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Cloning and sequence analysis of human calcyphosine complementary DNA1
Biochimica et Biophysica Acta 1352 Ž1997. 249–252
Short-sequence paper
Cloning and sequence analysis of human calcyphosine complementary DNA 1 Hakim El Housni, Adrian Radulescu, Raymond Lecocq, Jacques E. Dumont, Daniel Christophe ) IRIBHN, UniÕersite´ Libre de Bruxelles, Faculte´ de Medecine, 808 route de Lennik, Bat. ´ ˆ C, 1070 Brussels, Belgium Received 4 April 1997; accepted 15 April 1997
Abstract Calcyphosine, initially identified as thyroid protein p24, is a calcium-binding protein containing four EF-hand domains. It was first cloned and characterized in the dog and corresponds to R2D5 antigen in rabbit. Using the canine calcyphosine cDNA sequence as a probe, we have isolated its human counterpart from a thyroid cDNA library. The two sequences display a high degree of conservation, both at nucleotide and deduced amino acid levels. Sequence comparison with other proteins showed that the closest homologue of calcyphosine is the crustacean CCBP-23 protein. Northern blot analysis revealed that calcyphosine messenger RNA is much less abundant in human than in canine thyrocytes. Western blot experiments indicated that the amount of protein is also dramatically reduced in man compared to dog. q 1997 Elsevier Science B.V. Keywords: Calcyphosine; Calcium; cAMP; Thyroid
Calcyphosine was cloned from a dog thyroid cDNA library by Lefort et al. w1x. It is a calcium-binding protein containing four EF-hand domains whose synthesis and phosphorylation is up-regulated by cAMP agonists in thyrocytes. Based on sequence similarities, calcyphosine is closely related to calmodulin w2,3x. It distinguishes itself by the presence of two insertions, of 2 and 17 amino acids respectively, in the fourth EF-hand domain canonical motif and by a restricted tissue distribution. Cloning of the rabbit R2D5 antigen from olfactory epithelium revealed that
)
Corresponding author. Fax: q32 2 555 4655. E-mail: [email protected] 1 Accession number: X97966.
it constitutes the homologue of calcyphosine in this species w4x. R2D5 antigen presents 86% identity with dog calcyphosine and has been reported to be phosphorylated by protein kinase A and Ca2qrcalmodulin-dependent protein kinase II Ž CaM kinase II. in vitro. The calcium-binding property and phosphorylation characteristics of calcyphosine potentially allow it to integrate signals originating from both calcium and cAMP cascades, which could constitute a privileged point of cross-signalling between these two regulatory pathways in selected cell types. However, experimental evidence supporting this view is still lacking. The presence of the calcyphosine gene in the human genome has been demonstrated w5x. It was localized to the p13.3 region of chromosome 19 by in situ hybridization using a partial human genomic
0167-4781r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 0 7 3 - 0
250
H. El Housni et al.r Biochimica et Biophysica Acta 1352 (1997) 249–252
clone. We now report here the cloning of the complete calcyphosine cDNA from man. An oligoŽdT.-primed, directional cDNA library in lambda ZAP II vector ŽZAP-cDNA synthesis and cloning system, Stratagene, La Jolla, CA, USA. was constructed using mRNA isolated from primary cultured human thyrocytes maintained in the presence of the cAMP agonist forskolin. 2.5 = 10 5 pfu were screened using the dog calcyphosine coding sequence as a probe. Three positive clones were obtained and subjected to in vivo excision as described by the manufacturer of the cloning system ŽStratagene. . The longest insert Žsize: ; 1.1 kb. from the resulting plasmid clones was subcloned in bacteriophage M13mp18r19 and sequenced using an Applied Biosystems 370A DNA sequencer according to the instructions of the manufacturer ŽApplied Biosystems.. Sequence comparison with the dog calcyphosine cDNA revealed that about 100 bp of the coding sequence were lacking at the 5X end of the human clone. The incomplete human cDNA was then used as a probe in further screenings of the library. A complete human calcyphosine cDNA clone Žinsert size: ; 1.3 kb. was isolated after screening of a total of 2.5 = 10 6 pfu. The complete coding sequence was
determined on both strands as described above. This n u c le o tid e se q u e n c e is a v a ila b le fro m EMBLrGenBank database under accession number X97966. The deduced human calcyphosine protein sequence was compared to its counterparts in dog and rabbit ŽFig. 1. . All three sequences display extensive sequence conservation, including in the two insertions that disrupt the fourth EF-hand motif. The presence of an arginine residue instead of a glycine residue in central position in the first EF-hand domain is also conserved in the human sequence. This arginine residue is part of the RSRS motif in the dog calcyphosine sequence Žpositions 37 to 40. which constitutes a putative phosphorylation site for protein kinase A w6x. The RSRS motif is not entirely conserved in man, where the first arginine residue Žposition 37. is replaced by a glycine residue. This substitution is likely to decrease the affinity of protein kinase A for this site w7x. However, the rabbit protein, which also contains a glycine at this position, seems to be still phosphorylated by protein kinase A in vitro w4x. Comparison of the calcyphosine sequence with that of other proteins ŽFig. 2. showed that its closest relative is the crustacean CCBP-23 protein w8x which
Fig. 1. Sequence alignment of human Žhum., dog and rabbit Žrab. calcyphosine proteins. The four ‘EF-hand’ domains, delimited by vertical arrows, are aligned under the ‘EF-hand’ consensus sequence Žn s hydrophobic residue, ) s oxygen-containing residue, ys any residue.. Conserved residues appear in light-shaded boxes and the two insertions in the fourth canonical EF-hand motif are indicated by deep-shaded boxes. The putative phosphorylation site for protein kinase A is underlined.
