Eur. J . Riochem. 64, 607 - 61 3 (1 976)
The Complete Amino-Acid Sequence of Rat Thyrocalcitonin Daniel RAULAIS. John HAGAMAN, David A. ONTJES, Roger L. LUNDBLAD. and Henry S. K I N G D O N Laboraloire Associe 163 du Centre National de la Recherche Scientifique, Faculte de inedecine Saint-Antoine, Paris. and the Dental Research Center and the Departments of Medicine, Pharmacology, Pathology, and Biochemistry, The School of Medicine. University of North Carolina at Chapel Hill (Received November 7, 1975/January 26. 1976)
The complete amino acid sequence of rat thyrocalcitonin has been determined by automated Edman degradations of the intact molecule, a cyanogen bromide fragment, and by degradations of mixtures of peptides produced by hydrolysis of the hormone with trypsin and chymotrypsin. The 1
5
10
15
sequence determined was H,N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp20
25
30
Leu-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ser-Ile-Gly-Val-G~y-A~a-Pro-NH~ . This sequence differs in only two positions from that found in the human hormone, i. e. leucine-16 in the rat us phenylalanine-16 in the human, and serine-26 in the rat cs alanine-26 in the human. These similarities and differences are consistent with the previously reported immunological properties of the hormones isolated from these two species.
Calcitonins are polypeptide hormones secreted and stored in the parafollicular C cells of the mammalian thyroid gland or in the ultimobranchial body in lower vertebrates [I]. These hormones provoke hypocalcemia in rats when injected intravenously or subcutaneously [2]. Calcitonins have been isolated from many species, and the amino acid sequences of several have been determined [3 - 131. The purification and amino acid analysis of rat thyrocalcitonin have been reported previously [14]. The purification of rat thyrocalcitonin was made possible through a massive collection of rat thyroid glands by the Rat Pituitary Hormone Program, National Institute of Arthritis and Metabolic and Digestive Diseases, under the direction of Dr A. F. Parlow. This paper describes the determination of the amino acid sequence of the rat hormone. The rat thyroid gland contains a high concentration of calcitonin per weight of tissue [2], but the glands are so small that in fact over 90000 rat thyroid glands had to be processed in order to obtain enough peptide to complete the analysis of amino acid sequence which we are now reporting. However, in view of the recently reported similarities in immunological properties of the rat hormone and fragments of the human hormone [14, 151 it seemed desirable to determine directly the sequence of the rat hormone in order to obtain a better understanding of the relationE n z y m v ~ .Trypsin (EC 3.4.21.4); chymotrypsin (EC 3.4.21.1).
ship between the structures of the two hormones and their immunological and biological activities.
MATERIALS A N D METHODS Rat thyrocalcitonin was prepared as described previously [I41 except for the following modification of the extraction procedure. Defatted rat thyroid powder, 15 g, was added gradually with constant stirring to 300 ml of the acetone/HCl solvent [I41 in an N, atmosphere for 40-60 min at 25 "C. The mixture was then filtered through a 150-ml sintered glass funnel (70 - 100-nm pore size) with positive N, pressure. The filtrate was collected on ice; the retained material was dispersed, washed for 5 - 10 min at 25 'C with 100 ml fresh solvent and re-filtered. Combined filtrates from four separately prepared 15-g batches were pooled, and the volume reduced, by adding 75 ml of benzene (AR grade, thiol-free) to 200-ml aliquots of the extract in a 500-ml separatory funnel and shaking vigorously. The resulting lower aqueous phase retained the biological activity in a markedly reduced volume. The combined aqueous phases were evaporated further by N2 flushing under reduced pressure, and then lyophilized. This modification reduced preparation time and yielded a much lighter, less gummy, and more easily solubilized product for application to the Sephadex G-50 column [14].
