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Differential structural requirements for the MSH and MCH activities of melanin concentrating hormone
Life Sciences, Vol. 40, pp. 1139-1145 Printed in the U.S.A.
Pergamon Journals
DIFFERENTIAL STRUCTURAL REQUIREMENTS FOR THE MSH AND MCH ACTIVITIES OF MELANIN CONCENTRATING HORMONE Mac E. ItsdleyI, Christian Zechel 2, Brian C. Wilkes 2, Ana M. de L. Castruccl 3 Maria A. Viscontl 3, Manuel Pozo-Alonso I, and V i c t o r J. Bruby 2# Departments of Anatomy I and Chemistry 2 University o f Arizona, Tucson, Arizona 85721 U.S.A. and Ikmpartmento de F i s l o l o g i a 3, U n i v e r s i d a d e de Sao Paulo CPII176, Sao Paulo, Srasll (Received in final form December 18, 1986)
Summary H-Asp-Thr-Me t-At g - ~ a - M e t-Val-Oly-- Ar g-Va l-Tyr -At g-Pro--~s-TrpClu-Val-OR, melanin concentrating hormone (MCH), exhibits both melanin granule concentrating and dispersing (MSH-like) activities. Fragment analogues of MCH ~ r e synthesized as described herein and the melanotropic activities of the peptides ware determined. In the frog (Rana plplens) and lizard (Anolis carollnensls) skin blnassays, the 5-i~-a'~-5-14 fragments o - - ~ were inactive (at concentrations of I0-5M or less), whereas the 1-14 sequence exhibited minimal (about I0%) MSH-llke activity compared to MCH, which, as reported previously, was about 600 times less active than a-4qSH. In the teleost (fish) skin bloassay, the MCTI5_17 analogue was equlpotent to MCH, whereas the 1-14 analogue was 10-30 times and the cycllc N- and C- termlnal truncated analogue, MCHs_I4 , was about 300 times less active than MCH. These results suggest that the N-termlnal sequence is particularly critical to MSH-like activity in the tetrapod species studied, whereas other structural regions of MCH m particularly in the C-terminal, are more related to MCH activity in teleosts. A putative melanin ~ranule (melanosome) concentrating (aggregating) hormone, HCH (Figure I), hls been isolated from the salmon pituitary gland and its primary structure was elucidated (1,2). This cyclic heptadecapeptlde, !
l
H-Asp-Thr-Met-Arg-Cys-Mat-Val-Oly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH, was synthesized and its biological activity has been determined (3,4). The peptide not only causes melanosome aggregation within teleost (fish) melanophores, but also induces melanosome dispersion within tetrapod (frog and lizard) melanophores (3). In the present report we document the melauotropic actions of N-and C-termlnally truncated analogues of MCH on teleost and tetrapod melanophores, and demonstrate that the N-and C-termlnal residues are essential, respectively, for the MSH and MCH activities of the hormone.
# To whom reprint requests should be addressed at the Department of Chemistry, University of Arizona, Tucson, Arizona 85721 U.S.A. 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1140
MCH: Structural Activity Studies
1
3
5
7
9
Vol. 40, No. 12, 1987
11
15
13
I
17
!
MCHI-17
Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val
MCHI-14
Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys
!
l
!
!
MCHs-17
Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val
MCHs-14
C~s-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-C~s FIGURE I.
Primary structure of MCH analogues.
Materials and Methods Synthesis: MCH was prepared as previously reported ( 4 ) . The following three fragment analogues of MC~ were prepared: the N-term/hal tetradecapeptlde sequence, MCHI_I4 , the C-termlnal tridecapeptide MCHb_I7 sequence, and the Nand C-terminal truncated analogue MCH5_I4 (Figure I). Melting points were determined on a Thomas-Hoover melting point apparatus. Thln-layer chromatography (TLC) was performed on Silufol plates (Kavaller, Czechoslovakia) using the following solvent systems: A) l-butanol/acetlc acid/H20 (4:1:5, upper phase only); B) 1-butanol/acetlc acld/pyrldine/~20 (15:3:10:12); C) l-butanol/pyridine/acetic acld/R20 (5:5:1:4), Detection was by iodine vapor. High pressure liquid chromatography (RPLC) was performed on a Perkln-Elmer instrument with detection at 214 nm using a Vydac C18 column (25 x 1.0 cm). Aqueous trlfluoroacetlc acid (0.1%) was the buffer wlth acetonltrile as the organic modifier. Fast atom bombardment mass spectra (FAB/MS) were aqulred on a Varian 311A spectrometer equipped with an Ion Tech Ltd. source, wlth Xenon as the bombarding gas. NU-Boc protected amino acids and amino acid derivatives were purchased from Vega Biotechnologles (Tucson, AZ), Peninsula Laboratories (San Carlos, CA), Bachem (Torrence, CA), or were prepared using published procedures. Before use, all amino acid derivatives were tested for homogeneity by TLC in solvent systems A, B, and C, by melting point determinations, and by the nlnhydrln test (5). Solvents used for chromatography were redlstilled prior to use.
