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Sang-Hyun Kim,
Su Hyang Ryu,
Sang-Ho Lee,
Yong-Hoon Lee,
Sang-Rae Lee,
Jae-Won Huh,
Sun-Uk Kim, Ekyune Kim,
Sunghyun Kim,
Sangyong Jon,
Russell E Bishop,
Kyu-Tae Chang
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ABSTRACT: Shiga toxin (STx) belongs to the AB(5) toxin family and is transiently localized in the periplasm before secretion into the extracellular milieu. While producing outer membrane vesicles (OMVs) containing only A subunit of the toxin (STxA), we created specific STx1B- and STx2B-deficient mutants of E. coli O157:H7. Surprisingly, STxA subunit was absent in the OMVs and periplasm of the STxB-deficient mutants. In parallel, the A subunit of heat-labile toxin (LT) of enterotoxigenic E. coli (ETEC) was absent in the periplasm of the LT-B-deficient mutant, suggesting that instability of toxin A subunit in the absence of the B subunit is a common phenomenon in the AB(5) bacterial toxins. Moreover, STx2A was barely detectable in the periplasm of E. coli JM109 when stx2A was overexpressed alone, while it was stably present when stxB was co-expressed. Compared with STx2 holotoxin, purified STx2A was degraded rapidly by periplasmic proteases when assessed for in vitro proteolytic susceptibility, suggesting that the B subunit contributes to stability of the toxin A subunit in the periplasm. We propose a novel role for toxin B subunits of AB(5) toxins in protection of the A subunit from proteolysis during holotoxin assembly in the periplasm.
Biochimica et Biophysica Acta 10/2011; 1808(10):2359-65. · 4.66 Impact Factor
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ABSTRACT: V-set and immunoglobulin domain containing 1 (VSIG1) is a newly discovered member of the junctional adhesion molecule (JAM) family; it is encoded by a gene located on human chromosome X and preferentially expressed in a variety of cancers in humans. Little is known about its physiological function. To determine the role(s) of VSIG1 in mammalian spermatogenesis, we first generated a specific antibody against mouse VSIG1 and examined the presence and localization of the protein in tissues. RTRCR and Western blot analysis of the mouse tissues indicated that VSIG1 was specifically expressed in the testis. Furthermore, the results of our trypsinization and biotinylation assays strongly support the assumption that VSIG1 is localized on the testicular germ cell surface. In order to determine whether VSIG1 is capable of participation in homotypic interactions, we performed a GST-pull down assay by using recombinant GST-fusion and Histagging proteins. The pull-down assay revealed that each GST-fusion Ig-like domain shows homotypic binding. We further show that mVSIG1 can adhere to the Sertoli cells through its first Ig-like domain. To identify the protein that interacted with cytoplasmic domain, we next performed co-immunoprecipitation analysis. This analysis showed that ZO-1, which is the central structural protein of the tight junction, is the binding partner of the cytoplasmic domain of mouse VSIG1. Our findings suggest that mouse VSIG1 interacts with Sertoli cells by heterophilic adhesion via its first Ig-like domain. In addition, its cytoplasmic domain is critical for binding to ZO-1.