Amino-Acid Sequence of Rat Thyrocalcitonin
608
Automatic Edman degradations were performed in the Beckman model 890C sequencer, using a dimethylbenzylamine buffer program which included two phenylisothiocyanate coupling steps of 10 min each, and a single heptafluorobutyric acid cleavage step [16,171.The temperature of the reaction chamber was set at 57 "C. The phenylthiazolinones liberated in the degradation were converted to phenylthiohydantoins in 1 .0N HCl at 80 "C for 10 min. After extraction with ethyl acetate, the organic phase was analyzed for phenylthiohydantoin derivatives by gas chromatography [18]using a Hewlett-Packard 5710-Aequipped with a column of Chromasorb-W loaded with 10% SP-400.Identifications were confirmed when necessary by repeat gas chromatographic analysis after silylation. In addition, the presence or absence of certain residues was established by thin-layer chromatography using Eastman 6060 plastic sheets coated with silica gel containing fluorescent indicator with the solvent systems X P and XM described by Inagami and Murakami [19]. The aqueous phases containing water-soluble amino acid derivatives were analyzed on the same sheets of silica gel [20]. Histidine phenylthiohydantoin was detected by its unique color using the test described by Easley (221;cysteic acid and histidine derivatives from the aqueous phase were also detected by their maximum absorption in ethanol at 269 nm using a Beckman Acta CIII ultraviolet spectrophotometer
[=I. Reduction and alkylation of the hormone with i~do['~C]acetic acid was performed by the procedure of Hirs et al, [23], and the presence of ['4C]carboxymethyl cysteine at positions 1 and 7 was confirmed in the organic phase using a Packard Tricarb scintillation counter. The oxidation and enzymic degradation procedures were those described by Neher et al. [3] for human thyrocalcitonin. Trypsin (TRTPCK 35 E 694, treated with I,-I -tosyl-amido-2-phenylethyl chloromethyl ketone) and a-chymotrypsin (CDS 2 CB) were supplied by Worthington Biochemical Corporation. Cyanogen bromide degradation of the hormone was carried out as described by Gross [28]. The dansylation method described by Gray [24] was used to detect the presence of prolinamide at the end of the chain ; the dansylprolinamide obtained was identified by two-dimensional thin-layer chromatography on polyamide sheets [25],first in a solvent consisting of 1.51%; formic acid in water, and then a system of benzene/acetic acid (9/l).The presence of one free carboxyl group in the intact molecule was confirmed by the carbodiimide coupling of arginine methyl ester according to the method of Gibson and Anderson [26]. Because the amount of sample was somewhat limited, some of the Edman degradations were per-
Table 1. Sequence analysis of reduced and ulkyluted rut thyroculcitonin Cycle 1 2 3 4 5 6
I 8 9 10 11 12 13 14
Amino acid
Cycle
CY s Gly Asn Leu Ser Thr CYS Met Leu G'Y Thr TY r Thr Gln
15 16 17 18 19
Amino acid -
Leu -
Phe
20 21 22 23
Phe Pro
-
-
~
-
-
-
~
~
21
Leu or Ile
formed on mixtures of peptides generated by specific cleavages as proposed by Gray [27].
R ES U LTS The complete amino acid sequence of rat thyrocalcitonin was deduced by degradation of only 3.0 mg (0.8 pmol) of purified hormone. The first automated sequential degradation was begun on 0.6 mg of hormone reduced and alkylated with i~ do['~ C] acetic acid. Residues were identified unequivocally up to and including methionine-8, but the yields were much lower than expected because partial blocking of the amino terminal had taken place during alkylation. Therefore this run was terminated after cycle 8. The peptide remaining in the reaction cup was removed and submitted to cyanogen bromide cleavage to generate a fragment with a free amino terminal corresponding to residue 9 of the native hormone. The sample was desalted by gel filtration on Biogel P2. A second automated sequential degradation was performed on 0.3 mg of native hormone through cycle 8, confirming the sequence of this part of the molecule, in particular the presence of methionine-8. At this point the cyanogen bromide fragment described above was added and the sequential degradation was continued. Table 1 shows the 19 residues identified in this determination, including residues 1 - 14 inclusive, leucine in position 16, phenylalanine in position 19 and 22,proline in position 23, and leucine or isoleucine in position 27. Due to the small amount of peptide available, we then used the technique of sequential degradation of fragment mixtures as proposed by Gray [27].1.2 mg of rat thyrocalcitonin was oxidized with performic acid, and then digested with 10 pg of trypsin. Since the rat hormone contains only one lysine and no arginine, this yielded a mixture of two peptides containing
D. Raulais, J. Hagaman, D. A. Ontjes, R. L. Lundblad, and H. S. Kingdon Table 2. Sryuence analysis of trypticfragment mixture oj oxidized rat thyrocalcitonin Cycle
Amino acids
Cycle
Amino acids
Cys, Phe Gly, His Asn, Thr Leu, Phe Ser, Pro Thr, Gln Cys, Thr Met, Ser Leu
10 11 12 13 14 15 16 17
GIY Thr, Val Tyr, Gly Thr, Ala Gln, Pro-NH, Leu Asn
609
9 corresponding to residue 28. Also, lysine was found in cycle 6, confirming its presence at position 18 of the intact hormone. At this point the sequential analysis indicated the presence of only one free carboxyl group, aspartic acid at position 15. To confirm this, 0.2 mg of intact hormone was reacted with arginine methyl ester and water-soluble carbodiimide, purified by chromatography on a column of Bio-Gel P-6, and then hydrolyzed with 6 N HCI and the arginine content determined [26]. One mole of arginine was found per mole of intact hormone, confirming the presence of one free carboxyl group. This makes it likely that the amide assigments made during the sequential analysis of the intact hormone and the mixtures are correct.
