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Molecular Cloning and Mapping of a Novel ADAM Gene (ADAM29) to Human Chromosome 4
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SHORT COMMUNICATION Molecular Cloning and Mapping of a Novel ADAM Gene (ADAM29) to Human Chromosome 4 Rener Xu, 1 Jinli Cai, 1 Tian Xu, Weigou Zhou, Beibei Ying, Kejin Deng,* Shouyuan Zhao, and Changben Li 2 State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai, 200433, People’s Republic of China; and *Shanghai Research Centre of Life Science, Shanghai 200031, People’s Republic of China Received July 13, 1999; accepted October 18, 1999
Members of the ADAM family (type I integral membrane protein with a disintegrin and metalloprotease domain) have been implicated in many important biological processes involving cell– cell and cell–matrix interactions, such as fertilization and myoblast fusion. We report here the cDNA sequence of a novel human ADAM gene (ADAM29) that contains a putative fusion peptide. Northern blot analysis revealed that the mRNA of ADAM29 is highly expressed in the testis. By radiation hybrid panel mapping, the ADAM29 gene was assigned to human chromosome 4q34.2– qter. © 1999 Academic Press
The ADAM molecules are a rapidly growing family of cell surface proteinases that contain multiple domains including a disintegrin and a metalloproteinase (2, 3, 5, 10, 11). Members of the ADAM family have been shown to be involved in a variety of important biological processes, such as fertilization, muscle development, and neurogenesis (12). Recently, several ADAM molecules have been shown to mediate prolytic activation of peptide ligands. For example, TACE (TNF-␣ converting enzyme) was isolated due to its ability to release tumor necrosis factor-␣ from the plasma membrane (1, 6), and the Kuzbanian protein is involved in processing the Notch ligand, Delta (7, 8). Among the ADAM family members, ADAM20 and 21 are the two most recently identified human ADAMs and are related to fertilin-␣, fertilin-, and meltrin-␥ (ADAM1, 2, and 9). Both ADAM20 and 21 contain a putative fusion peptide, suggesting that they could be involved in cell fusion (4). We report here the identification and characterization of a full-length cDNA encoding a novel ADAM protein (ADAM29, Accession No. AF134708), which is Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF134708. 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed. Telephone and fax: (86)21-65642428. E-mail: [email protected].
closely related to human ADAM20 and 21. At first, we used the human ADAM20 coding sequence as a probe to search the homologues in the expressed sequence tag (EST) database and identified a 129-bp EST sequence (GenBank Accession No. X85598). The putative amino acid sequence deduced from this EST sequence suggested that it is a novel ADAM member and was named ADAM29. To clone the full-length cDNA, a PCR-based cDNA “walking” strategy was used. The 3⬘ cDNA of ADAM29 was obtained by PCR amplified from a human testis cDNA library (Clontech HL3024a) with an ADAM29-specific primer (M855F1, 5⬘-tgcgcctgcaccaaaaatcc-3⬘) and a gt10 vector primer (gt10 RV1, 5⬘cttgaggcgttcagaaagag-3⬘). The PCR products were further amplified with a pair of nested primers (M855F2, 5⬘-tcacctaaggtgttcaattctttgtagcaagtctcacttgcagtatt-3⬘; gt10 RV2, 5⬘-gcttatgagtatttcttccaggg-3⬘), and the 795-bp cDNA fragment was cloned in the pGEM-T vector (Promega). To recover 5⬘ sequences, ADAM29-specific primer M855R2 (5⬘-attcccacagcgcttcacatta-3⬘) and gt10-specific primer gt10 FW1 (5⬘-gaatcagaggtgccattgag-3⬘) were used in the primary PCR, and gt10 FW2 (5⬘-gcagccagtcaacacttacg-3⬘) and M855R1 (5⬘-ttcccaaaaatcaccataactac-3⬘) were used as nested primers before cloning of the amplified cDNAs. Finally, we directly amplified and cloned the entire ADAM29 coding