precursor protein, pro-hAKAP82, bind the RII subunit of PK-A and are the major polypeptides of a limited subset of proteins that become tyrosine-phosphorylated in a time-dependent manner after incubation in a medium supporting capacitation. In human sperm, capacitation is associated with several cellular changes, such as the tyrosine phosphorylation of specific proteins (20Carrera A. Moos J. Ning X. Gerton G. Tesarik J. Kopf G. Moss S. Dev. Biol. 1996; 180: 284-296Crossref PubMed Scopus (284) Google Scholar, 21Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar, 22Galantino-Homer H.L. Visconti P.E. Kopf G.S. Biol. Reprod. 1997; 56: 707-719Crossref PubMed Scopus (357) Google Scholar), that must take place before fertilization can occur. Capacitation also is associated with alterations in sperm motility patterns characteristic of sperm hyperactivation (23Morales P. Overstreet J.W. Katz D.F. J. Reprod. Fertil. 1988; 83: 119-128Crossref PubMed Scopus (72) Google Scholar). Because the evidence to date is supportive of both structural and functional roles for pro-mAKAP82 and mAKAP82 in mouse sperm motility, we hypothesize that pro-hAKAP82 and hAKAP82 play central roles in human sperm motility. The hypothesis is based on the observation that these proteins are phosphoproteins that are likely to be involved in the regulation of phosphorylation of other proteins in the sperm flagellum; such phosphorylation events are known to be critical for regulating motility (7Tash J.S. Means A.R. Biol. Reprod. 1982; 26: 745-763Crossref PubMed Scopus (194) Google Scholar, 8Brokaw C.J. J. Cell. Biochem. 1987; 35: 175-184Crossref PubMed Scopus (130) Google Scholar, 9Tash J.S. Krinks M. Patel J. Means R.L. Klee C.B. Means A.R. J. Cell Biol. 1988; 106: 1626-1633Crossref Scopus (173) Google Scholar, 10Tash J.S. Bracho G.E. J. Androl. 1994; 15: 505-509PubMed Google Scholar, 18Carrera A. Gerton G.L. Moss S.B. Dev. Biol. 1994; 165: 272-284Crossref PubMed Scopus (189) Google Scholar, 20Carrera A. Moos J. Ning X. Gerton G. Tesarik J. Kopf G. Moss S. Dev. Biol. 1996; 180: 284-296Crossref PubMed Scopus (284) Google Scholar). Support for this hypothesis requires further characterization of hAKAP82 and its precursor. In this paper, we report that pro-hAKAP82 and hAKAP82 were the human homologues of pro-mAKAP82 and mAKAP82. We provide evidence that two alternative transcripts of the gene were made during both human and mouse spermatogenesis because of the use of different donor/acceptor splice junctions. Additionally, both pro-hAKAP82 and hAKAP82 localized specifically to the entire length of the FS of ejaculated sperm. Finally, we show that the pro-hAKAP82 gene mapped to human Xp11.2, a finding that has significant implications for germ cell development and male infertility. To isolate a cDNA clone corresponding topro-hAKAP82, a random-primed human testis cDNA library in a λgt11 vector (CLONTECH, Palo Alto, CA) was screened by filter hybridization with a radiolabeled 1.9-kilobase pair cDNA fragment representing the 5′ end of the pro-mAKAP82 cDNA (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). After multiple rounds of screening, a 1.4-kilobase pair partial pro-hAKAP82 cDNA clone homologous to the 5′-UTR and the first 1222 bp of coding sequence of pro-mAKAP82 was isolated and subcloned into the EcoRI site of pGEM-3™ (Promega, Madison, WI). A human testis Expressed Sequence Tag (EST) clone containing an approximately 750-bp insert, which was over 90% homologous to bases 1948–2520 of the pro-mAKAP82 cDNA coding region and the pro-mAKAP82 3′-UTR, was identified by performing a BLAST (basic local alignment search tool (25Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (72097) Google Scholar)) search on the GenBank EST data base with the pro-mAKAP82 cDNA sequence. The human EST clone was obtained from the I.M.A.G.E. consortium (LLNL) (I.M.A.G.E. consortium clone 726916, Research Genetics, Inc. Huntsville, AL) (26Lennon G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Crossref PubMed Scopus (1089) Google Scholar, 27Adams M.D. Kelley J.M. Gocayne J.D. Dubnick M. Polymeropoulos M.H. Xiao H. Merril C.R. Wu A. Olde B. Moreno R.F. Kerlavage A.R. McCombie W.R. Venter J.C. Science. 