Table 3. Sequence unulysis of the chyrnotryptic fragment mixture of oxidizrd rat thyrocalcitonin Cycle 1 2 3 4 5 6
7 8 9 10 11 12
Amino acid
DISCUSSION
Cys, Thr, His Gly, Gln, Thr A m , Asp, Phe Leu, Leu, Pro Ser, Asn, Gln Thr, Lys, Thr Cys, Ser Met, -, Ile Leu. -. Gly Gly. -, Val -, Gly
The complete amino acid sequence of rat thyrocalcitonin, determined as described above, is presented in Table 4, with the previously determined sequence of the human hormone [3] for comparison. Previous work [14] had shown that rat thyrocalcitonin has immunological properties and an amino acid composition similar to human thyrocalcitonin, suggesting that most portions of the sequence of the two hormones are very similar. The studies of Moukhtar et d. [IS] indicated that the middle portions of the two chains probably differ in sequence whereas the carboxyterminal fragments 17 - 32 appeared to be very similar. The sequences presented in Table 4 show that the two hormones differ only at two positions, 16 and 26. The absence of an aromatic residue in position 16 of the rat hormone or the presence of the t w o differences in the fragment 11 - 32 could explain the different binding characteristics of human and rat thyrocalcitonins with an antibody which was shown to be directed against the 11 -32 portion of human thyrocalcitonin [15]. In contrast, the very similar affinity of the human and rat hormones for an antibody directed against the 17 - 32 fragment of human thyrocalcitonin would be due to the similarity in sequences in this position, with only a minor difference occurring at position 26. The amino acid composition of rat calcitonin inferred from the sequence deduced in the current studies differs slightly from that previously reported [14]. A comparison is presented in Table 5. The differences could be due to the limited amount of material which was available, meaning that the conventional analysis performed previously was near the detection limits of the method, and/or to a small amount of contaminant in the sample. As it stands, only the excess of one mole each of glutamic acid and alanine are disturbing, since the underestimation of threonine and cysteine would be expected.
- ?
-.
-
sequences 1 - 18 and 19 - 32. This mixture was submitted to automated Edman degradation in the sequencer. As presented in Table 2, two distinct amino acid derivatives were detected in each of the first 13 cycles except that we detected only leucine at position 9 and only glycine at position 10. Since the sequence of the first 14 residues was already known (Table l), it was possible to deduce the sequence of the second fragment, 19 - 32, with uncertainty only at residues 27 and 28. After the 13th cycle the single amino acid derivative detected at each cycle was attributed to positions 14 - 18 of the intact hormone. The aqueous phase at cycle 13 contained the carboxyterminal residue of the intact hormone, prolinamide, which was identified as the dansyl derivative. Performic-acid-oxidized rat thyrocalcitonin (0.4 mg) was digested with chymotrypsin, yielding only three fragments, 1 - 12, 13 - 19, and 20- 32, since the phenylalanine-proline bond at position 22 - 23 was not cleaved. Edman degradation of the mixture of these fragments gave three amino acid derivatives at each of the first six cycles (Table 3). From the sequences deduced in Table 2, we then confirmed the presence of isoleucine in cycle 8 corresponding to residue 27 of the intact hormone, and glycine in cycle
Amino-Acid Sequence of Rat Thyrocalcitonin
610
Table 5 Comparison of previous conventional anitno acid analysis with that deduced from sequence anaiysis The previous conventional amino acid analysis results were taken from Burford et al [I41 Amino acid
Number of residues deduced from ~
Lysine Histidine Arginine Aspartate/asparagine Threonine Serine Glutamate/glutamine Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
present sequence analysis
amino acid analysis measured
estimated
1 1 0 3
1.5 0.8 0.4 3.1 3.6 2.2 3.2 2.1 3.5 1.9 0.6 1.5 0.7 1.o 3.3 0.9 1.6
1-2 I 0 3 4 2 3 2 3 -4 2 2 1-2 1 1 3 1 2
5 2 2 2 4 1 2 1 1 1 3 1 2
-
The structures of calcitonins obtained from six different species have already been established. While each consists of a peptide chain of 32 amino acid residues, which a disulfide bond between residues 1 and 7, the sequences differ considerably; only nine positions contain residues common to all species. However, the hormones can be divided into three distinct types on the basis of the similarities in their sequences, immunological properties, and biological potencies. One type is represented by human thyrocalcitonin [3], and now by the rat hormone. The second type, also obtained from mammals, includes porcine [4-61, bovine [7, 81 and ovine [9] hormones. The third group, isolated from ultimobranchial glands, possesses a much higher biological potency than those of the other types and includes salmon [lo, 111 and eel calcitonins [12]; very likely chicken calcitonin [13] could be added to this group although the sequence of the latter is not yet known. One interesting feature of the current work on rat thyrocalcitonin, and the comparison of its amino acid sequence with that of human thyrocalcitonin, is that only two amino acid substitutions have been documented. The substitution of leucine for phenylalanine at position 16 and substitution of serine for alanine at position 26 could be explained on the basis of a single nucleotide change in the gene coding for each amino acid in evolving from the rat to the human or vice versa. One of the intriguing aspects of this research is the apparent close evolutionary relationship between these dissimilar mammalian species.