The MCH analogues were prepared using Merrifleld resins substituted with the C-termlnal amino acid by standard methods ( 6 ) . Boc-Val-Merrifield resin had an amino acid substitution of 0.47 mmol/gram resin and Boc-Cys(PMB)Merrifield resin had an amino acid substitution of 0.77 mmol/gram resin as determined by the method of Gisln ( 7 ) . NU-Boc amino acid derivatives were coupled to the resin using a three-fold excess of each amino acid derivative, a 2.4 fold excess of dicyclohexylcarbodiimide (DCC) and a 2.4 fold excess of l-hydroxybenzotriazole (HOBT). Removal of the NU-Boc protecting group was accomplished with 45% trlfluoroacetlc acid (TFA) in dichloromethane (CH2CI 2) containing 2% anlsole. A cycle for the incorporation of each amino acid derivative consisted of the following: I) washing the resin with three 20-ml portions of CN2CI 2, one min per wash; 2) washing with two 30-ml portions of Et0H, 2 mln per wash; 3) washing with two 20-ml portions of CH2C12, one mln per wash; 4) cleavage of the NU-Boc protecting group with 45% TFA in C~2C12, one wash for 2 mln, one
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MCH: Structural Activity Studies
1141
wash for 20 min; 5) washing with three 20-ml portions of CH2C12, one min per wash; 6) neutralization of the resin with two 30-ml protlons of i0% dlisopropylethylamlne in CH2C12, 2 mln per wash; 7) washing with two 20-ml portions of CH2CI 2, one min per wash; 8) addition of the next Na-Boc amino acid derivative along with DCC and HOBT in 30 ml N,N-dimethylformamide (DMF). Coupling of each amino acid derivative was complete In 30 to 60 min as monitored through the ninhydrln test. After coupling of the final amino acid derivative, the resulting peptide resin was dried in vacuo. Synthesis of MC_~_5_17: Starting with 2.10 g Boc-Val resin (I.0 mmol total), the f o l l o w l n g - ~ - B o c amino acid derivatives were coupled (in order of their coupling): Glu(Bzl); Trp(For); Cys(PMB); Pro; Arg(Tos); Tyr(2,6-Cl2-Bzl); Val; Arg(Tos); Gly; Val; Met and Cys(PMB). The resulting peptide resin was dried and weighed (5.35 g). To one fourth of this resin (1.33 g, 0.25 mmol), an HF cleavage of the peptide from the resin was accomplished using the procedure of Metsueda (8). The mixture was washed with 3 x 30-ml of ethyl acld and the product extracted with 3 x 30-ml 30% acetic acid and lyophillzed. The crude [Cys(R) 5, Cys(N)I4]HCHI_I4 was diluted in 1 liter deaerated H20 and cycllzed as before giving 250 mg of crude product. A portion of this product (24 mg) was purified by CMC chromatography as before, and preparative HPLC, giving 2.0 mg of the title compound: NPLC - k" (18% acetonltrile, 82% - 0.1% aqueous trlfluoroacetlc acld) ffi 2.1; FAB/MS - 1684 (Cald. 1683.74). Synthesls of HCHI_14: To 1.00 g of Boc-Cys(PHB) resin (0.5 mmol), the following NU-Boc amino acid derivatives were coupled (in order of their coupling): Pro; Arg(Tos); Tyr(2,6-Cl 2 Bz;); Val; Arg(Tos); Gly; Val; Met; Cys(PMB); Arg(Tos); Met; Thr(Bzl); and Asp(Bzl) giving 1.50 g of peptlde resin. Half of the resulting peptide resin (0.75 g) was subjected to HF cleavage as before. The mixture was washed with 3 x 30-ml ethyl acetate and the product extracted with 3 x 30-ml 30% acetic acld and lyophillzed. The crude [Cys(R) 5, Cys(H)I4]MCHI_I4 was diluted in 1 liter deaerated H20 and cycllzed as before giving 250 mg of crude product. A portion of thls product (24 mg) was purified by CMC chromatography as before, and preparative tIPLC, giving 2.0 mg of the title compound: HPLC - ~ (18% acetonitile, 82% - 0.1% aqueous trlfluoroacetlc acld) = 2.1; FAB/MS - 1684 (Cald. 1683.74). Synthesis of MCHs_I4: Starting with 1.00 g of Boc-Cys(PMB)-resin (0.5 mmol), the fol~'owlng N~-Boc amino acld derivatives were coupled to the growing peptlde chain (in order of their coupling): Pro; Arg (Tos); Tyr(2,6-Cl2Bzl) ; Val; Arg (Tos); Gly; Val; Met; and Cys(PMB) give 1.40 g of peptide resin. The resultant peptlde resin was subjected to HF cleavage as before. The HF was removed in vacuo, the resulting mixture was washed with 3 x 30-ml of ethyl acetate and the ~roduct extract with 3 x 35-mi of 30% acetic acid. The crude [Cys(H) 5, Cys(H)I4]MCH5_I4 was diluted in 2 i of deaerated H20 , the pH adjusted to 8.5, and the peptide cyclized as before, giving the crude product. The peptlde was purified by gel filtration on Sephadex G-25 and CMC ion exchange chromatography, followed by preparative HPLC: HPLC - k" (15% acetonitrile, 85% - 0.1% aqueous trlfluoroacetic acid) ffi 4.05; FAB/MS - 1181 (Calcd. 1180.5). Bioassays: The bioassays for MSH-IIke activity of MCH and related analogues utilized skins from the frog (Rana pipiens) and the lizard (Anolis carolinensis). Skins were mounted on metal rings and held in place by an outer plastic ring as described originally for the frog (i0) and lizard skin bio-assays and as detailed in a recent report (II). Changes in reflectance from the outer (epidermal) surface of the skins as induced by the peptides were monitored photometrically. The melanin concentrating activities of MCH, MCHI_14 , and MCH5_I7 were determined on isolated scales from a number of teleost species (Table I). The minimal effective concentrations of the pep-
1142
MCH: Structural Activity Studies
Vol. 40, No. 12, 1987
tides to elicit melanin granule aggregation within melanophores as subjectively under a dissecting microscope are reported (Table I).