1991; 252: 1651-1656Crossref PubMed Scopus (1883) Google Scholar). To map the chromosomal location of the pro-hAKAP82 gene by fluorescencein situ hybridization (FISH), a genomic clone containingpro-hAKAP82 was isolated from a genomic human bacterial artificial chromosome (BAC) library (in vector pBeloBAC 11) by Research Genetics using a PCR-based technique. PCR primers were designed based on the cDNA sequence for pro-hAKAP82. Sequence analysis of the BAC clone showed that it contained pro-hAKAP82. A genomic clone containing the 5′-flanking region of pro-mAKAP82 was isolated from a mouse genomic library in λGEM™-11 (Promega) using filter hybridization with a radiolabeled PCR fragment from the mouse cDNA as a probe (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A second genomic clone containing the full-length genomic sequence of pro-mAKAP82 together with its 5′- and 3′-flanking regions was isolated from a mouse P1 embryonic stem cell library using a PCR-based technique and primers derived from the pro-mAKAP82 cDNA sequence (Genome Systems Inc., St. Louis, MO). A 1.6-kilobase pair PCR product corresponding to the region of pro-hAKAP82 that was not included in the human cDNA or EST clones (homologous to bases 437 to 2092 of the pro-mAKAP82 cDNA) was amplified by PCR from a human testis cDNA library using primers based on the pro-hAKAP82 cDNA sequence (corresponding to bases 437 to 457 of the pro-hAKAP82 cDNA coding region) and the pro-hAKAP82 EST sequence (corresponding to bases 2097 to 2077 of the pro-hAKAP82 cDNA coding region). To assay for the presence of an alternative splice variant of pro-hAKAP82, a DNA fragment was amplified by PCR from a human testis cDNA library using primers corresponding to a region in the 5′-UTR of mouse Fsc1 (GenBank accession number U10341 (28Fulcher K.D. Mori C. Welch J.E. O'Brien D.A. Klapper D.G. Eddy E.M. Biol. Reprod. 1995; 52: 41-49Crossref PubMed Scopus (83) Google Scholar)) and to a region in the 5′ end of the coding region of the pro-hAKAP82 cDNA. PCR products were purified with the Wizard™ PCR preps kit (Promega) before sequencing. All sequencing was done with the AmpliTaq®, FS dye terminator cycle sequencing kit chemistry or the BigDye™ terminator cycle sequencing kit chemistry and the appropriate primers using a 373A DNA sequencer (PE Applied Biosystems, Foster City, CA). Ambiguities were resolved by sequencing the opposite strand. DNA and protein sequence analyses were performed using the MacVector™ (Kodak Scientific Imaging Systems, New Haven, CT) and Sequencher™ (Gene Codes Corp., Ann Arbor, MI) software programs. Samples of human semen were obtained by masturbation from normal, healthy donors with good sperm motility (total sperm motility greater than 75%, progressive motility greater than 60%). Ejaculate volume, percentage of total and progressively motile sperm, and sperm concentration were determined for each ejaculate. Sperm were washed 3× in PBS. Before final centrifugation, the volume, sperm concentration, and total sperm numbers again were determined for each sample. After the final wash, sperm pellets were dissolved in SDS sample buffer containing 40 mm dithiothreitol and boiled for 5 min. The amount of protein in each sample was determined by the Amido Black procedure (29Schaffner W. Weissman C. Anal. Biochem. 1973; 56: 502-504Crossref PubMed Scopus (1955) Google Scholar). An antiserum against the predicted pro domain of pro-hAKAP82 (anti-hpro) was prepared as follows. A peptide corresponding to residues 131–145 (NH2-VGDTEGDYHRASSEN-COOH, the hpro peptide) of the pro-hAKAP82 protein was synthesized with a cysteine added to the amino terminus (Quality Control Biochemicals, Hopkinton, MA). The peptide was conjugated to keyhole limpet hemocyanin via the cysteine and then used for immunization. The antiserum was characterized by immunoblotting using protein extracts from ejaculated human sperm. A portion of the antiserum was affinity-purified, eluted, neutralized, and dialyzed in PBS. Proteins