D . Raulais, J. Hagaman, D. A. Ontjes, R. L. Lundblad, and H . S. Kingdon
The determination of sequences of calcitonins from other species and the comparison of sequence to specific biological activities may provide more information on the relationship between structure and biological potency. '
The authors gratefully acknowledge the help of D r A. F. Parlow who provided us with the rat thyroid glands used to prepare the hormone. D r Raulais also acknowledges the cooperation of the Centre National de la Recherche Scient$que, during his tenure as a visiting scientist in the United States. We also thank D r Claudia Noyes for assistance in performing sequencer runs and gas-liquid chromatographic analyses. and Gerald Lominac for technical assistance. The authors also wish to thank Drs Philip Hirsch, Paul Munson, and Gerard Milhaud for their support and encouragement during various stages of this work. This research was supported in part by National Institutes of Health grants D E 02668, R R 05333, HL 16633, AM 13587, AM 10558, and contract AM-9 0056.
REFERENCES I . Copp, D. H., Cockroft, D. W. & Kueh, Y. (1967) Science (Wash. D.C.) 158, 924-925. 2. Hirsch, P. F. & Munson. P. L. (1969) Physiol. Rev. 49, 548622. 3. Neher, R.. Riniker, B.. Rittel, W. & Zuber, H. (1968) Helv. Chim. Acta, 51, 1900-1905. 4. Neher, R., Riniker, B., Zuber, H., Rittel, W. & Kahnt, F. W. (1968) Hell;. Chim. Actu, 51, 917 -924. 5. Potts, J . T., Jr, Niall, H. D., Keutmann, H. T., Brewer, H. B., Jr & Deftos. L. J. (1968) Proc. Nut1 Acud. Sci. U.S.A. 59, 1321 1328. 6 . Bell, P. H.. Barg, W. F., Jr, Colucci, D. F., Davies, M . C., Dziobkowski. C., Englert, M. E., Heyder, E.. Paul, R . & Snedeker, E. H. (1968) J . Am. Chenz. 90, 2704-2706. 7. Niall. H. D., Penhasi, H.. Gilbert, P., Myers, R. C., Williams. F. G . & Potts. J . T.. Jr (1969) Fed. Proc. 28, 661. -
61 1
8. Brewer. H. B., J r Ronan, R. (1969) Proc. Nail Acud. Sci. U.S.A. 63,940-947. 9. Sauer, R., Niall, H. D. & Potts, J. T., J r (1970) Fed. Proc. 29, 728. 10. Niall, H. D., Keutman. H. T., Copp, D. H. & Potts, J. T., Jr (1969) Proc. Nut1 Acud. Sci. U.S.A. 64, 771 -778. 11. Keutmann, H. T., Leguin, R., Habener, R. M., Singer, J. F., Niall, H . D. & Potts. J. T., Jr (1971) Endocrinol. Proc. 3th Int. Symp. pp. 316 - 323, Heinemann, London. 12. Otani, M., Noda, T., Yamauchi, H., Watanabe, S., Matsuda, T., Orimo, H . & Narita, K. (1974) Proc. 5th Parathyroid C'onjierence (R. V. Talmage, M. Owen, J . A. Parsons, eds), pp. 111 - 115, Excerpta Medica, Amsterdam. 13. Nieto, A,, Moya, F. & Candela, J. L. R. (1973) Biochim. Biophys. Acta, 322, 383 - 391. 14. Burford. H. J., Ontjes, D. A,, Cooper, C. W., Parlow, A. F. & Hirsch, P. F. (1975) Endocrinology, 96, 340-348. 15. Moukhtar, M . S., Tharaud, D.. Jullienne, A., Raulais, D., Calmettes, C . & Milhaud, G. (1974) Experientia (Easel) 30, 552 - 555. 16. Hermodson, M. A., Ericsson. L. H., Titani, K., Neurath, H. & Walsh, K. A. (1972) Biochemistry, I!, 4493-4502. 17. Raulais, D. (1973) C.R. Hebd. Seances Acud. Sci. Sir. D. Sci. Nut. (Paris) 277, 219 -222. 18. Pisano, J. J. & Bronzert, T. J. (1969) J . B i d . Chem. 244, 5597 - 5607. 19. Inagami, T. & Murakami, K. (1972) Anal. Biochenl. 47, 501 504. 20. Inagami, T. (1973) Anal. Biochem. 52, 318-321. 21. Easley, C. W. (1965) Biochim. Biophys. Actu, 107, 386-388. 22. Edman, P. (1970) in Protein Sequence Determination (S. B. Needleman, ed), pp. 21 6 - 219, Springer-Verlag, BerlinHeidelberg-New York. 23. Hirs, C. H. W. (1967) Methods Enzymol. 11, 199-203. 24. Gray, W. R. (3972) Methods Enzymol. 25, 121 - 338. 25. Woods. K. R. & Wang. K. T. (1967) Biochinz. Biophys. Aclcr, 133, 369 - 370. 26. Gibson, D. & Anderson, P. J . (1972) Biochern. Biophys. Rcs. Cornmun. 49, 453 -459. 27. Gray, W. R . (1968) Nature (Lond.) 220, 1300-1304 28. Gross. E. (1967) Methods Enzymol. 11, 238-255.