Molecules and Cells 10/2010; 30(5):443-8. · 2.18 Impact Factor
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Ekyune Kim,
Youngjeon Lee,
Hyun-Ju Lee,
Ji Su Kim,
Bong-Seok Song,
Jae-Won Huh,
Sang-Rae Lee,
Sun-Uk Kim,
Sang-Hyun Kim,
Yonggeun Hong,
Insop Shim,
Kyu-Tae Chang
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ABSTRACT: The retromer complex, which mediates retrograde transport from endosomes to the trans-Golgi network, is a heteropentameric complex that contains a multifunctional cargo recognition heterotrimer consisted of the vacuolar protein sorting (Vps) subunits Vps26, Vps29, and Vps35. In mammals, there are two different isoforms of Vps26, Vps26a and Vps26b, that localize to the endosome, and to the plasma membrane, respectively. To elucidate the biological significance of the Vps26b isoform, we generated Vps26b knockout mice and studied their molecular, histological, and behavioral phenotypes. We found that the loss of Vps26b results in no significant defects in the behavior, body size, and health of the mice. Vps26b-deficient mice showed a severe reduction of Vps35 protein at cellular level and lacked the Vps26b-Vps29-Vps35 retromer complex, despite the normal presence of the Vps26a-Vps29-Vps35 retromer complex. Relatively, the amount of sortilin was increased approximately 20% in the Vps26b-deficient mice, whereas the sorLA was normal. These results suggest that mouse Vps26b-Vps29-Vps35 retromer complex is implicated in the transport of sortilin from endosomes to the trans-Golgi network (TGN).
Biochemical and Biophysical Research Communications 10/2010; 403(2):167-71. · 2.48 Impact Factor
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Jae-Won Huh,
Young-Hyun Kim,
Dae-Soo Kim,
Sang-Je Park,
Sang-Rae Lee,
Sang-Hyun Kim, Ekyune Kim,
Sun-Uk Kim,
Myeong-Su Kim,
Heui-Soo Kim,
Kyu-Tae Chang
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ABSTRACT: The leptin receptor (LEPR) is a crucial regulatory protein that interacts with Leptin. In our analysis of LEPR, novel AluJb-derived alternative transcripts were identified in the genome of the rhesus monkey. In order to investigate the occurrence of AluJb-derived alternative transcripts and the mechanism underlying exonization events, we conducted analyses using a number of primate genomic DNAs and adipose RNAs of tissue and primary cells derived from the crab-eating monkey. Our results demonstrate that the AluJb element has been integrated into our common ancestor genome prior to the divergence of simians and prosimians. The lineage-specific exonization event of the LEPR gene in chimpanzees, orangutans, and Old World monkeys appear to have been accomplished via transition mutations of the 5' splicing site (second position of C to T). However, in New World monkeys and prosimians, the AluJb-related LEPR transcript should be silenced by the additional transversion mutation (fourth position of T to G). The AluJb-related transcript of human LEPR should also be silenced by a mutation of the 5' splicing site (first position of G to A) and the insertion of one nucleotide sequence (minus fourth position of A). Our data suggests that lineage-specific exonization events should be determined by the combination event of the formation of splicing sites and protection against site-specific mutation pressures. These evolutionary mechanisms could be major sources for primate diversification.
Molecules and Cells 09/2010; 30(3):201-7. · 2.18 Impact Factor
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ABSTRACT: Shiga toxins (STx) are secreted extracellularly through the outer membrane vesicles (OMVs) of Escherichia coli O157:H7. In an attempt to produce STxA-deficient OMVs from E. coli O157:H7, site-specific deletions of the stx1A and stx2A subunit genes were carried out. The STxA-deficient phenotype of the stx1A/stx2A mutant was confirmed by Vero cell cytotoxicity and VTEC-RPLA assay. Western blot analyses showed that the B (STxB) subunits were present without coupling to STxA in the OMVs of the STxA-deficient mutant. Furthermore, STxB was located in its homo-pentameric complexes, as revealed by immunoprecipitation and immunoblotting with anti-STxB antibodies. These results suggest that STxB alone can be oligomerized into the B pentamer in the periplasm, and subsequently entrapped into the OMVs. Determination of the median lethal dose concentration for the OMV preparations suggests that the STxA-deficient OMVs containing STxB complex could be safely used as vaccine delivery vehicles.