observed
The teleost fish, Synbranchus marmoratus, an eel obtained from the Pantanal (Big Swamp) of Brazil were also used. Skins were removed and prepared as originally described for the frog (10) and lizard (II) skin bioassays. A unique feature of this fish skin bloassay is that, unlike the frog and lizard skin bloassays, the "resting" (unstimulated) state of the melanophores Is characterized by dispersed melanosomes. Therefore, the assay is particularly appropriate for the study of melanosome aggregating agents such as MCH, and thus was used for determination of quantitative dose response curves for all peptldes (Figure 2).
Results and Discussion As demonstrated in our earlier publications (3,4), synthetic salmon melanin concentrating hormone, MCH, stimulated melanin granule (melanosome) dispersion (MSR-IIke activity) within Integumental melanocytes of both the frog (R._y..pipiens) and the lizard (A__~.carolinensls). In the present experiments, it was shown that the fragment analogue, MCHI_I4, exhibited MSH-like activity about 10% that of the native peptide MCH In these two bloassays (data not shown). The C-terminal fragment analogues MCH5_I7 and MCHs_I4 lacked melanosome dispersing activity even at the highest concentrations employed (I0-5M). Furthermore, MCH5_I7 and MCHs_I4 did not inhibit melanosome dispersion stimulated by =-MSH (a-melanocyte stimulating hormone, a-melanotropln) even when used at a concentration (10-SM) I00,000 times greater than that of ~-MSH (10-10M). These results suggest that one or more amino acids within the Nterminal 1-4 sequence of MCH (Asp-Thr-Met-Arg) is required for MSH-llke activity. The low MSH-like activity of MCH 1 14 also suggests that in tetrapods soma structural component of the C-terminal sequence of MCH (Trp-Glu-Val) is important for melanosome dispersion activity. In the teleost species studied, it was determined by light microscopic observations that all three truncated analogues possessed MCH-llke activity. By this method it was determined that HCHs_I7 was almost equipotent to the native hormone, HCHI_I7 (Table I). MCHI_I4 was also quite active, being about i/I0 less active than MCHI_I7. The activity of the central cyclic analogue, MCR5_I4, was quite minimal (data not shown), being about 1/300th that of MCH. Therefore, the potency ranking of the MCR analogues on teleost melanophores was as follows: HCHI_I7 = MCH5_I7 > MCRI_I4 > MCH5_I4. This same potency ranking was obtained by a more objective method using skins of the teleost Synbranchus marmoratus (Figure 2). Two laboratories have now reported that salmon MCH does indeed stimulate melanosome dispersion within amphibian melanophores (3,12). MCH, like MSH, stimulates frog skin darkening, a response which involves both the centrifugal movement of melanosomes within melanophores and the perlnuclear aggregation of reflecting organelles (platelets) within irldophores, the functional unit of the chromatic response being referred to as a dermal chromatophore unit (13). In both the frog and the lizard, MCH action on skin darkening is truly MSH-like in action. Isolated cultured frog melanophores as well as reflecting cells (iridophores) also respond to MC~ in a similar manner as they do to a-MSH (12). It will be important to determine the structural features of the peptide that account for the two contrasting melanosome mobilizing activities of MCH. There are no readily apparent similarities between the structure of MCH (Fig. I) and a-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-ProVal-Nl{2). The present results suggest that the MSH-llke and the MCH-like
Vol. 40, No. 12, 1987
MCH: Structural Activity Studies
o Control • MCH v MCH547 • MCHI-14
80
0
1143
T
,~~
MCHs-,4
70 60 50 0
~
40 n~ ec..~
:50
V
20 I0 0 I
I
I
I
I
12
II
I0
9
8
-
Log [Concentration] (M)
FIGURE 2. D o s e - r e s p o n s e c u r v e s d e n o t i n g MCH-like a c t i v i t i e s MCHI_I4 , and HCH5_I4 compared to MC~I_I7 on melanophores of $ynbranchus marmoratus.