H. El Housni et al.r Biochimica et Biophysica Acta 1352 (1997) 249–252
251
Fig. 2. Similarities between calcyphosine and the most related CCBP-23 protein sequences. The dendrogram has been constructed using the GCG-PILEUP program ŽUniversity of Wisconsin.. Conservation is expressed as percentages of identity of each sequence relative to human calcyphosine.
differs in the amino terminal end and contains an additional insertion of one amino acid residue in the fourth EF-hand domain. To compare the level of expression of the calcyphosine gene in human and canine thyroids, Northern blot experiments were conducted, using either the human or the canine sequence to probe total RNA preparations from thyroid tissue of both species. A standard protocol w9x was followed and the last washes were done in 0.1 = SSC at 558C, taking into account the existence of a modest divergence in the sequences from the two species. Similar results were obtained with both probes, showing a largely reduced expression of the calcyphosine mRNA in man as compared to dog ŽFig. 3A.. To verify that this situation is conserved at the protein level, Western blot experiments were conducted using an antiserum raised against the dog protein w1x. Protein extracts were prepared from primary cultured thyrocytes w10x of both species maintained in the presence of 10 m M forskolin for 22 h Žcultured cells were preferred here because protein extracts from thyroid tissue contain very large quantities of colloidal thyroglobulin, which severely restricts the amounts of extracts that can be loaded on the gel.. The Western analysis was performed as described previously w1x and the immuno-
Fig. 3. A: Northern blot analysis of calcyphosine mRNA in human and canine thyroid tissue. Identical amounts Ž8 m g. of total RNA from human and canine thyroids were probed with calcyphosine cDNA from dog Žleft panel. and man Žright panel.. The arrow points at the signal corresponding to canine calcyphosine mRNA Ž1.4 kb.. B: Western blot analysis of calcyphosine in primary cultured human and canine thyrocytes. 50 m g of proteins from human thyrocytes Žleft lane. and 40 m g of proteins from dog thyrocytes Žright lane. were probed with a polyclonal antibody raised against canine calcyphosine. Binding was revealed using 125 I-labeled protein A. The positions of size standards are indicated on the left. Staining of gel lanes run in parallel confirmed that the extracts from both cell types contained intact proteins essentially Žnot shown..
252
H. El Housni et al.r Biochimica et Biophysica Acta 1352 (1997) 249–252
complexes were revealed by using 125 I-labeled protein A Ž purchased from Amersham.. As shown in Fig. 3B, an intense signal at the expected size for calcyphosine Ž24 kDa. was readily detected in the lane corresponding to the dog cell extract, but no clear signal appeared at that position in the lane corresponding to the human cell extract. Similar negative results were also obtained for the human protein in immuno-precipitation experiments from 35S-labeled cell extracts Žnot shown.. In view of the very high similarity of the primary sequence of human and canine calcyphosine, it is very unlikely that the polyclonal antiserum used here would not be able to detect the human protein if present in amounts comparable to the amounts contained in the dog cells. This result thus indicates that calcyphosine, if present in the human thyrocytes, is certainly much less abundant here than in the canine thyrocytes. Further investigation of the possible physiological consequences of this difference will have to await the characterization of the cellular functionŽs. of calcyphosine. It is noteworthy that attempting to target the calcyphosine gene in mouse led to the unexpected finding that rodents lack a functional calcyphosine gene w11x. Thus, this gene is highly expressed in dog thyroid w6x, very poorly expressed at best in human thyroid Žlow level of mRNA detected but no protein found in a standard analysis. as shown here, and not expressed at all in several rodents w11x. This raises indeed serious questions about the exact role the protein plays in thyroid physiology. We thank A. Allgeier and S. Clement for help in ´ the Northern blot experiment and C. Govaerts for
help in the use of GCG software. This work was supported by the Belgian program ‘Poles ˆ d’Attraction Inter-universitaires’ Ž Prime Minister’s office, Science Policy Programming. and European Community ‘Human Capital’ grant Ž ERBCHRXCT940573. and contract Ž CT94.0513.. The scientific responsibility is assumed by the authors. H.E.H. is recipient of a FRIA doctoral fellowship and D.C. is Senior Research Associate of the Belgian FNRS.