D. Raulais. 83 Boulevard Saint-Michel, F-75005 Paris, France J. Hagaman, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill. North Carolina, U.S.A. 27514
D. A. Ontjes. Division of Endocrinology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. 27514 K. L. Lundhlad, Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill. North Carolina, U.S.A. 275 14 H. S. Kingdon. Division of Haematology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. 27514
-
The Complete Amino-Acid Sequence of Rat Thyrocalcitonin Daniel RAULAIS. John HAGAMAN, David A. ONTJES, Roger L. LUNDBLAD. and Henry S. K I N G D O N Laboraloire Associe 163 du Centre National de la Recherche Scientifique, Faculte de inedecine Saint-Antoine, Paris. and the Dental Research Center and the Departments of Medicine, Pharmacology, Pathology, and Biochemistry, The School of Medicine. University of North Carolina at Chapel Hill (Received November 7, 1975/January 26. 1976)
The complete amino acid sequence of rat thyrocalcitonin has been determined by automated Edman degradations of the intact molecule, a cyanogen bromide fragment, and by degradations of mixtures of peptides produced by hydrolysis of the hormone with trypsin and chymotrypsin. The 1
5
10
15
sequence determined was H,N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp20
25
30
Leu-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ser-Ile-Gly-Val-G~y-A~a-Pro-NH~ . This sequence differs in only two positions from that found in the human hormone, i. e. leucine-16 in the rat us phenylalanine-16 in the human, and serine-26 in the rat cs alanine-26 in the human. These similarities and differences are consistent with the previously reported immunological properties of the hormones isolated from these two species.
Calcitonins are polypeptide hormones secreted and stored in the parafollicular C cells of the mammalian thyroid gland or in the ultimobranchial body in lower vertebrates [I]. These hormones provoke hypocalcemia in rats when injected intravenously or subcutaneously [2]. Calcitonins have been isolated from many species, and the amino acid sequences of several have been determined [3 - 131. The purification and amino acid analysis of rat thyrocalcitonin have been reported previously [14]. The purification of rat thyrocalcitonin was made possible through a massive collection of rat thyroid glands by the Rat Pituitary Hormone Program, National Institute of Arthritis and Metabolic and Digestive Diseases, under the direction of Dr A. F. Parlow. This paper describes the determination of the amino acid sequence of the rat hormone. The rat thyroid gland contains a high concentration of calcitonin per weight of tissue [2], but the glands are so small that in fact over 90000 rat thyroid glands had to be processed in order to obtain enough peptide to complete the analysis of amino acid sequence which we are now reporting. However, in view of the recently reported similarities in immunological properties of the rat hormone and fragments of the human hormone [14, 151 it seemed desirable to determine directly the sequence of the rat hormone in order to obtain a better understanding of the relationE n z y m v ~ .Trypsin (EC 3.4.21.4); chymotrypsin (EC 3.4.21.1).
ship between the structures of the two hormones and their immunological and biological activities.
MATERIALS A N D METHODS Rat thyrocalcitonin was prepared as described previously [I41 except for the following modification of the extraction procedure. Defatted rat thyroid powder, 15 g, was added gradually with constant stirring to 300 ml of the acetone/HCl solvent [I41 in an N, atmosphere for 40-60 min at 25 "C. The mixture was then filtered through a 150-ml sintered glass funnel (70 - 100-nm pore size) with positive N, pressure. The filtrate was collected on ice; the retained material was dispersed, washed for 5 - 10 min at 25 'C with 100 ml fresh solvent and re-filtered. Combined filtrates from four separately prepared 15-g batches were pooled, and the volume reduced, by adding 75 ml of benzene (AR grade, thiol-free) to 200-ml aliquots of the extract in a 500-ml separatory funnel and shaking vigorously. The resulting lower aqueous phase retained the biological activity in a markedly reduced volume. The combined aqueous phases were evaporated further by N2 flushing under reduced pressure, and then lyophilized. This modification reduced preparation time and yielded a much lighter, less gummy, and more easily solubilized product for application to the Sephadex G-50 column [14].