of the
MCH5_I7, teleost,
1144
MCH: Structural Activity Studies
Vol. 40, No. 12, 1987
TABLE I Minimal effective concentrations of melanin concentrating hormone and related fragment analogues on melanosome aggregation within melanophores of a number of teleost species. MCH Peptide Activities a Teleost MCHI-I7
MCHI-14
MCH5-17
Carassius auratus
10-9
10-8
10-8
Xiphophorus hellerl
10-9
NR
10-8
Poecilla retlculata
I0-II
NR
I0-II
Cichlasoma nisrofasclatum
10-8
NR
10-8
Barbus tetteya
10-9
10-7
10-7
Brachydanio framkey
i0-I0
10-8
10-8
Astronotus ocellatus
10-8
10-7
10-8
Pterophyllum scalare
10-9
10-7
10-8
Thayerla sanctae-marlae
10-9
10-8
10-9
Labeo erythrurus
10-8
10-7
10-8
Poecilia sphenope
10-10
10-7
10-8
Gyrinocheilus aymonieri
l0 -I0
10 -8
10 -8
aMinlmal molar response.
concentration
to
give
an
observable
melanin
concentrating
activities of MCH reside in different molecular regions of the hormone. The significant MSH-llke activity for MCH, the reduced but still significant activity of MCHI_I4 , but the lack of MSH-llke activity of MCHb_I7 and MCHb_I4 indicate that the N-termlnal region (MCHI_14) of MCH is required for ellcltlng MSH-llke activity. On the other hand, the full MCH-like activity of MCH and MCH5_I7 , but greatly reduced activity of MCHI_I4 and MCN5_I4 suggest that the C-termlnal region (MCHI5_I7) of MC~ is important but not essential for MCH-llke activity. Therefore it may be possible to Eurther enhance either of these two biological activities more selectively by the design of synthetic analogues of MCH. Salmon MCH activity on teleost melanophores can be reversed by ~-MSH (4). In teleosts, amphlhlans and reptiles, e-MSH clearly functions as a melanosome dispersing hormone. The actions of MCH in stimulating melanosome aggregation are at present restricted to teleosts. Left unresolved is whether the opposing actions of MCH and MSH in teleosts are mediated through the same or different melanophore receptors. Since the actions of the two hormones on teleost malanophores are opposite relative to melanosome movements, it might be expected that separate receptors are involved. In addition, the melano-
Vol. 40, No. 12, 1987
MCH: Structural Activity Studies
1145
some dispersing actlvitles of MSH in teleosts require Ca2+ (as in amphibians and reptiles), whereas the melanosome aggregating activity of MCH is independent of such a divalent cation requirement (14). MCH-like immunoreactivity has now been localized to teleost pituitaries (15) and rat brain (16). It might be expected, as demonstrated for most other peptide hormones, that MCH may regulate a number of diverse physiological functions depending on the particular neuronal or cellular types within which it may be localized. Salmon MCH has now been reported to inhibit corticotropin releasing hormone (CRH) induced corticotropin (ACTH) secretion from the rat pituitary gland in vitro (17). Therefore, it is possible that an MCH-related peptide may function in some vertebrate species as a CRIH (corticotropin release fnhibiting hormone)i It will be important, therefore, to determine whether such a factor (a corticostatin) mediates its action through CNS melanotropin receptors or through MCH receptors as may be present on teleost melanophores.
Acknowledgement Supported in part hy U.S. Public Health Service grant AM17420 and by National Science Foundation grants PCM-8112200 and PCM-8412084. M.A. Visconti is a Fellow of Fundaczo de Amparo A Pesquisa do Estado de Sgo Paulo, Brazil, Grant 84/1263-O.