region from the same library. Sequence analysis of these cDNAs revealed that ADAM29 is closely related to ADAM21 and ADAM20 (Fig. 1). ADAM29 contains all the domains in a typical ADAM molecule including a signal peptide, a proprotein domain, a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, a transmembrane region, and a cytoplasmic tail. The zinc binding site in the ADAM29 metalloprotease domain differs at only one position from the conserved catalytic sequence HEXXHXXGXXH, with the critical glutamic acid residue being converted to histidine in ADAM29. This makes it unlikely that ADAM29 can function as a protease. Within the cysteine-rich domain of ADAM29, a short (24 amino acid) hydropho-
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Genomics 62, 537–539 (1999) Article ID geno.1999.6029 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
538
SHORT COMMUNICATION
bic stretch can be found that can be modeled into an amphipathic helix with a central proline residue (data not shown), a characteristic of the putative fusion peptide identified in the membrane-anchored subunit (9),
FIG. 2. Northern analysis of ADAM29 expression. Human multiple tissue Northern blots (Clontech) were hybridized with a 1.8-kb human ADAM29 cDNA fragment. Positions of the RNA markers (in kilobases) are indicated on the left and the right. An additional large transcript was detected in skeletal muscle. Lanes 1–16: heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocyte.
suggesting that ADAM29 may play a role in mediating cell– cell fusion. We further examined the expression pattern of ADAM29 by probing multitissue Northern blots (human MTN I and II, Clontech) with a 1.8-kb ADAM29 cDNA fragment. A single transcript of approximately 4.0 kb was highly expressed in human testis, but was detected only slightly or not at all in the 15 other tissues examined (Fig. 2). These data suggest that ADAM29 may be a novel ADAM molecule involved in human spermatogenesis. By radiation hybrid panel mapping, the human ADAM29 gene was mapped to chromosome 4q34.2– qter. Primers at unique positions (5⬘-gaggccagaaacacattatc-3⬘, 554 –573 bp; 5⬘-tgaaaacccccaaaacag-3⬘, 749 – 732 bp) that amplify the 196-bp DNA band were used to type the Stanford Human Genome Center GB4 radiation hybrid panel (Research Genetics). In the panel, 93 radiation hybrid clones were used to detect the presence or absence of this PCR fragment. Comparison of the result with previously defined markers (using the radiation hybrid server at http://www-shgc. stanford.edu/RH/index.html) indicated that the ADAM29 gene was placed 22.44 cR 3000 distal to marker AFMa239xa5 near the long terminal of human chromosome 4 (4q34.2– qter) with very high probability (LOD ⬎ 15). ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (39880018) and the Chinese Southern Research Center for Human Genome (22049).
REFERENCES 1. FIG. 1. Comparison of the predicted amino acid sequences of human ADAM29 with human ADAM20 and 21. Identical amino acid residues are shaded. The potential disintegrin loop triplexes are indicated in boldface type, the putative fusion peptides are boxed, and the catalytic domain is underlined. The boundaries between the various domains are predicted and labeled according to ADAM20 and 21.
Black, R. A., Rauch, C. T., Kozlosky, C. J., Peschon, J. J., Slack, J. L., Wolfsson, M. F., Castner, B. J., Stooking, K. L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K. A., Gerhart, M., Davis, R., Fitzner, J. N., Johnson, R. S., Paxton, R. J., March, C. J., and Gerietti, D. P. (1997). A metalloprotease disintegrin that releases tumor necrosis factor-␣ from cells. Nature 385: 729 –733. 2. Black, R. A., and White, J. M. (1998). ADAMs: Focus on the protease domain. Curr. Opin. Cell Biol. 10: 654 – 659.
SHORT COMMUNICATION 3. 4.
5. 6.
7.
