Predicted structure of rabbit N-terminal, calcitonin and katacalcin peptides

Vol. 171, No. 3, 1990 September 28, 1990

PREDICTED

STRUCTURE

K. Martial,

S. Minvielle,

U 113 INSERM

Received

BIOCHEMICAL

July

5,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1111-1114

OF RABBIT KATACALClN A. Jullienne,

N-TERMINAL, PEPTIDES N. Segond,

G. Milhaud

and URA 163 CNRS, CHU St. Antoine, 75571 Paris CCdex 12, France

CALCITONIN

AND

and F. Lasmoles 27 Rue Chaligny,

1990

Poly A rich RNA was extracted from rabbit thyroid and cDNA obtained by the action of reverse transcriptase. The cDNA was used to construct a library in lambda GT 11. Screening of the library with a radio-labelled probe specific for human calcitonin allowed the isolation of a clone containing an open reading frame with a high homology with human and murine exon 4 of calcitonin/calcitonin gene-related peptide gene. This sequence codes for a typical calcitonin precursor. We deduced the amino acid sequence of rabbit Nterminal peptide, calcitonin and katacalcin. a1990 Academic west. Inc.

Calcitonin (1,2) a hormone produced by the “c” cells situated in the thyroid in mammals and in the ultimobranchial bodies in non mammalian vertebrates is involved in the regulation of calcium and bone metabolism. Due to differences in the sequence of the midand C-terminal regions of the molecules, calcitonins can be divided into three groups: - human, murine - ovine, bovine, porcine - chicken, eel, salmon. Calcitonin is cleaved from a larger polyprotein precursor which comprises in addition to calcitonin co-secreted N- and C-terminal peptides. While the sequence of calcitonin is established in 10 species the sequence of the N- and Cterminal peptides is only elucidated in four species, man (3), rat (4), chicken (5) and salmon (6). We have determined the sequence of calcitonin mRNA in another mammalian species, the rabbit, and deduced the sequence of calcitonin and the N- and C-terminal peptides.

MATERIAL

AND METHODS

RNA was extracted from rabbit thyroids by the single step guanidine thiocyanate method of Chomczynski (7). Poly A rich RNA was separated by oligo-dT chromatography (8). A cDNA copy was obtained by the action of

1111

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

171, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

reverse transcriptase using oligo-dT as a primer. Double stranded cDNA was synthesized by the Gubler and Hoffman modification (9) of the Okayama and Berg method (10). Double stranded cDNA ends were flushed with T4 DNA polymerase, and after treatment with EcoR I methylase to protect the putative EcoR I restriction sites, EcoR I linkers were added. The double stranded cDNA was then cut with EcoRI and ligated with the phosphatased arms of GT 11 phage lambda and packaged. The obtained phages were propagated in Y 1090 bacteria strain and screened with a cDNA probe specific for exon 4 of human calcitonin gene labelled with [32P] dCTP. A single colony showing a strong signal with the specific probe was purified and the insert obtained by the action of EcoR I. Sequencing strategy: the EcoR I/EcoR I insert was cleaved by Bgl II and the resulting Bgl II/Bgl II and Bgl II/EcoR I fragments subcloned in mp18 Ml3 using the BamH I and EcoR I sites. Sequencing of these clones revealed the presence of a Nsi I restriction site in the 3’ region of the calcitonin messenger. This site was used to insert EcoR I/Nsi I fragments in Pst I and Eco RI cloning sites of mp18 Ml3 and mp19 M13. The sequence was established using the dideoxy chain termination method (11) using both universal and custom primers. Data shown have been derived from overlapping sequence determined on both strands of the clones. RESULTS

AND DISCUSSION

The nucleotide sequence and the deduced amino acid sequence of an almost complete rabbit calcitonin mRNA are reported in figure 1. In figure 2 the sequences of the procalcitonin peptides in rabbit are compared to the human and murine sequences. The nucleotide sequence shows a high sequence similarity with human (81%) and murine (77%) calcitonin messengers. It contains an open reading frame coding for typical calcitonin molecule, N-terminal, and C-terminal peptides. In the precursor, rabbit calcitonin as all other known calcitonins is preceded by the dibasic dipeptide lys-arg and followed by the cleavage and amidation sequence gly-arg-arg-arg. As originally suggested by us on the basis of immunological data (12) and the high hybridization between probes specific for human calcitonin and poly A rich RNA extracted from the rabbit thyroid gland, the sequence of calcitonin in rabbit is highly similar to that of human and murine calcitonins. The two differences are the following: in position 16 leucine replaces phenylalanine in human calcitonin and in position 30 valine replaces glycine. These differences do not modify the secondary structure of the molecule. It should be remembered that in all calcitonins glycine is located in position 2, with the only exception of chicken calcitonin. Recently much attention has been focused on the amino terminal peptide (13) of the calcitonin precursor, which has been shown to have a high mitotic activity on bone osteoblasts (14). The rabbit N-terminal peptide is a 56 a.a peptide, sharing 51 a.a in common with the human N-terminal peptide (57 a.a). 1112

Vol.