FEMS Immunology & Medical Microbiology 01/2010; 58(3):412-20. · 2.44 Impact Factor
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ABSTRACT: To develop low endotoxic and multi-immunogenic outer membrane vesicles (OMVs), a deletion mutant of the msbB gene in Salmonella enterica serovar Typhimurium (S. Typhimurium) was used as a source of low endotoxic OMV, and an expression vector of the canine parvovirus (CPV) VP2 epitope fused to the bacterial OmpA protein was constructed and transformed into the Salmonella deltaAmsbB mutant. In a lethality test, BALB/c mice injected intraperitoneally with the Salmonella deltaAmsbB mutant survived for 7 days, whereas mice injected intraperitoneally with the wild type survived for 3 days. Moreover, all mice inoculated orally with the deltaAmsbB mutant survived for 30 days, but 80% of mice inoculated orally with the wild type survived. The OmpA::CPV VP2 epitope fusion protein was expressed successfully and associated with the outer membrane and OMV fractions from the mutant S. Typhimurium transformed with the fusion protein-expressing vector. In immunogenicity tests, sera obtained from the mice immunized with either the Salmonella msbB mutant or its OMVs containing the OmpA::CPV VP2 epitope showed bactericidal activities against wild-type S. Typhimurium and contained specific antibodies to the CPV VP2 epitope. In the hemagglutination inhibition (HI) assay as a measurement of CPV-neutralizing activity in the immune sera, there was an 8-fold increase of HI titer in the OMV-immunized group compared with the control. These results suggested that the CPV-neutralizing antibody response was raised by immunization with OMV containing the OmpA::CPV VP2 epitope, as well as the protective immune response against S. Typhimurium in BALB/c mice.
Journal of Microbiology and Biotechnology 10/2009; 19(10):1271-9. · 1.38 Impact Factor
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ABSTRACT: In an effort to devise a safer and more effective vaccine delivery system, outer membrane vesicles (OMVs) were engineered to have properties of intrinsically low endotoxicity sufficient for the delivery of foreign antigens. Our strategy involved mutational inactivation of the MsbB (LpxM) lipid A acyltransferase to generate OMVs of reduced endotoxicity from Escherichia coli (E. coli) O157:H7. The chromosomal tagging of a foreign FLAG epitope within an OmpA-fused protein was exploited to localize the FLAG epitope in the OMVs produced by the E. coli mutant having the defined msbB and the ompA::FLAG mutations. It was confirmed that the desired fusion protein (OmpA::FLAG) was expressed and destined to the outer membrane (OM) of the E. coli mutant from which the OMVs carrying OmpA::FLAG are released during growth. A luminal localization of the FLAG epitope within the OMVs was inferred from its differential immunoprecipitation and resistance to proteolytic degradation. Thus, by using genetic engineering-based approaches, the native OMVs were modified to have both intrinsically low endotoxicity and a foreign epitope tag to establish a platform technology for development of multifunctional vaccine delivery vehicles.
Biochimica et Biophysica Acta 09/2009; 1788(10):2150-9. · 4.66 Impact Factor
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ABSTRACT: Although sperm entry into the oocyte-cumulus complex and subsequent sperm penetration through the cumulus matrix to reach the oocyte zona pellucida are essential for mammalian fertilization, the molecular mechanism remains controversial. Previously, we have shown that mouse sperm lacking SPAM1 are capable of penetrating the cumulus matrix despite a delayed dispersal of cumulus cells. We also have identified another sperm hyaluronidase, HYAL5, as a candidate enzyme involved in sperm penetration through the cumulus. In the present study, we produced HYAL5-deficient mice to uncover the functional roles of HYAL5 and SPAM1 in fertilization. The HYAL5-deficient mice were fully fertile and yielded normal litter sizes. In vitro fertilization assays demonstrated that HYAL5-deficient epididymal sperm is functionally normal. We thus conclude that HYAL5 may be dispensable for fertilization. Comparative analysis among wild-type, HYAL5-deficient, and SPAM1-deficient epididymal sperm revealed that only SPAM1 is probably involved in sperm penetration through the cumulus matrix. Notably, the loss of SPAM1 resulted in a remarkably increased accumulation of sperm on the surface or outer edge of the cumulus. These data suggest that SPAM1 may function in sperm entry into the cumulus and sperm penetration through the cumulus matrix.