Amino-Acid Sequence of Rat Thyrocalcitonin
608
Automatic Edman degradations were performed in the Beckman model 890C sequencer, using a dimethylbenzylamine buffer program which included two phenylisothiocyanate coupling steps of 10 min each, and a single heptafluorobutyric acid cleavage step [16,171.The temperature of the reaction chamber was set at 57 "C. The phenylthiazolinones liberated in the degradation were converted to phenylthiohydantoins in 1 .0N HCl at 80 "C for 10 min. After extraction with ethyl acetate, the organic phase was analyzed for phenylthiohydantoin derivatives by gas chromatography [18]using a Hewlett-Packard 5710-Aequipped with a column of Chromasorb-W loaded with 10% SP-400.Identifications were confirmed when necessary by repeat gas chromatographic analysis after silylation. In addition, the presence or absence of certain residues was established by thin-layer chromatography using Eastman 6060 plastic sheets coated with silica gel containing fluorescent indicator with the solvent systems X P and XM described by Inagami and Murakami [19]. The aqueous phases containing water-soluble amino acid derivatives were analyzed on the same sheets of silica gel [20]. Histidine phenylthiohydantoin was detected by its unique color using the test described by Easley (221;cysteic acid and histidine derivatives from the aqueous phase were also detected by their maximum absorption in ethanol at 269 nm using a Beckman Acta CIII ultraviolet spectrophotometer
[=I. Reduction and alkylation of the hormone with i~do['~C]acetic acid was performed by the procedure of Hirs et al, [23], and the presence of ['4C]carboxymethyl cysteine at positions 1 and 7 was confirmed in the organic phase using a Packard Tricarb scintillation counter. The oxidation and enzymic degradation procedures were those described by Neher et al. [3] for human thyrocalcitonin. Trypsin (TRTPCK 35 E 694, treated with I,-I -tosyl-amido-2-phenylethyl chloromethyl ketone) and a-chymotrypsin (CDS 2 CB) were supplied by Worthington Biochemical Corporation. Cyanogen bromide degradation of the hormone was carried out as described by Gross [28]. The dansylation method described by Gray [24] was used to detect the presence of prolinamide at the end of the chain ; the dansylprolinamide obtained was identified by two-dimensional thin-layer chromatography on polyamide sheets [25],first in a solvent consisting of 1.51%; formic acid in water, and then a system of benzene/acetic acid (9/l).The presence of one free carboxyl group in the intact molecule was confirmed by the carbodiimide coupling of arginine methyl ester according to the method of Gibson and Anderson [26]. Because the amount of sample was somewhat limited, some of the Edman degradations were per-
Table 1. Sequence analysis of reduced and ulkyluted rut thyroculcitonin Cycle 1 2 3 4 5 6
I 8 9 10 11 12 13 14
Amino acid
Cycle
CY s Gly Asn Leu Ser Thr CYS Met Leu G'Y Thr TY r Thr Gln
15 16 17 18 19
Amino acid -
Leu -
Phe
20 21 22 23
Phe Pro
-
-
~
-
-
-
~
~
21
Leu or Ile
formed on mixtures of peptides generated by specific cleavages as proposed by Gray [27].
R ES U LTS The complete amino acid sequence of rat thyrocalcitonin was deduced by degradation of only 3.0 mg (0.8 pmol) of purified hormone. The first automated sequential degradation was begun on 0.6 mg of hormone reduced and alkylated with i~ do['~ C] acetic acid. Residues were identified unequivocally up to and including methionine-8, but the yields were much lower than expected because partial blocking of the amino terminal had taken place during alkylation. Therefore this run was terminated after cycle 8. The peptide remaining in the reaction cup was removed and submitted to cyanogen bromide cleavage to generate a fragment with a free amino terminal corresponding to residue 9 of the native hormone. The sample was desalted by gel filtration on Biogel P2. A second automated sequential degradation was performed on 0.3 mg of native hormone through cycle 8, confirming the sequence of this part of the molecule, in particular the presence of methionine-8. At this point the cyanogen bromide fragment described above was added and the sequential degradation was continued. Table 1 shows the 19 residues identified in this determination, including residues 1 - 14 inclusive, leucine in position 16, phenylalanine in position 19 and 22,proline in position 23, and leucine or isoleucine in position 27. Due to the small amount of peptide available, we then used the technique of sequential degradation of fragment mixtures as proposed by Gray [27].1.2 mg of rat thyrocalcitonin was oxidized with performic acid, and then digested with 10 pg of trypsin. Since the rat hormone contains only one lysine and no arginine, this yielded a mixture of two peptides containing
D. Raulais, J. Hagaman, D. A. Ontjes, R. L. Lundblad, and H. S. Kingdon Table 2. Sryuence analysis of trypticfragment mixture oj oxidized rat thyrocalcitonin Cycle
Amino acids
Cycle
Amino acids
Cys, Phe Gly, His Asn, Thr Leu, Phe Ser, Pro Thr, Gln Cys, Thr Met, Ser Leu
10 11 12 13 14 15 16 17
GIY Thr, Val Tyr, Gly Thr, Ala Gln, Pro-NH, Leu Asn
609
9 corresponding to residue 28. Also, lysine was found in cycle 6, confirming its presence at position 18 of the intact hormone. At this point the sequential analysis indicated the presence of only one free carboxyl group, aspartic acid at position 15. To confirm this, 0.2 mg of intact hormone was reacted with arginine methyl ester and water-soluble carbodiimide, purified by chromatography on a column of Bio-Gel P-6, and then hydrolyzed with 6 N HCI and the arginine content determined [26]. One mole of arginine was found per mole of intact hormone, confirming the presence of one free carboxyl group. This makes it likely that the amide assigments made during the sequential analysis of the intact hormone and the mixtures are correct.