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171, No. 3, 1990 1 (CTC LEU 46 CAG GLN 91 AGC SER 136 CTG LEV 181 GAG GLV 226 TCC SER 271 ACA THR 316 GCG ALA 361 AAC ASN 406 ATC ILE 462 CA

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

31 16 AAG TTC TCC CCT TTC CTG GCT CTT AGC ATC TTG GTC LYS PHE SER PRO PHE LEV ALA LEV SER ILE LEV VAL 61 76 GCA GAC AGC CTC CAC GCA GTG CCA TTC AGA TCT GTC ALA ASP SER LEV HIS ALA VAL PRO PHE ARG SER VAL 106 121 AGC CCA GAC CTG GCC ACA CTC AGT GAG GAG GAA GCG SER PRO ASP LEV ALA THR LEV SER GLU GLV GLV ALA 151 166 CTG GCT GCA CTG GTG CAG GAC TAT GTG CAG ATG AAG LEV ALA ALA LEU VAL GLN ASP TYR VAL GLN MET LYS 211 196 CTG GAA CAG CAG CAG GAG ACA GAA GGC TCC AGC CTG LEU GLV GLN GLN GLN GLV THR GLV GLY SER SER LEv 241 256 AGA TCT AAG CGA TGT GGT AAT CTG AGT ACC TGC ATG ARG SER LYS CYS GLY ASN LEU SER THR CYS MET 286 301 TAC ACC CAG GAT CTC AAC AAG TTT CAC ACG TTC CCC TYR THR GLN ASP LEU ASN LYS PHE HIS THR PHE PRO 331 346 ATT GGG GTC GTA GCA CCT GGC AAG AAA AGA GAT ATG ILE GLY VAL VAL ALA PRO GLy LYS LYS BBG ASP MET 376 391 TTG GAT GTG GAC CAC CGC CCT CAA TTT GGC ATG CCT LEV ASP VAL ASP HIS ARG PRO GLN PHE GLY MET PRO 421 436 AAC TAA ATTCTCCTCTCTMTTTCCCTTTTTGCTACCTTTCTATAAATTGATG ASN ***

CTG TAC LEU TYR TTG GAG LEU GLV CGC CTC ARG LEV GCC AGT ALA SER GCC AGC ALA SER CTG'GGC LEV GLY CAA ACT GLN THR GCC AAC ALA ASN CAA AAC GLN ASN

F;ie.l Nucleotide sequence of rabbit calcitonin messenger RNA and predicted amino acid sequence of rabbit calcitonin polyprotein. Cl eavage sites of calcitonin in the precursor are underlined.

Differences with the murine N-terminal peptide are higher as only 47 amino acid residues are conserved. The C-terminal peptide in contrast is less well preserved. It should be noted however that the rabbit C-terminal peptide is of N-terminal

DeDtidp

rabbit

VPFRSVLESSP

human

A----A-----A-P-----D-----------------------E--R

murine

--L--T-----

rabbit

CGNLSTCMLGTYTQDLNKFHTFPQTAIGVVAP

human

---------------F-------------G--

murine Km

-------------------------S---G--

rabbit

DMANNLDVDHRPQFGMPQNIN

human

--SSD-ER----HVS----A-

murine

---KD-ETN-H-Y--N

1

DLATLSEEEARLLLAALVQDYVQMKASELEQQQET

GM----------10

EGSSLASSRS

-----D-P--

-----N-M---vR----EE-qEA-----D-P--D-p--

20

30

Fig.2. Sequences of rabbit N-terminal, calcitonin, and katacalcin compared with the human and murine peptides. 1113

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the same size and shares much more sequence similarities with human calcitonin than with murine calcitonin. In conclusion the elucidation of the sequence of the calcitonin precursor peptides in the rabbit opens the way to the realization of specific radioimmunoassays for these peptides in this species and for the study of structure-activity relationships in this laboratory animal, which is a highly used model for renal studies, as well as in other species. ACKNOWLEDGMENTS We are very indebted interest in this work.

to

Dr.

M.S.

Moukhtar

for

constant

advice,

help

and

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9. Gubler U. and Hoffman B.J. (1983). Gene, 52, 263-268. 10. Okayama H. and Berg P. (1982). Mol. Cell. Biol., 2, 161-170. 11. Sanger F., Nicklen S. and Coulson A.R. (1977). Proc. Natl. Acad. Sci., USA, 74, 5463-5467. 12. Jullienne A., Garel J.M., Calmettes C., Raulais D., Milhaud G. and Moukhtar M.S. (1979). Experientia, 35, 112-l 13. 13. Burns D.M., Birnbaum R.S. and Roos B.A. (1989). Mol. Endocrinol., 3, 140-147. 14. Burns D.M., Forstrom J.M., Friday K.E., Howard G.A. and Roos B.A. (1989). Proc. Natl. Acad Sci., 86, 9519-9523.

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