References w1x A. Lefort, R. Lecocq, F. Libert, F. Lamy, S. Swillens, G. Vassart, J.E. Dumont, EMBO J. 8 Ž1989. 111–116. w2x P. James, T. Vorherr, E. Carafoli, Trends Biochem. Sci. 20 Ž1995. 38–42. w3x M. Ikura, Trends Biochem. Sci. 21 Ž1996. 14–17. w4x Y. Nemoto, J. Ikeda, K. Katoh, H. Koshimoto, Y. Yoshihara, K. Mori, J. Cell Biol. 123 Ž1993. 963–976. w5x A. Lefort, E. Passage, F. Libert, J. Szpirer, G. Vassart, M.-G. Mattei, Cytogenet. Cell Genet. 54 Ž1990. 154–155. w6x R. Lecocq, F. Lamy, C. Erneux, J.E. Dumont, Biochem. J. 306 Ž1995. 147–151. w7x B.E. Kemp, R.B. Pearson, Trends Biochem. Sci. 15 Ž1990. 342–346. w8x A. Sauter, W. Staudenmann, G.J. Hughes, C.W. Heizmann, Eur. J. Biochem. 227 Ž1995. 97–101. w9x T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York, 1982. w10x P.P. Roger, J.E. Dumont, Mol. Cell. Endocrinol. 36 Ž1984. 79–93. w11x S. Clement, J.E. Dumont, S. Schurmans, Biochem. Biophys. ´ Res. Commun. Ž1997. in press.
Short-sequence paper
Cloning and sequence analysis of human calcyphosine complementary DNA 1 Hakim El Housni, Adrian Radulescu, Raymond Lecocq, Jacques E. Dumont, Daniel Christophe ) IRIBHN, UniÕersite´ Libre de Bruxelles, Faculte´ de Medecine, 808 route de Lennik, Bat. ´ ˆ C, 1070 Brussels, Belgium Received 4 April 1997; accepted 15 April 1997
Abstract Calcyphosine, initially identified as thyroid protein p24, is a calcium-binding protein containing four EF-hand domains. It was first cloned and characterized in the dog and corresponds to R2D5 antigen in rabbit. Using the canine calcyphosine cDNA sequence as a probe, we have isolated its human counterpart from a thyroid cDNA library. The two sequences display a high degree of conservation, both at nucleotide and deduced amino acid levels. Sequence comparison with other proteins showed that the closest homologue of calcyphosine is the crustacean CCBP-23 protein. Northern blot analysis revealed that calcyphosine messenger RNA is much less abundant in human than in canine thyrocytes. Western blot experiments indicated that the amount of protein is also dramatically reduced in man compared to dog. q 1997 Elsevier Science B.V. Keywords: Calcyphosine; Calcium; cAMP; Thyroid
Calcyphosine was cloned from a dog thyroid cDNA library by Lefort et al. w1x. It is a calcium-binding protein containing four EF-hand domains whose synthesis and phosphorylation is up-regulated by cAMP agonists in thyrocytes. Based on sequence similarities, calcyphosine is closely related to calmodulin w2,3x. It distinguishes itself by the presence of two insertions, of 2 and 17 amino acids respectively, in the fourth EF-hand domain canonical motif and by a restricted tissue distribution. Cloning of the rabbit R2D5 antigen from olfactory epithelium revealed that
)
Corresponding author. Fax: q32 2 555 4655. E-mail: [email protected] 1 Accession number: X97966.