from ejaculated human sperm were separated under reducing conditions by SDS-polyacrylamide gel electrophoresis on a 10% (w/v) gel and electrophoretically transferred to nitrocellulose membranes. Equal amounts of protein were analyzed in each lane. The blots were blocked, probed with anti-hpro (1:2000 (v/v)), processed, and developed using an ECL kit (Amersham Pharmacia Biotech) as described previously (19Johnson L. Foster J. Haig-Ladewig L. VanScoy H. Rubin C. Moss S. Gerton G. Dev. Biol. 1997; 192: 340-350Crossref PubMed Scopus (104) Google Scholar) before being exposed to Reflection™ film (NEN Life Science Products). For the preabsorption experiments, the antiserum was incubated with the hpro peptide (1 mg/ml) in PBS containing 0.1% (v/v) Tween 20 and 3% (w/v) bovine serum albumin for 1 h at room temperature and then used to probe the immunoblots as described above. One-ml aliquots of sperm diluted in PBS to 3 × 106 cells/ml were permeabilized in 0.1% (v/v) Triton X-100 for 15 min and then washed once in PBS. Pellets were resuspended in 1 ml of PBS, and the cells were transferred onto coverslips. After settling, cells were fixed in 4% (w/v) paraformaldehyde, incubated in −20 °C methanol, washed, and blocked in normal goat serum as described previously (19Johnson L. Foster J. Haig-Ladewig L. VanScoy H. Rubin C. Moss S. Gerton G. Dev. Biol. 1997; 192: 340-350Crossref PubMed Scopus (104) Google Scholar). Sperm then were incubated in anti-hpro diluted 1:10 (v/v) in 10% goat serum overnight at 4 °C and washed in PBS. Sperm were incubated for 1 h at 37 °C in the secondary antibody (fluorescein isothiocyanate-conjugated goat anti-rabbit IgG, Jackson Immunoresearch Laboratories, Inc., West Grove, PA), diluted 1:50 (v/v) in 10% goat serum, and again washed in PBS before being mounted on slides with mounting media (Fluoromount-G, Southern Biotechnology Associates Inc., Birmingham. AL). Slides were viewed with a Zeiss Photomicroscope III equipped with epifluorescence. Photographs were taken with Kodak T-Max film, 3200 ASA. Paired sample and control photographs were exposed for equal amounts of time. Localization of the pro-hAKAP82 gene to a single chromosome band was accomplished using FISH mapping. The entire human genomic BAC clone containing an insert of approximately 100 kilobase pairs, including pro-hAKAP82, was labeled with digoxigenin-11-dUTP (Boehringer Mannheim) by nick translation and hybridized to metaphase chromosome spreads prepared from peripheral blood lymphocytes from a normal man. Labeled probe (300 ng) was incubated overnight at 37 °C with 3 μg of human cot-1 DNA (Amersham Pharmacia Biotech) in Hybrisol VII (Oncor Inc., Gaithersburg, MD). The probe then was denatured at 72 °C for 5 min and pre-annealed for 30 min at 37 °C. Slides were dehydrated and denatured before hybridizing overnight at 37 °C with the probe in a humidified chamber. Slides were washed in 50% formamide,1× SSC (0.15 M NaCl and 0.015 M sodium citrate) for 10 min and then twice in 2× SSC for 4 min each. All washes were done at 40 °C with gentle shaking. Slides were transferred to a solution of 1× phosphate-buffered detergent (Oncor). Detection was performed with rhodamine-labeled anti-digoxigenin antibody, and chromosomes were counterstained with diamidinophenylindole. Metaphase chromosome spreads were visualized using a Zeiss universal microscope with a Photometrics™ cooled-CCD camera and Quips™ Imaging Software (Vysis™ Inc., Downers Grove, IL). Twenty metaphase spreads with signals on both chromatids at the same band position were used to determine chromosomal location. We determined the full-length sequence of the pro-hAKAP82 cDNA by aligning and sequencing the pro-hAKAP82 cDNA clone, PCR product, and EST clone (see "Experimental Procedures"; GenBank accession number AF072756). The composite cDNA sequence contained an initiator methionine with an in-frame, upstream stop codon. The coding region of pro-hAKAP82 was 2535 bases long and contained one open reading frame, which predicted a protein of 845 amino acids and concluded with an in-frame stop codon. Although no