Biology of Reproduction 08/2009; 81(5):939-47. · 4.01 Impact Factor
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ABSTRACT: Fertilin, a heterodimeric protein complex composed of ADAM1 and ADAM2 located on the sperm surface, is involved in sperm-egg interaction. In our study, we examined the physiological processing and subcellular localization of M. fascicularis ADAM2 during spermatogenesis in the testis and epididymal tract. M. fascicularis ADAM2 was initially synthesized as a 100 kDa precursor in testicular germ cells. After passing into 50 kDa intermediate form in the epididymal tracts, the precursor form was finally processed into a 47 kDa protein in sperm. We found that M. fascicularis ADAM2 is localized on the sperm surface and contributes to the formation of a candidate fertilin complex. In particular, Far-Western blot analysis revealed that M. fascicularis ADAM2 cystein-rich domain may be related to protein-protein interaction. Therefore, the cystein-rich domain of ADAM2 could provide a mechanism to form a fertilin complex.
Animal reproduction science 05/2009; 117(1-2):155-9. · 1.56 Impact Factor
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Ekyune Kim,
Ki-Eun Park,
Ji-Su Kim,
Dong Chul Baek,
Jae-Woong Lee,
Sang-Rae Lee,
Myeong-Su Kim,
Sang-Hyun Kim,
Chan-Shick Kim,
Deog-Bon Koo,
Han-Seok Kang,
Zae-Young Ryoo,
Kyu-Tae Chang
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ABSTRACT: In the mouse, ADAM3, a well-characterized testis-specific protein of the A disintegrin and metalloprotease (ADAM) family, has a crucial role in fertilization by mediating sperm binding to the egg zona pellucida. However, little is known about ADAM3 in other species, such as domestic pigs. We have identified porcine ADAM3 and analyzed the protein. RT-PCR and trypsinization of sperm surface proteins revealed that porcine ADAM3 is expressed at high levels in the testis and on the sperm surface. Furthermore, an IVF inhibition assay with a recombinant porcine ADAM3 disintegrin domain showed that treatment of the disintegrin domain effectively prevented pig sperm-egg interactions. In the present study, we demonstrated the presence of ADAM3a and ADAM3b molecules in the pig and examined their roles in fertilization.
Journal of Reproduction and Development 01/2009; 55(2):156-62. · 1.46 Impact Factor
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ABSTRACT: A family of vacuolar protein sorting (Vps) proteins, which are components of mammalian retromer complex, has been studied in the mouse. Vps26a is known as a retromer component that plays an important role in embryonic development: however, its cell-type expression and precise role remain to be elucidated. In this study, we identified a new isoform of Vps26a, called Vps26aT, which was expressed specifically in the mouse testis. Diverse expression patterns of Vps26 variants in mouse tissues were determined by Western blot and RT-PCR analyses, and the direct interaction of Vps26aT with Vps35 was also demonstrated by immunoprecipitation and pull-down assay using antibodies raised against each Vps component. Our results revealed that the retromer complex could be formed from different Vps26 isoforms in a tissue-specific manner, resulting in more than two types of the retromer complex, including the Vps26a-Vps29-Vps35, Vps26aT-Vps29-Vps35, and Vps26b-Vps29-Vps35 complexes in mouse tissues.
Biochemical and Biophysical Research Communications 10/2008; 375(1):16-21. · 2.48 Impact Factor
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ABSTRACT: Mammalian fertilization requires sperm to penetrate the cumulus mass and egg zona pellucida prior to fusion with the egg. Although sperm penetration through these physical barriers is essential, the molecular mechanism has not yet been completely elucidated. In addition to sperm motility, hyaluronan-hydrolyzing and proteolytic enzymes of sperm have been suggested to participate in the penetration events. Here we focus on the functional roles of hyaluronidase and protease in sperm passage through the cumulus mass and zona pellucida.