References TSUBOKAWA, M. KISHIDA, and B.I. BAKER, KAWAZOE, M. H. KAWAUCHI, I. Nature 305, 321-323 (1983). 2. M. ENAMI, Science 121, 36-37 (1955). SHERBROOKE, A.M.L. CASTRUCCI and M.E. 3. ~~,EYw1~~~c."*,";4,H~~LlIW;3C<1984). 4. B.C. GILKES, V.J. HRUBY, A.M.L. CASTRUCCI, W.C. SHERBROOKE and M.E. 613-619 (1984). HADLEY, Biophys. Res. Commun. Biochem. 122, 5. E. KAISER, R.L. COLESCOTT, C.C. BOSSINGER and P.I. COOK, Anal. Biochem. 2, 594-598 (1970). GISIN, Helv. 1476-1482 (1973). Chim. Acta, 6. B.F. 56, 248-249 (1974). GISIN, Anal. Chim. Acta, 7. B.F. 58, 26-34 (1982). Int. J. Peptide Protein Res., 20, 8. G.R. MATSUEDA, 9. B.C. WILKES, T.K. SAWYER, V.J. HRUBY and M.E. HADLEY, Int. J. Pept. Protein Res. 2, 313-324 (1983). 553-560 10. K. SHIZUME, A.B. LERNER and T.B. FITZPATRICK, Endocrinology, 2, (1954). 11. A.H.L. CASTRUCCI, M.E. HADLEY and V.J. HRUBY, Gen. Comp. Endocr1nol. 55, 104-111 (1984). 486-490 12. H. IDE, I. KAWAZOE and H. KAWAUCHI, Gen. Comp. Endocrinol. 2, (1985). 67-79 (1968). 13. J.T. BAGNARA, M.E. HADLEY and J.D. TAYLOR, Cell Biol. 2, 14. N. OSHIMA, H. KASUKAWA, R. FUJII, B.C. WILKES, V.J. HRUBY, A.M.L. CASTRUCCI Zool. 235, 175-180 (1985). and M.E. HADLEY, J. Exp. Y. NAKAI, H. KAWAUCHI and Y. HAYASHI, Cell Tissue Res. 242, 15. 4"i-4"sAI;pp6s5). 16. N. ZAMIR, G. SKOFITSCH, M.J. BANNON and D.M. JACOBOWITZ, Proc. Natl. Acad. Sci., 1J.S.A.g, 1528-1531 (1986). 17. B.I. BAKER, D.J. BIRD and J.C. BUCKINGHAM, J. Endocrinol. 106, R5-R8 (1985). 1.
Pergamon Journals
DIFFERENTIAL STRUCTURAL REQUIREMENTS FOR THE MSH AND MCH ACTIVITIES OF MELANIN CONCENTRATING HORMONE Mac E. ItsdleyI, Christian Zechel 2, Brian C. Wilkes 2, Ana M. de L. Castruccl 3 Maria A. Viscontl 3, Manuel Pozo-Alonso I, and V i c t o r J. Bruby 2# Departments of Anatomy I and Chemistry 2 University o f Arizona, Tucson, Arizona 85721 U.S.A. and Ikmpartmento de F i s l o l o g i a 3, U n i v e r s i d a d e de Sao Paulo CPII176, Sao Paulo, Srasll (Received in final form December 18, 1986)
Summary H-Asp-Thr-Me t-At g - ~ a - M e t-Val-Oly-- Ar g-Va l-Tyr -At g-Pro--~s-TrpClu-Val-OR, melanin concentrating hormone (MCH), exhibits both melanin granule concentrating and dispersing (MSH-like) activities. Fragment analogues of MCH ~ r e synthesized as described herein and the melanotropic activities of the peptides ware determined. In the frog (Rana plplens) and lizard (Anolis carollnensls) skin blnassays, the 5-i~-a'~-5-14 fragments o - - ~ were inactive (at concentrations of I0-5M or less), whereas the 1-14 sequence exhibited minimal (about I0%) MSH-llke activity compared to MCH, which, as reported previously, was about 600 times less active than a-4qSH. In the teleost (fish) skin bloassay, the MCTI5_17 analogue was equlpotent to MCH, whereas the 1-14 analogue was 10-30 times and the cycllc N- and C- termlnal truncated analogue, MCHs_I4 , was about 300 times less active than MCH. These results suggest that the N-termlnal sequence is particularly critical to MSH-like activity in the tetrapod species studied, whereas other structural regions of MCH m particularly in the C-terminal, are more related to MCH activity in teleosts. A putative melanin ~ranule (melanosome) concentrating (aggregating) hormone, HCH (Figure I), hls been isolated from the salmon pituitary gland and its primary structure was elucidated (1,2). This cyclic heptadecapeptlde, !
l
H-Asp-Thr-Met-Arg-Cys-Mat-Val-Oly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH, was synthesized and its biological activity has been determined (3,4). The peptide not only causes melanosome aggregation within teleost (fish) melanophores, but also induces melanosome dispersion within tetrapod (frog and lizard) melanophores (3). In the present report we document the melauotropic actions of N-and C-termlnally truncated analogues of MCH on teleost and tetrapod melanophores, and demonstrate that the N-and C-termlnal residues are essential, respectively, for the MSH and MCH activities of the hormone.
# To whom reprint requests should be addressed at the Department of Chemistry, University of Arizona, Tucson, Arizona 85721 U.S.A. 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1140
MCH: Structural Activity Studies
1
3
5
7
9
Vol. 40, No. 12, 1987
11
15
13
I
17
!
MCHI-17
Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val
MCHI-14
Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys
!
l
!
!
MCHs-17
Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val
MCHs-14
C~s-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-C~s FIGURE I.
Primary structure of MCH analogues.