consensus polyadenylation signals (AATAAA) were present upstream of the putative poly(A) tract, two less well conserved potential polyadenylation signals (AATACA) were present at bases 2807 and 2827. The predicted molecular weights for the various forms of the human protein were 93,500 for pro-hAKAP82 and, assuming that the cleavage site in pro-hAKAP82 is the same as in pro-mAKAP82, 73,272 for hAKAP82 and 20,244 for the pro domain (hpro). Two cDNA sequences, pro-mAKAP82 and Fsc1, have been reported for the major mouse FS protein (18Carrera A. Gerton G.L. Moss S.B. Dev. Biol. 1994; 165: 272-284Crossref PubMed Scopus (189) Google Scholar, 28Fulcher K.D. Mori C. Welch J.E. O'Brien D.A. Klapper D.G. Eddy E.M. Biol. Reprod. 1995; 52: 41-49Crossref PubMed Scopus (83) Google Scholar). The predicted amino acid sequence of pro-hAKAP82 was highly homologous to both of these proteins. Specifically, at least 79% of the amino acids were identical, and 91% were conserved between the mouse (both pro-mAKAP82 and Fsc1) and human sequences. Critical functional domains that have been defined previously within the mouse protein; specifically, the RII binding region (FYVNRLSSLVIQMA) and the pro-mAKAP82/mAKAP82 cleavage site (KNTNNNQSPS) (11Visconti P. Johnson L. Oyaski M. Fornes M. Moss S. Gerton G. Kopf G. Dev. Biol. 1997; 192: 351-363Crossref PubMed Scopus (175) Google Scholar, 18Carrera A. Gerton G.L. Moss S.B. Dev. Biol. 1994; 165: 272-284Crossref PubMed Scopus (189) Google Scholar) were identical in the human homologue. The protein coding regions and 3′-UTRs of the pro-mAKAP82 and Fsc1 cDNAs are essentially identical; however, the 5′-UTRs of the two sequences share no homology. Furthermore, the Fsc1 5′-UTR contains an alternative in-frame start codon (27 bases upstream of the start codon for pro-mAKAP82) that could result in a protein containing an additional 9 amino acids at its amino terminus. These observations suggested that the two transcripts arise from alternative splicing of the same gene. To determine whether this was the case, genomic clones of pro-mAKAP82 and its 5′- and 3′-flanking regions were isolated and sequenced. The 5′-UTRs of both pro-mAKAP82 and Fsc1 were present in a single genomic clone and were separated from each other by 585 bp (Figs. 1, A and B), indicating that the two cDNA clones (pro-mAKAP82 and Fsc1) resulted from alternative splicing of the pro-mAKAP82 gene. The coding region of pro-mAKAP82 contained 5 exons and 4 introns with consensus splice donor/acceptor sites present at most exon/intron boundaries (Table I; GenBank accession numbers AF087516 and AF087517). An overview of the structure of pro-mAKAP82 is shown in Fig. 1 ATable INucleotide sequences of splice junctions in the pro-mAKAP82 gene5′ ExonIntron3′ ExonNumberSizeSequenceDonorSizeAcceptorSequenceNumberbpbp1′142ACAACGgtagag585ctgcagATGTCT21"115ATCAAGgtaatg>1 kbp1-aThis intron contains a highly repetitive region that has been intractable to sequencing. kbp, kilobase pairs.ctgcagATGTCT2296AAAGTGgtaaga1308atgtagATATGC3348GAAGATgattcc360tcaccaAAAGAT44102TCTAAGgtgacc1985ttctagACGGAG552115GAGAAGgtaaga354tcacagCTCCCT6The sequences of exons and introns are indicated by capital and small letters, respectively.1-a This intron contains a highly repetitive region that has been intractable to sequencing. kbp, kilobase pairs. Open table in a new tab The sequences of exons and introns are indicated by capital and small letters, respectively. The 5′-UTR of the pro-hAKAP82 cDNA was highly homologous to the 5′-UTR of pro-mAKAP82 but shared no homology with the 5′-UTR of Fsc1. To determine whether the pro-hAKAP82 gene, like the mouse homologue, was alternatively spliced, we used a PCR-based approach to search for a human cDNA sequence homologous to the Fsc1 5′-UTR. Using primers corresponding to regions in the 5′-UTR of Fsc1 and the 5′ end of the coding region of the pro-hAKAP82 cDNA, an approximately 400-bp product (5′-UTRf) was amplified from a human testis cDNA library. The sequence of 5′-UTRf was highly homologous to the 5′-UTR/5′ end of the coding region of Fsc1 (67% identical bases, Fig. 1 