Blobel, C. P.(1997). Metalloprotease-disintegrins: Links to cell adhesion and cleavage of TNF-␣ and Notch. Cell 90: 589 –592. Hooft van Huijsduijnen, R. (1998). ADAM 20 and 21; two novel human testis-specific membrane metalloproteases with similarity to fertilin-␣. Gene 206: 273–282. Huovila, A. P. J., Almeida, E. A., and White, J. M. (1996). ADAMs and cell fusion. Curr. Opin. Cell Biol. 8: 692– 699. Moss, M. L., Jin, S. L., Milla, M. E., Burkhart, W., Carter, H. L., Chen, W. J., Clay, W. C., Didsbury, J. R., Hassler, D., Hoffman, C. R., Kost, T. A., Lambert, M. H., Leesnitzer, M. A., McCauley, P., McGeehan, G., Mitchell, J., Moyer, M., Pahel, G., Rocque, W., Overton, L. K., Schoenen, F., Seaton, T., Su, J. F., Warner, J., Willard, D., et al. (1997). Cloning of a disintegrin metalloprotease that processes precursor tumor necrosis factor-␣. Nature 385: 733–736. Qi, H., Rand, M. D., Wu, X., Sestan, N., Wang, W., Rakic, P., Xu, T., and Artavanis-Tsakonas, S. (1999). Processing of the Notch ligand delta by the metalloprotease Kuzbanian. Science 283: 91–94.
8.
9. 10.
11.
12.
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Rooke, J., Pan, D., Xu, T., and Rubin, G. M. (1996). KUZ, a conserved metalloprotease– disintegrin protein with two roles in Drosophila neurogenesis. Science 273: 1227– 1231. White, J. M.(1992). Membrane fusion. Science 258: 917– 924. Wolfsberg, T. G., Primacoff, P., Myles, D. G., and White, J. M. (1995). ADAM, a novel family of membrane proteins containing disintegrin and metalloprotease domain: Multipotential functions in cell– cell and cell–matrix interactions. J. Cell Biol. 131: 275–278. Wolfsberg, T. G., Straight, P. D., Gerena, R. L., Huovila, A. P., Primacoff, P., Myles, D. G., and White, J. M. (1995). ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain. Dev. Biol. 169: 378 –383. Wolfsberg, T. G., and White, J. M. (1996). ADAMs in fertilization and development. Dev. Biol. 180: 389 – 401.
SHORT COMMUNICATION Molecular Cloning and Mapping of a Novel ADAM Gene (ADAM29) to Human Chromosome 4 Rener Xu, 1 Jinli Cai, 1 Tian Xu, Weigou Zhou, Beibei Ying, Kejin Deng,* Shouyuan Zhao, and Changben Li 2 State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai, 200433, People’s Republic of China; and *Shanghai Research Centre of Life Science, Shanghai 200031, People’s Republic of China Received July 13, 1999; accepted October 18, 1999
Members of the ADAM family (type I integral membrane protein with a disintegrin and metalloprotease domain) have been implicated in many important biological processes involving cell– cell and cell–matrix interactions, such as fertilization and myoblast fusion. We report here the cDNA sequence of a novel human ADAM gene (ADAM29) that contains a putative fusion peptide. Northern blot analysis revealed that the mRNA of ADAM29 is highly expressed in the testis. By radiation hybrid panel mapping, the ADAM29 gene was assigned to human chromosome 4q34.2– qter. © 1999 Academic Press
The ADAM molecules are a rapidly growing family of cell surface proteinases that contain multiple domains including a disintegrin and a metalloproteinase (2, 3, 5, 10, 11). Members of the ADAM family have been shown to be involved in a variety of important biological processes, such as fertilization, muscle development, and neurogenesis (12). Recently, several ADAM molecules have been shown to mediate prolytic activation of peptide ligands. For example, TACE (TNF-␣ converting enzyme) was isolated due to its ability to release tumor necrosis factor-␣ from the plasma membrane (1, 6), and the Kuzbanian protein is involved in processing the Notch ligand, Delta (7, 8). Among the ADAM family members, ADAM20 and 21 are the two most recently identified human ADAMs and are related to fertilin-␣, fertilin-, and meltrin-␥ (ADAM1, 2, and 9). Both ADAM20 and 21 contain a putative fusion peptide, suggesting that they could be involved in cell fusion (4). We report here the identification and characterization of a full-length cDNA encoding a novel ADAM protein (ADAM29, Accession No. AF134708), which is Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF134708. 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed. Telephone and fax: (86)21-65642428. E-mail: [email protected].