Table 3. Sequence unulysis of the chyrnotryptic fragment mixture of oxidizrd rat thyrocalcitonin Cycle 1 2 3 4 5 6
7 8 9 10 11 12
Amino acid
DISCUSSION
Cys, Thr, His Gly, Gln, Thr A m , Asp, Phe Leu, Leu, Pro Ser, Asn, Gln Thr, Lys, Thr Cys, Ser Met, -, Ile Leu. -. Gly Gly. -, Val -, Gly
The complete amino acid sequence of rat thyrocalcitonin, determined as described above, is presented in Table 4, with the previously determined sequence of the human hormone [3] for comparison. Previous work [14] had shown that rat thyrocalcitonin has immunological properties and an amino acid composition similar to human thyrocalcitonin, suggesting that most portions of the sequence of the two hormones are very similar. The studies of Moukhtar et d. [IS] indicated that the middle portions of the two chains probably differ in sequence whereas the carboxyterminal fragments 17 - 32 appeared to be very similar. The sequences presented in Table 4 show that the two hormones differ only at two positions, 16 and 26. The absence of an aromatic residue in position 16 of the rat hormone or the presence of the t w o differences in the fragment 11 - 32 could explain the different binding characteristics of human and rat thyrocalcitonins with an antibody which was shown to be directed against the 11 -32 portion of human thyrocalcitonin [15]. In contrast, the very similar affinity of the human and rat hormones for an antibody directed against the 17 - 32 fragment of human thyrocalcitonin would be due to the similarity in sequences in this position, with only a minor difference occurring at position 26. The amino acid composition of rat calcitonin inferred from the sequence deduced in the current studies differs slightly from that previously reported [14]. A comparison is presented in Table 5. The differences could be due to the limited amount of material which was available, meaning that the conventional analysis performed previously was near the detection limits of the method, and/or to a small amount of contaminant in the sample. As it stands, only the excess of one mole each of glutamic acid and alanine are disturbing, since the underestimation of threonine and cysteine would be expected.
- ?
-.
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sequences 1 - 18 and 19 - 32. This mixture was submitted to automated Edman degradation in the sequencer. As presented in Table 2, two distinct amino acid derivatives were detected in each of the first 13 cycles except that we detected only leucine at position 9 and only glycine at position 10. Since the sequence of the first 14 residues was already known (Table l), it was possible to deduce the sequence of the second fragment, 19 - 32, with uncertainty only at residues 27 and 28. After the 13th cycle the single amino acid derivative detected at each cycle was attributed to positions 14 - 18 of the intact hormone. The aqueous phase at cycle 13 contained the carboxyterminal residue of the intact hormone, prolinamide, which was identified as the dansyl derivative. Performic-acid-oxidized rat thyrocalcitonin (0.4 mg) was digested with chymotrypsin, yielding only three fragments, 1 - 12, 13 - 19, and 20- 32, since the phenylalanine-proline bond at position 22 - 23 was not cleaved. Edman degradation of the mixture of these fragments gave three amino acid derivatives at each of the first six cycles (Table 3). From the sequences deduced in Table 2, we then confirmed the presence of isoleucine in cycle 8 corresponding to residue 27 of the intact hormone, and glycine in cycle
Amino-Acid Sequence of Rat Thyrocalcitonin
610
Table 5 Comparison of previous conventional anitno acid analysis with that deduced from sequence anaiysis The previous conventional amino acid analysis results were taken from Burford et al [I41 Amino acid
Number of residues deduced from ~
Lysine Histidine Arginine Aspartate/asparagine Threonine Serine Glutamate/glutamine Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
present sequence analysis
amino acid analysis measured
estimated
1 1 0 3
1.5 0.8 0.4 3.1 3.6 2.2 3.2 2.1 3.5 1.9 0.6 1.5 0.7 1.o 3.3 0.9 1.6
1-2 I 0 3 4 2 3 2 3 -4 2 2 1-2 1 1 3 1 2
5 2 2 2 4 1 2 1 1 1 3 1 2
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The structures of calcitonins obtained from six different species have already been established. While each consists of a peptide chain of 32 amino acid residues, which a disulfide bond between residues 1 and 7, the sequences differ considerably; only nine positions contain residues common to all species. However, the hormones can be divided into three distinct types on the basis of the similarities in their sequences, immunological properties, and biological potencies. One type is represented by human thyrocalcitonin [3], and now by the rat hormone. The second type, also obtained from mammals, includes porcine [4-61, bovine [7, 81 and ovine [9] hormones. The third group, isolated from ultimobranchial glands, possesses a much higher biological potency than those of the other types and includes salmon [lo, 111 and eel calcitonins [12]; very likely chicken calcitonin [13] could be added to this group although the sequence of the latter is not yet known. One interesting feature of the current work on rat thyrocalcitonin, and the comparison of its amino acid sequence with that of human thyrocalcitonin, is that only two amino acid substitutions have been documented. The substitution of leucine for phenylalanine at position 16 and substitution of serine for alanine at position 26 could be explained on the basis of a single nucleotide change in the gene coding for each amino acid in evolving from the rat to the human or vice versa. One of the intriguing aspects of this research is the apparent close evolutionary relationship between these dissimilar mammalian species.