B). The 5′-UTRf sequence, like the sequence of Fsc1, contained an alternative in-frame start codon 27 bp upstream of the start codon for pro-hAKAP82, which could result in a protein containing an additional 9 amino acids at its amino terminus compared with pro-hAKAP82. Five of these 9 deduced amino acids were identical to the 9 predicted additional amino acids of Fsc1. This finding is strong support for the concept that, like the mouse, there are at least two alternative splice variants of the pro-hAKAP82 gene, one with a 5′-UTR homologous to pro-mAKAP82 and one with a 5′-UTR homologous to Fsc1. A polyclonal antibody generated against the mature mAKAP82 protein recognizes two bands in ejaculated human sperm; one at M r 82,000 (hAKAP82) and another at M r 97,000 (20Carrera A. Moos J. Ning X. Gerton G. Tesarik J. Kopf G. Moss S. Dev. Biol. 1996; 180: 284-296Crossref PubMed Scopus (284) Google Scholar). These findings suggest that, like the mouse, hAKAP82 is formed by proteolytic cleavage of a higher M r precursor, pro-hAKAP82. An antibody (anti-hpro) raised against a peptide sequence in the predicted processed (hpro) region of pro-hAKAP82 (a region presumed to be absent from mature hAKAP82) was used to probe immunoblots of ejaculated human sperm protein. Anti-hpro identified a polypeptide atM r 97,000, which is pro-hAKAP82 (Fig. 2 A). In addition, aM r 18,000 protein was detected. This protein approximates the size predicted for the hpro domain of pro-hAKAP82 (M r 20,244) and indicated that some of this fragment persisted in mature sperm. As expected, because the hpro peptide has been removed from the mature protein, no band was recognized at M r 82,000 (hAKAP82). Preabsorption of anti-hpro with the hpro peptide abolished the immunoreactivity, demonstrating that the antiserum reacted specifically with the hpro sequence. The sizes of hAKAP82 (20Carrera A. Moos J. Ning X. Gerton G. Tesarik J. Kopf G. Moss S. Dev. Biol. 1996; 180: 284-296Crossref PubMed Scopus (284) Google Scholar), pro-hAKAP82, and hpro are consistent with the hypothesis that the pro-hAKAP82/hAKAP82 cleavage site is similar, if not identical, to that of the mouse. Immunoreactivity was seen along the entire length of the principal piece when human sperm were probed with anti-hpro (Fig. 2, Band C). All sperm labeled in a similar fashion. This result is in contrast to the findings in mature cauda epididymal mouse sperm in which pro-mAKAP82 and mouse pro are found only in the proximal portion of the principal piece (19Johnson L. Foster J. Haig-Ladewig L. VanScoy H. Rubin C. Moss S. Gerton G. Dev. Biol. 1997; 192: 340-350Crossref PubMed Scopus (104) Google Scholar). Anti-hpro was specific for the FS as it reacted exclusively with the principal piece of the flagellum. The antiserum reacted specifically with the hpro sequence as immunoreactivity was not seen in samples in which anti-hpro was preabsorbed with the hpro peptide. Additionally, no staining was seen with the preimmune serum. To map the chromosomal location of the pro-hAKAP82 gene and to determine whether the gene is a candidate for any previously mapped human genetic diseases involving male infertility, we analyzed chromosomes from a normal man by FISH using a digoxigenin-11-dUTP-labeled BAC genomic clone of pro-hAKAP82. Results showed that pro-hAKAP82mapped to Xp11.2, adjacent to the centromere (Fig. 3). This region of the human X chromosome is syntenic to the most proximal end of the mouse X chromosome, the location of the pro-mAKAP82 gene (30Moss S. VanScoy H. Gerton G. Mamm. Genome. 1997; 8: 37-38Crossref PubMed Scopus (44) Google Scholar). The high homology of the amino acid sequences of pro-hAKAP82, pro-mAKAP82/Fsc1, and a 75-kDa rat FS protein (76% identical to and 89% conserved with pro-hAKAP82 at the amino acid level) indicates that the structure and function of AKAP82 is highly conserved in sperm of a number of mammalian species. Of particular interest is the finding that the amino acid sequence of the RII binding site of pro-mAKAP82 (11Visconti P. Johnson L. Oyaski M. Forn
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