closely related to human ADAM20 and 21. At first, we used the human ADAM20 coding sequence as a probe to search the homologues in the expressed sequence tag (EST) database and identified a 129-bp EST sequence (GenBank Accession No. X85598). The putative amino acid sequence deduced from this EST sequence suggested that it is a novel ADAM member and was named ADAM29. To clone the full-length cDNA, a PCR-based cDNA “walking” strategy was used. The 3⬘ cDNA of ADAM29 was obtained by PCR amplified from a human testis cDNA library (Clontech HL3024a) with an ADAM29-specific primer (M855F1, 5⬘-tgcgcctgcaccaaaaatcc-3⬘) and a gt10 vector primer (gt10 RV1, 5⬘cttgaggcgttcagaaagag-3⬘). The PCR products were further amplified with a pair of nested primers (M855F2, 5⬘-tcacctaaggtgttcaattctttgtagcaagtctcacttgcagtatt-3⬘; gt10 RV2, 5⬘-gcttatgagtatttcttccaggg-3⬘), and the 795-bp cDNA fragment was cloned in the pGEM-T vector (Promega). To recover 5⬘ sequences, ADAM29-specific primer M855R2 (5⬘-attcccacagcgcttcacatta-3⬘) and gt10-specific primer gt10 FW1 (5⬘-gaatcagaggtgccattgag-3⬘) were used in the primary PCR, and gt10 FW2 (5⬘-gcagccagtcaacacttacg-3⬘) and M855R1 (5⬘-ttcccaaaaatcaccataactac-3⬘) were used as nested primers before cloning of the amplified cDNAs. Finally, we directly amplified and cloned the entire ADAM29 coding region from the same library. Sequence analysis of these cDNAs revealed that ADAM29 is closely related to ADAM21 and ADAM20 (Fig. 1). ADAM29 contains all the domains in a typical ADAM molecule including a signal peptide, a proprotein domain, a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, a transmembrane region, and a cytoplasmic tail. The zinc binding site in the ADAM29 metalloprotease domain differs at only one position from the conserved catalytic sequence HEXXHXXGXXH, with the critical glutamic acid residue being converted to histidine in ADAM29. This makes it unlikely that ADAM29 can function as a protease. Within the cysteine-rich domain of ADAM29, a short (24 amino acid) hydropho-
537
Genomics 62, 537–539 (1999) Article ID geno.1999.6029 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
538
SHORT COMMUNICATION
bic stretch can be found that can be modeled into an amphipathic helix with a central proline residue (data not shown), a characteristic of the putative fusion peptide identified in the membrane-anchored subunit (9),
FIG. 2. Northern analysis of ADAM29 expression. Human multiple tissue Northern blots (Clontech) were hybridized with a 1.8-kb human ADAM29 cDNA fragment. Positions of the RNA markers (in kilobases) are indicated on the left and the right. An additional large transcript was detected in skeletal muscle. Lanes 1–16: heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocyte.
suggesting that ADAM29 may play a role in mediating cell– cell fusion. We further examined the expression pattern of ADAM29 by probing multitissue Northern blots (human MTN I and II, Clontech) with a 1.8-kb ADAM29 cDNA fragment. A single transcript of approximately 4.0 kb was highly expressed in human testis, but was detected only slightly or not at all in the 15 other tissues examined (Fig. 2). These data suggest that ADAM29 may be a novel ADAM molecule involved in human spermatogenesis. By radiation hybrid panel mapping, the human ADAM29 gene was mapped to chromosome 4q34.2– qter. Primers at unique positions (5⬘-gaggccagaaacacattatc-3⬘, 554 –573 bp; 5⬘-tgaaaacccccaaaacag-3⬘, 749 – 732 bp) that amplify the 196-bp DNA band were used to type the Stanford Human Genome Center GB4 radiation hybrid panel (Research Genetics). In the panel, 93 radiation hybrid clones were used to detect the presence or absence of this PCR fragment. Comparison of the result with previously defined markers (using the radiation hybrid server at http://www-shgc. stanford.edu/RH/index.html) indicated that the ADAM29 gene was placed 22.44 cR 3000 distal to marker AFMa239xa5 near the long terminal of human chromosome 4 (4q34.2– qter) with very high probability (LOD ⬎ 15). ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (39880018) and the Chinese Southern Research Center for Human Genome (22049).