D . Raulais, J. Hagaman, D. A. Ontjes, R. L. Lundblad, and H . S. Kingdon
The determination of sequences of calcitonins from other species and the comparison of sequence to specific biological activities may provide more information on the relationship between structure and biological potency. '
The authors gratefully acknowledge the help of D r A. F. Parlow who provided us with the rat thyroid glands used to prepare the hormone. D r Raulais also acknowledges the cooperation of the Centre National de la Recherche Scient$que, during his tenure as a visiting scientist in the United States. We also thank D r Claudia Noyes for assistance in performing sequencer runs and gas-liquid chromatographic analyses. and Gerald Lominac for technical assistance. The authors also wish to thank Drs Philip Hirsch, Paul Munson, and Gerard Milhaud for their support and encouragement during various stages of this work. This research was supported in part by National Institutes of Health grants D E 02668, R R 05333, HL 16633, AM 13587, AM 10558, and contract AM-9 0056.
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61 1
8. Brewer. H. B., J r Ronan, R. (1969) Proc. Nail Acud. Sci. U.S.A. 63,940-947. 9. Sauer, R., Niall, H. D. & Potts, J. T., J r (1970) Fed. Proc. 29, 728. 10. Niall, H. D., Keutman. H. T., Copp, D. H. & Potts, J. T., Jr (1969) Proc. Nut1 Acud. Sci. U.S.A. 64, 771 -778. 11. Keutmann, H. T., Leguin, R., Habener, R. M., Singer, J. F., Niall, H . D. & Potts. J. T., Jr (1971) Endocrinol. Proc. 3th Int. Symp. pp. 316 - 323, Heinemann, London. 12. Otani, M., Noda, T., Yamauchi, H., Watanabe, S., Matsuda, T., Orimo, H . & Narita, K. (1974) Proc. 5th Parathyroid C'onjierence (R. V. Talmage, M. Owen, J . A. Parsons, eds), pp. 111 - 115, Excerpta Medica, Amsterdam. 13. Nieto, A,, Moya, F. & Candela, J. L. R. (1973) Biochim. Biophys. Acta, 322, 383 - 391. 14. Burford. H. J., Ontjes, D. A,, Cooper, C. W., Parlow, A. F. & Hirsch, P. F. (1975) Endocrinology, 96, 340-348. 15. Moukhtar, M . S., Tharaud, D.. Jullienne, A., Raulais, D., Calmettes, C . & Milhaud, G. (1974) Experientia (Easel) 30, 552 - 555. 16. Hermodson, M. A., Ericsson. L. H., Titani, K., Neurath, H. & Walsh, K. A. (1972) Biochemistry, I!, 4493-4502. 17. Raulais, D. (1973) C.R. Hebd. Seances Acud. Sci. Sir. D. Sci. Nut. (Paris) 277, 219 -222. 18. Pisano, J. J. & Bronzert, T. J. (1969) J . B i d . Chem. 244, 5597 - 5607. 19. Inagami, T. & Murakami, K. (1972) Anal. Biochenl. 47, 501 504. 20. Inagami, T. (1973) Anal. Biochem. 52, 318-321. 21. Easley, C. W. (1965) Biochim. Biophys. Actu, 107, 386-388. 22. Edman, P. (1970) in Protein Sequence Determination (S. B. Needleman, ed), pp. 21 6 - 219, Springer-Verlag, BerlinHeidelberg-New York. 23. Hirs, C. H. W. (1967) Methods Enzymol. 11, 199-203. 24. Gray, W. R. (3972) Methods Enzymol. 25, 121 - 338. 25. Woods. K. R. & Wang. K. T. (1967) Biochinz. Biophys. Aclcr, 133, 369 - 370. 26. Gibson, D. & Anderson, P. J . (1972) Biochern. Biophys. Rcs. Cornmun. 49, 453 -459. 27. Gray, W. R . (1968) Nature (Lond.) 220, 1300-1304 28. Gross. E. (1967) Methods Enzymol. 11, 238-255.
D. Raulais. 83 Boulevard Saint-Michel, F-75005 Paris, France J. Hagaman, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill. North Carolina, U.S.A. 27514
D. A. Ontjes. Division of Endocrinology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. 27514 K. L. Lundhlad, Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill. North Carolina, U.S.A. 275 14 H. S. Kingdon. Division of Haematology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. 27514
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