it constitutes the homologue of calcyphosine in this species w4x. R2D5 antigen presents 86% identity with dog calcyphosine and has been reported to be phosphorylated by protein kinase A and Ca2qrcalmodulin-dependent protein kinase II Ž CaM kinase II. in vitro. The calcium-binding property and phosphorylation characteristics of calcyphosine potentially allow it to integrate signals originating from both calcium and cAMP cascades, which could constitute a privileged point of cross-signalling between these two regulatory pathways in selected cell types. However, experimental evidence supporting this view is still lacking. The presence of the calcyphosine gene in the human genome has been demonstrated w5x. It was localized to the p13.3 region of chromosome 19 by in situ hybridization using a partial human genomic
0167-4781r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 0 7 3 - 0
250
H. El Housni et al.r Biochimica et Biophysica Acta 1352 (1997) 249–252
clone. We now report here the cloning of the complete calcyphosine cDNA from man. An oligoŽdT.-primed, directional cDNA library in lambda ZAP II vector ŽZAP-cDNA synthesis and cloning system, Stratagene, La Jolla, CA, USA. was constructed using mRNA isolated from primary cultured human thyrocytes maintained in the presence of the cAMP agonist forskolin. 2.5 = 10 5 pfu were screened using the dog calcyphosine coding sequence as a probe. Three positive clones were obtained and subjected to in vivo excision as described by the manufacturer of the cloning system ŽStratagene. . The longest insert Žsize: ; 1.1 kb. from the resulting plasmid clones was subcloned in bacteriophage M13mp18r19 and sequenced using an Applied Biosystems 370A DNA sequencer according to the instructions of the manufacturer ŽApplied Biosystems.. Sequence comparison with the dog calcyphosine cDNA revealed that about 100 bp of the coding sequence were lacking at the 5X end of the human clone. The incomplete human cDNA was then used as a probe in further screenings of the library. A complete human calcyphosine cDNA clone Žinsert size: ; 1.3 kb. was isolated after screening of a total of 2.5 = 10 6 pfu. The complete coding sequence was
determined on both strands as described above. This n u c le o tid e se q u e n c e is a v a ila b le fro m EMBLrGenBank database under accession number X97966. The deduced human calcyphosine protein sequence was compared to its counterparts in dog and rabbit ŽFig. 1. . All three sequences display extensive sequence conservation, including in the two insertions that disrupt the fourth EF-hand motif. The presence of an arginine residue instead of a glycine residue in central position in the first EF-hand domain is also conserved in the human sequence. This arginine residue is part of the RSRS motif in the dog calcyphosine sequence Žpositions 37 to 40. which constitutes a putative phosphorylation site for protein kinase A w6x. The RSRS motif is not entirely conserved in man, where the first arginine residue Žposition 37. is replaced by a glycine residue. This substitution is likely to decrease the affinity of protein kinase A for this site w7x. However, the rabbit protein, which also contains a glycine at this position, seems to be still phosphorylated by protein kinase A in vitro w4x. Comparison of the calcyphosine sequence with that of other proteins ŽFig. 2. showed that its closest relative is the crustacean CCBP-23 protein w8x which
Fig. 1. Sequence alignment of human Žhum., dog and rabbit Žrab. calcyphosine proteins. The four ‘EF-hand’ domains, delimited by vertical arrows, are aligned under the ‘EF-hand’ consensus sequence Žn s hydrophobic residue, ) s oxygen-containing residue, ys any residue.. Conserved residues appear in light-shaded boxes and the two insertions in the fourth canonical EF-hand motif are indicated by deep-shaded boxes. The putative phosphorylation site for protein kinase A is underlined.
H. El Housni et al.r Biochimica et Biophysica Acta 1352 (1997) 249–252
251
Fig. 2. Similarities between calcyphosine and the most related CCBP-23 protein sequences. The dendrogram has been constructed using the GCG-PILEUP program ŽUniversity of Wisconsin.. Conservation is expressed as percentages of identity of each sequence relative to human calcyphosine.