The International Journal of Developmental Biology 01/2008; 52(5-6):677-82. · 2.82 Impact Factor
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ABSTRACT: Fertilin, a heterodimeric protein complex composed of alpha (ADAM1) and beta (ADAM2) subunits on the sperm surface, is believed to mediate adhesion and fusion between the sperm and egg plasma membranes. Here we have shown that mutant male mice lacking ADAM1b are fertile and that the loss of ADAM1b results in no significant defect in sperm functions such as migration from the uterus into oviduct, binding to egg zona pellucida, and fusion with zona pellucida-free eggs. ADAM1b-deficient epididymal sperm showed a severe reduction of ADAM2 on the cell surface, despite the normal presence of ADAM2 in testicular germ cells. The appearance of ADAM1b and ADAM2 on the sperm surface depended on formation and abundance of ADAM1b/ADAM2 fertilin in testicular germ cells. These results suggest that mouse ADAM1b/ADAM2 fertilin may play a crucial role not in the sperm/egg fusion but in the appearance of these two ADAMs on the sperm surface.
Journal of Biological Chemistry 04/2006; 281(9):5634-9. · 4.77 Impact Factor
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ABSTRACT: A glycosylphosphatidylinositol (GPI)-anchored hyaluronidase, PH-20, on the sperm surface has long been believed to assist sperm penetration through the cumulus mass surrounding the eggs. However, mouse sperm lacking PH-20 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus cells. Intriguingly, a 55-kDa hyaluronan-hydrolyzing protein was abundantly present in wild-type and PH-20-deficient mouse sperm. In this study, we purified the 55-kDa mouse protein from soluble protein extracts released from epididymal sperm by acrosome reaction and identified as a hyaluronidase, Hyal5. Hyal5 was exclusively expressed in the testis and formed a 160-kbp gene cluster together with Hyalp1, Hyal4, and Ph-20 on mouse chromosome 6. Hyal5 was a single-chain hyaluronidase present on the plasma and acrosomal membranes of sperm presumably as a GPI-anchored protein. Moreover, hyaluronan zymography revealed that Hyal5 is enzymatically active in the pH range 5-7 and inactive at pH 3 and 4. Both Hyal5-enriched PH-20-free soluble protein extracts and PH-20-deficient mouse sperm were capable of dispersing cumulus cells from the cumulus mass. Cumulus cell dispersal was strongly inhibited by the presence of a hyaluronidase inhibitor, apigenin. These results suggest that in the mouse, Hyal5 may function principally as a "cumulus matrix depolymerase" in the sperm penetration through the cumulus mass and in the local hyaluronan hydrolysis near or on the surface of the egg zona pellucida to enable the proximal region of sperm tail to move freely. PH-20 may compensate in part for the functional roles of Hyal5.
Proceedings of the National Academy of Sciences 01/2006; 102(50):18028-33. · 9.68 Impact Factor
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ABSTRACT: To elucidate synthesis, processing, and subcellular localization of mouse ADAM3 (cyritestin) during spermatogenesis and epididymal sperm transport, we carried out immunoblotting and immunohistochemical analysis of testicular germ cells, and epididymal and vas deferens sperm, using affinity-purified anti-ADAM3 antibody. ADAM3 was initially synthesized as a 110-kDa precursor in round spermatids, and the precursor was then processed into a 42-kDa mature protein during the sperm transport into and/or once in the epididymis. The mature ADAM3 was localized on the anterior part of capacitated sperm heads and was rapidly removed from the head region during the calcium ionophore A23187-induced acrosome reaction. These results demonstrate that the mature form of ADAM3 is involved in the binding of sperm to the egg zona pellucida, not in the membrane fusion between sperm and egg.