Materials and Methods Synthesis: MCH was prepared as previously reported ( 4 ) . The following three fragment analogues of MC~ were prepared: the N-term/hal tetradecapeptlde sequence, MCHI_I4 , the C-termlnal tridecapeptide MCHb_I7 sequence, and the Nand C-terminal truncated analogue MCH5_I4 (Figure I). Melting points were determined on a Thomas-Hoover melting point apparatus. Thln-layer chromatography (TLC) was performed on Silufol plates (Kavaller, Czechoslovakia) using the following solvent systems: A) l-butanol/acetlc acid/H20 (4:1:5, upper phase only); B) 1-butanol/acetlc acld/pyrldine/~20 (15:3:10:12); C) l-butanol/pyridine/acetic acld/R20 (5:5:1:4), Detection was by iodine vapor. High pressure liquid chromatography (RPLC) was performed on a Perkln-Elmer instrument with detection at 214 nm using a Vydac C18 column (25 x 1.0 cm). Aqueous trlfluoroacetlc acid (0.1%) was the buffer wlth acetonltrile as the organic modifier. Fast atom bombardment mass spectra (FAB/MS) were aqulred on a Varian 311A spectrometer equipped with an Ion Tech Ltd. source, wlth Xenon as the bombarding gas. NU-Boc protected amino acids and amino acid derivatives were purchased from Vega Biotechnologles (Tucson, AZ), Peninsula Laboratories (San Carlos, CA), Bachem (Torrence, CA), or were prepared using published procedures. Before use, all amino acid derivatives were tested for homogeneity by TLC in solvent systems A, B, and C, by melting point determinations, and by the nlnhydrln test (5). Solvents used for chromatography were redlstilled prior to use.
The MCH analogues were prepared using Merrifleld resins substituted with the C-termlnal amino acid by standard methods ( 6 ) . Boc-Val-Merrifield resin had an amino acid substitution of 0.47 mmol/gram resin and Boc-Cys(PMB)Merrifield resin had an amino acid substitution of 0.77 mmol/gram resin as determined by the method of Gisln ( 7 ) . NU-Boc amino acid derivatives were coupled to the resin using a three-fold excess of each amino acid derivative, a 2.4 fold excess of dicyclohexylcarbodiimide (DCC) and a 2.4 fold excess of l-hydroxybenzotriazole (HOBT). Removal of the NU-Boc protecting group was accomplished with 45% trlfluoroacetlc acid (TFA) in dichloromethane (CH2CI 2) containing 2% anlsole. A cycle for the incorporation of each amino acid derivative consisted of the following: I) washing the resin with three 20-ml portions of CN2CI 2, one min per wash; 2) washing with two 30-ml portions of Et0H, 2 mln per wash; 3) washing with two 20-ml portions of CH2C12, one mln per wash; 4) cleavage of the NU-Boc protecting group with 45% TFA in C~2C12, one wash for 2 mln, one
Vol. 40, No. 12, 1987
MCH: Structural Activity Studies
1141
wash for 20 min; 5) washing with three 20-ml portions of CH2C12, one min per wash; 6) neutralization of the resin with two 30-ml protlons of i0% dlisopropylethylamlne in CH2C12, 2 mln per wash; 7) washing with two 20-ml portions of CH2CI 2, one min per wash; 8) addition of the next Na-Boc amino acid derivative along with DCC and HOBT in 30 ml N,N-dimethylformamide (DMF). Coupling of each amino acid derivative was complete In 30 to 60 min as monitored through the ninhydrln test. After coupling of the final amino acid derivative, the resulting peptide resin was dried in vacuo. Synthesis of MC_~_5_17: Starting with 2.10 g Boc-Val resin (I.0 mmol total), the f o l l o w l n g - ~ - B o c amino acid derivatives were coupled (in order of their coupling): Glu(Bzl); Trp(For); Cys(PMB); Pro; Arg(Tos); Tyr(2,6-Cl2-Bzl); Val; Arg(Tos); Gly; Val; Met and Cys(PMB). The resulting peptide resin was dried and weighed (5.35 g). To one fourth of this resin (1.33 g, 0.25 mmol), an HF cleavage of the peptide from the resin was accomplished using the procedure of Metsueda (8). The mixture was washed with 3 x 30-ml of ethyl acld and the product extracted with 3 x 30-ml 30% acetic acid and lyophillzed. The crude [Cys(R) 5, Cys(N)I4]HCHI_I4 was diluted in 1 liter deaerated H20 and cycllzed as before giving 250 mg of crude product. A portion of this product (24 mg) was purified by CMC chromatography as before, and preparative HPLC, giving 2.0 mg of the title compound: NPLC - k" (18% acetonltrile, 82% - 0.1% aqueous trlfluoroacetlc acld) ffi 2.1; FAB/MS - 1684 (Cald. 1683.74). Synthesls of HCHI_14: To 1.00 g of Boc-Cys(PHB) resin (0.5 mmol), the following NU-Boc amino acid derivatives were coupled (in order of their coupling): Pro; Arg(Tos); Tyr(2,6-Cl 2 Bz;); Val; Arg(Tos); Gly; Val; Met; Cys(PMB); Arg(Tos); Met; Thr(Bzl); and Asp(Bzl) giving 1.50 g of peptlde resin. Half of the resulting peptide resin (0.75 g) was subjected to HF cleavage as before. The mixture was washed with 3 x 30-ml ethyl acetate and the product extracted with 3 x 30-ml 30% acetic acld and lyophillzed. The crude [Cys(R) 5, Cys(H)I4]MCHI_I4 was diluted in 1 liter deaerated H20 and cycllzed as before giving 250 mg of crude product. A portion of thls product (24 mg) was purified by CMC chromatography as before, and preparative tIPLC, giving 2.0 mg of the title compound: HPLC - ~ (18% acetonitile, 82% - 0.1% aqueous trlfluoroacetlc acld) = 2.1; FAB/MS - 1684 (Cald. 1683.74). Synthesis of MCHs_I4: Starting with 1.00 g of Boc-Cys(PMB)-resin (0.5 mmol), the fol~'owlng N~-Boc amino acld derivatives were coupled to the growing peptlde chain (in order of their coupling): Pro; Arg (Tos); Tyr(2,6-Cl2Bzl) ; Val; Arg (Tos); Gly; Val; Met; and Cys(PMB) give 1.40 g of peptide resin. The resultant peptlde resin was subjected to HF cleavage as before. The HF was removed in vacuo, the resulting mixture was washed with 3 x 30-ml of ethyl acetate and the ~roduct extract with 3 x 35-mi of 30% acetic acid. The crude [Cys(H) 5, Cys(H)I4]MCH5_I4 was diluted in 2 i of deaerated H20 , the pH adjusted to 8.5, and the peptide cyclized as before, giving the crude product. The peptlde was purified by gel filtration on Sephadex G-25 and CMC ion exchange chromatography, followed by preparative HPLC: HPLC - k" (15% acetonitrile, 85% - 0.1% aqueous trlfluoroacetic acid) ffi 4.05; FAB/MS - 1181 (Calcd. 