REFERENCES 1. FIG. 1. Comparison of the predicted amino acid sequences of human ADAM29 with human ADAM20 and 21. Identical amino acid residues are shaded. The potential disintegrin loop triplexes are indicated in boldface type, the putative fusion peptides are boxed, and the catalytic domain is underlined. The boundaries between the various domains are predicted and labeled according to ADAM20 and 21.
Black, R. A., Rauch, C. T., Kozlosky, C. J., Peschon, J. J., Slack, J. L., Wolfsson, M. F., Castner, B. J., Stooking, K. L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K. A., Gerhart, M., Davis, R., Fitzner, J. N., Johnson, R. S., Paxton, R. J., March, C. J., and Gerietti, D. P. (1997). A metalloprotease disintegrin that releases tumor necrosis factor-␣ from cells. Nature 385: 729 –733. 2. Black, R. A., and White, J. M. (1998). ADAMs: Focus on the protease domain. Curr. Opin. Cell Biol. 10: 654 – 659.
SHORT COMMUNICATION 3. 4.
5. 6.
7.
Blobel, C. P.(1997). Metalloprotease-disintegrins: Links to cell adhesion and cleavage of TNF-␣ and Notch. Cell 90: 589 –592. Hooft van Huijsduijnen, R. (1998). ADAM 20 and 21; two novel human testis-specific membrane metalloproteases with similarity to fertilin-␣. Gene 206: 273–282. Huovila, A. P. J., Almeida, E. A., and White, J. M. (1996). ADAMs and cell fusion. Curr. Opin. Cell Biol. 8: 692– 699. Moss, M. L., Jin, S. L., Milla, M. E., Burkhart, W., Carter, H. L., Chen, W. J., Clay, W. C., Didsbury, J. R., Hassler, D., Hoffman, C. R., Kost, T. A., Lambert, M. H., Leesnitzer, M. A., McCauley, P., McGeehan, G., Mitchell, J., Moyer, M., Pahel, G., Rocque, W., Overton, L. K., Schoenen, F., Seaton, T., Su, J. F., Warner, J., Willard, D., et al. (1997). Cloning of a disintegrin metalloprotease that processes precursor tumor necrosis factor-␣. Nature 385: 733–736. Qi, H., Rand, M. D., Wu, X., Sestan, N., Wang, W., Rakic, P., Xu, T., and Artavanis-Tsakonas, S. (1999). Processing of the Notch ligand delta by the metalloprotease Kuzbanian. Science 283: 91–94.
8.
9. 10.
11.
12.
539
Rooke, J., Pan, D., Xu, T., and Rubin, G. M. (1996). KUZ, a conserved metalloprotease– disintegrin protein with two roles in Drosophila neurogenesis. Science 273: 1227– 1231. White, J. M.(1992). Membrane fusion. Science 258: 917– 924. Wolfsberg, T. G., Primacoff, P., Myles, D. G., and White, J. M. (1995). ADAM, a novel family of membrane proteins containing disintegrin and metalloprotease domain: Multipotential functions in cell– cell and cell–matrix interactions. J. Cell Biol. 131: 275–278. Wolfsberg, T. G., Straight, P. D., Gerena, R. L., Huovila, A. P., Primacoff, P., Myles, D. G., and White, J. M. (1995). ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain. Dev. Biol. 169: 378 –383. Wolfsberg, T. G., and White, J. M. (1996). ADAMs in fertilization and development. Dev. Biol. 180: 389 – 401.