Journal of Reproduction and Development 10/2004; 50(5):571-8. · 1.46 Impact Factor
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ABSTRACT: In mouse, two different isoforms of ADAM1 (fertilin alpha), ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells, whereas epididymal sperm contain only ADAM1b on the plasma membrane. In this study, we show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. However, epididymal sperm of ADAM1a-deficient mice were capable of fertilizing cumulus-intact, zona pellucida-intact eggs in vitro despite the delayed dispersal of cumulus cells and the reduced adhesion/binding to the zona pellucida. Among testis (sperm)-specific proteins examined, only the level of ADAM3 (cyritestin) was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 (fertilin beta) in testicular germ cells, although no direct interaction between the fertilin complex and ADAM3 was found. These results suggest that ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface. These proteins then can participate in sperm migration into the oviduct, the dispersal of cumulus cells, and sperm binding to the zona pellucida.
Journal of Biological Chemistry 09/2004; 279(33):34957-62. · 4.77 Impact Factor
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ABSTRACT: Although fertilin is a heterodimeric complex between ADAM1 (A Disintegrin And Metalloprotease 1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface, two different ADAM1 isoforms, ADAM1a and ADAM1b, are present in the mouse testis. In this study, we have examined the localization of ADAM1a and ADAM1b in testicular germ cells and epididymal sperm. ADAM1a was restrictedly present within the endoplasmic reticulum of germ cells, whereas epididymal sperm contained only ADAM1b on the cell surface. The precursors of ADAM1a and ADAM1b formed a heterodimeric complex with that of ADAM2 in the endoplasmic reticulum of germ cells. The heterodimeric complex between the mature forms of ADAM1b and ADAM2 was also found on the sperm surface. These data imply the potential roles of ADAM1a and ADAM1b in spermatogenesis and fertilization, respectively.
Biochemical and Biophysical Research Communications 06/2003; 304(2):313-9. · 2.48 Impact Factor
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ABSTRACT: Fertilin is reported to be a heterodimeric protein composed of A Disintegrin And Metalloprotease 1 (ADAM1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface. In the process of clarifying the molecular basis of mouse ADAM1, we have identified two intron-less mouse genes encoding different isoforms of ADAM1, termed ADAM1a and ADAM1b. The amino acid sequences of ADAM1a and ADAM1b deduced from the DNA sequences were homologous to each other (99% identity) in the pro- and metalloprotease domains, whereas the C-terminal half region of ADAM1a, including the disintegrin and Cys-rich domains, shared only a low degree of identity (37%) with that of ADAM1b. These two genes were both localized on mouse chromosome 5 as a single copy gene, and were expressed specifically in the testis. These data demonstrate the presence of the ADAM1a (Adam1a) and ADAM1b (Adam1b) genes in mouse, instead of the ADAM1 gene, and may imply different roles of ADAM1a and ADAM1b in spermatogenesis, sperm maturation, and/or fertilization.
Gene 06/2002; 291(1-2):67-76. · 2.34 Impact Factor
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ABSTRACT: Fertilin is reported to be a heterodimeric protein composed of isintegrin nd etalloprotease 1 (ADAM1, fertilin α) and ADAM2 (fertilin β) located on the sperm surface. In the process of clarifying the molecular basis of mouse ADAM1, we have identified two intron-less mouse genes encoding different isoforms of ADAM1, termed ADAM1a and ADAM1b. The amino acid sequences of ADAM1a and ADAM1b deduced from the DNA sequences were homologous to each other (99% identity) in the pro- and metalloprotease domains, whereas the C-terminal half region of ADAM1a, including the disintegrin and Cys-rich domains, shared only a low degree of identity (37%) with that of ADAM1b. These two genes were both localized on mouse chromosome 5 as a single copy gene, and were expressed specifically in the testis. These data demonstrate the presence of the ADAM1a (Adam1a) and ADAM1b (Adam1b) genes in mouse, instead of the ADAM1 gene, and may imply different roles of ADAM1a and ADAM1b in spermatogenesis, sperm maturation, and/or fertilization.
Gene.