1180.5). Bioassays: The bioassays for MSH-IIke activity of MCH and related analogues utilized skins from the frog (Rana pipiens) and the lizard (Anolis carolinensis). Skins were mounted on metal rings and held in place by an outer plastic ring as described originally for the frog (i0) and lizard skin bio-assays and as detailed in a recent report (II). Changes in reflectance from the outer (epidermal) surface of the skins as induced by the peptides were monitored photometrically. The melanin concentrating activities of MCH, MCHI_14 , and MCH5_I7 were determined on isolated scales from a number of teleost species (Table I). The minimal effective concentrations of the pep-
1142
MCH: Structural Activity Studies
Vol. 40, No. 12, 1987
tides to elicit melanin granule aggregation within melanophores as subjectively under a dissecting microscope are reported (Table I).
observed
The teleost fish, Synbranchus marmoratus, an eel obtained from the Pantanal (Big Swamp) of Brazil were also used. Skins were removed and prepared as originally described for the frog (10) and lizard (II) skin bioassays. A unique feature of this fish skin bloassay is that, unlike the frog and lizard skin bloassays, the "resting" (unstimulated) state of the melanophores Is characterized by dispersed melanosomes. Therefore, the assay is particularly appropriate for the study of melanosome aggregating agents such as MCH, and thus was used for determination of quantitative dose response curves for all peptldes (Figure 2).
Results and Discussion As demonstrated in our earlier publications (3,4), synthetic salmon melanin concentrating hormone, MCH, stimulated melanin granule (melanosome) dispersion (MSR-IIke activity) within Integumental melanocytes of both the frog (R._y..pipiens) and the lizard (A__~.carolinensls). In the present experiments, it was shown that the fragment analogue, MCHI_I4, exhibited MSH-like activity about 10% that of the native peptide MCH In these two bloassays (data not shown). The C-terminal fragment analogues MCH5_I7 and MCHs_I4 lacked melanosome dispersing activity even at the highest concentrations employed (I0-5M). Furthermore, MCH5_I7 and MCHs_I4 did not inhibit melanosome dispersion stimulated by =-MSH (a-melanocyte stimulating hormone, a-melanotropln) even when used at a concentration (10-SM) I00,000 times greater than that of ~-MSH (10-10M). These results suggest that one or more amino acids within the Nterminal 1-4 sequence of MCH (Asp-Thr-Met-Arg) is required for MSH-llke activity. The low MSH-like activity of MCH 1 14 also suggests that in tetrapods soma structural component of the C-terminal sequence of MCH (Trp-Glu-Val) is important for melanosome dispersion activity. In the teleost species studied, it was determined by light microscopic observations that all three truncated analogues possessed MCH-llke activity. By this method it was determined that HCHs_I7 was almost equipotent to the native hormone, HCHI_I7 (Table I). MCHI_I4 was also quite active, being about i/I0 less active than MCHI_I7. The activity of the central cyclic analogue, MCR5_I4, was quite minimal (data not shown), being about 1/300th that of MCH. Therefore, the potency ranking of the MCR analogues on teleost melanophores was as follows: HCHI_I7 = MCH5_I7 > MCRI_I4 > MCH5_I4. This same potency ranking was obtained by a more objective method using skins of the teleost Synbranchus marmoratus (Figure 2). Two laboratories have now reported that salmon MCH does indeed stimulate melanosome dispersion within amphibian melanophores (3,12). MCH, like MSH, stimulates frog skin darkening, a response which involves both the centrifugal movement of melanosomes within melanophores and the perlnuclear aggregation of reflecting organelles (platelets) within irldophores, the functional unit of the chromatic response being referred to as a dermal chromatophore unit (13). In both the frog and the lizard, MCH action on skin darkening is truly MSH-like in action. Isolated cultured frog melanophores as well as reflecting cells (iridophores) also respond to MC~ in a similar manner as they do to a-MSH (12). It will be important to determine the structural features of the peptide that account for the two contrasting melanosome mobilizing activities of MCH. There are no readily apparent similarities between the structure of MCH (Fig. I) and a-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-ProVal-Nl{2). The present results suggest that the MSH-llke and the MCH-like
Vol. 40, No. 12, 1987
MCH: Structural Activity Studies
o Control • MCH v MCH547 • MCHI-14
80
0
1143
T
,~~
MCHs-,4
70 60 50 0
~
40 n~ ec..~
:50
V
20 I0 0 I
I
I
I
I
12
II
I0
9
8
-
Log [Concentration] (M)
FIGURE 2. D o s e - r e s p o n s e c u r v e s d e n o t i n g MCH-like a c t i v i t i e s MCHI_I4 , and HCH5_I4 compared to MC~I_I7 on melanophores of $ynbranchus marmoratus.