differs in the amino terminal end and contains an additional insertion of one amino acid residue in the fourth EF-hand domain. To compare the level of expression of the calcyphosine gene in human and canine thyroids, Northern blot experiments were conducted, using either the human or the canine sequence to probe total RNA preparations from thyroid tissue of both species. A standard protocol w9x was followed and the last washes were done in 0.1 = SSC at 558C, taking into account the existence of a modest divergence in the sequences from the two species. Similar results were obtained with both probes, showing a largely reduced expression of the calcyphosine mRNA in man as compared to dog ŽFig. 3A.. To verify that this situation is conserved at the protein level, Western blot experiments were conducted using an antiserum raised against the dog protein w1x. Protein extracts were prepared from primary cultured thyrocytes w10x of both species maintained in the presence of 10 m M forskolin for 22 h Žcultured cells were preferred here because protein extracts from thyroid tissue contain very large quantities of colloidal thyroglobulin, which severely restricts the amounts of extracts that can be loaded on the gel.. The Western analysis was performed as described previously w1x and the immuno-
Fig. 3. A: Northern blot analysis of calcyphosine mRNA in human and canine thyroid tissue. Identical amounts Ž8 m g. of total RNA from human and canine thyroids were probed with calcyphosine cDNA from dog Žleft panel. and man Žright panel.. The arrow points at the signal corresponding to canine calcyphosine mRNA Ž1.4 kb.. B: Western blot analysis of calcyphosine in primary cultured human and canine thyrocytes. 50 m g of proteins from human thyrocytes Žleft lane. and 40 m g of proteins from dog thyrocytes Žright lane. were probed with a polyclonal antibody raised against canine calcyphosine. Binding was revealed using 125 I-labeled protein A. The positions of size standards are indicated on the left. Staining of gel lanes run in parallel confirmed that the extracts from both cell types contained intact proteins essentially Žnot shown..
252
H. El Housni et al.r Biochimica et Biophysica Acta 1352 (1997) 249–252
complexes were revealed by using 125 I-labeled protein A Ž purchased from Amersham.. As shown in Fig. 3B, an intense signal at the expected size for calcyphosine Ž24 kDa. was readily detected in the lane corresponding to the dog cell extract, but no clear signal appeared at that position in the lane corresponding to the human cell extract. Similar negative results were also obtained for the human protein in immuno-precipitation experiments from 35S-labeled cell extracts Žnot shown.. In view of the very high similarity of the primary sequence of human and canine calcyphosine, it is very unlikely that the polyclonal antiserum used here would not be able to detect the human protein if present in amounts comparable to the amounts contained in the dog cells. This result thus indicates that calcyphosine, if present in the human thyrocytes, is certainly much less abundant here than in the canine thyrocytes. Further investigation of the possible physiological consequences of this difference will have to await the characterization of the cellular functionŽs. of calcyphosine. It is noteworthy that attempting to target the calcyphosine gene in mouse led to the unexpected finding that rodents lack a functional calcyphosine gene w11x. Thus, this gene is highly expressed in dog thyroid w6x, very poorly expressed at best in human thyroid Žlow level of mRNA detected but no protein found in a standard analysis. as shown here, and not expressed at all in several rodents w11x. This raises indeed serious questions about the exact role the protein plays in thyroid physiology. We thank A. Allgeier and S. Clement for help in ´ the Northern blot experiment and C. Govaerts for
help in the use of GCG software. This work was supported by the Belgian program ‘Poles ˆ d’Attraction Inter-universitaires’ Ž Prime Minister’s office, Science Policy Programming. and European Community ‘Human Capital’ grant Ž ERBCHRXCT940573. and contract Ž CT94.0513.. The scientific responsibility is assumed by the authors. H.E.H. is recipient of a FRIA doctoral fellowship and D.C. is Senior Research Associate of the Belgian FNRS.
References w1x A. Lefort, R. Lecocq, F. Libert, F. Lamy, S. Swillens, G. Vassart, J.E. Dumont, EMBO J. 8 Ž1989. 111–116. w2x P. James, T. Vorherr, E. Carafoli, Trends Biochem. Sci. 20 Ž1995. 38–42. w3x M. Ikura, Trends Biochem. Sci. 21 Ž1996. 14–17. w4x Y. Nemoto, J. Ikeda, K. Katoh, H. Koshimoto, Y. Yoshihara, K. Mori, J. Cell Biol. 123 Ž1993. 963–976. w5x A. Lefort, E. Passage, F. Libert, J. Szpirer, G. Vassart, M.-G. Mattei, Cytogenet. Cell Genet. 54 Ž1990. 154–155. w6x R. Lecocq, F. Lamy, C. Erneux, J.E. Dumont, Biochem. J. 306 Ž1995. 147–151. w7x B.E. Kemp, R.B. Pearson, Trends Biochem. Sci. 15 Ž1990. 342–346. w8x A. Sauter, W. Staudenmann, G.J. Hughes, C.W. Heizmann, Eur. J. Biochem. 227 Ž1995. 97–101. w9x T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York, 1982. w10x P.P. Roger, J.E. Dumont, Mol. Cell. Endocrinol. 36 Ž1984. 79–93. w11x S. Clement, J.E. Dumont, S. Schurmans, Biochem. Biophys. ´ Res. Commun. Ž1997. in press.