of the
MCH5_I7, teleost,
1144
MCH: Structural Activity Studies
Vol. 40, No. 12, 1987
TABLE I Minimal effective concentrations of melanin concentrating hormone and related fragment analogues on melanosome aggregation within melanophores of a number of teleost species. MCH Peptide Activities a Teleost MCHI-I7
MCHI-14
MCH5-17
Carassius auratus
10-9
10-8
10-8
Xiphophorus hellerl
10-9
NR
10-8
Poecilla retlculata
I0-II
NR
I0-II
Cichlasoma nisrofasclatum
10-8
NR
10-8
Barbus tetteya
10-9
10-7
10-7
Brachydanio framkey
i0-I0
10-8
10-8
Astronotus ocellatus
10-8
10-7
10-8
Pterophyllum scalare
10-9
10-7
10-8
Thayerla sanctae-marlae
10-9
10-8
10-9
Labeo erythrurus
10-8
10-7
10-8
Poecilia sphenope
10-10
10-7
10-8
Gyrinocheilus aymonieri
l0 -I0
10 -8
10 -8
aMinlmal molar response.
concentration
to
give
an
observable
melanin
concentrating
activities of MCH reside in different molecular regions of the hormone. The significant MSH-llke activity for MCH, the reduced but still significant activity of MCHI_I4 , but the lack of MSH-llke activity of MCHb_I7 and MCHb_I4 indicate that the N-termlnal region (MCHI_14) of MCH is required for ellcltlng MSH-llke activity. On the other hand, the full MCH-like activity of MCH and MCH5_I7 , but greatly reduced activity of MCHI_I4 and MCN5_I4 suggest that the C-termlnal region (MCHI5_I7) of MC~ is important but not essential for MCH-llke activity. Therefore it may be possible to Eurther enhance either of these two biological activities more selectively by the design of synthetic analogues of MCH. Salmon MCH activity on teleost melanophores can be reversed by ~-MSH (4). In teleosts, amphlhlans and reptiles, e-MSH clearly functions as a melanosome dispersing hormone. The actions of MCH in stimulating melanosome aggregation are at present restricted to teleosts. Left unresolved is whether the opposing actions of MCH and MSH in teleosts are mediated through the same or different melanophore receptors. Since the actions of the two hormones on teleost malanophores are opposite relative to melanosome movements, it might be expected that separate receptors are involved. In addition, the melano-
Vol. 40, No. 12, 1987
MCH: Structural Activity Studies
1145
some dispersing actlvitles of MSH in teleosts require Ca2+ (as in amphibians and reptiles), whereas the melanosome aggregating activity of MCH is independent of such a divalent cation requirement (14). MCH-like immunoreactivity has now been localized to teleost pituitaries (15) and rat brain (16). It might be expected, as demonstrated for most other peptide hormones, that MCH may regulate a number of diverse physiological functions depending on the particular neuronal or cellular types within which it may be localized. Salmon MCH has now been reported to inhibit corticotropin releasing hormone (CRH) induced corticotropin (ACTH) secretion from the rat pituitary gland in vitro (17). Therefore, it is possible that an MCH-related peptide may function in some vertebrate species as a CRIH (corticotropin release fnhibiting hormone)i It will be important, therefore, to determine whether such a factor (a corticostatin) mediates its action through CNS melanotropin receptors or through MCH receptors as may be present on teleost melanophores.
Acknowledgement Supported in part hy U.S. Public Health Service grant AM17420 and by National Science Foundation grants PCM-8112200 and PCM-8412084. M.A. Visconti is a Fellow of Fundaczo de Amparo A Pesquisa do Estado de Sgo Paulo, Brazil, Grant 84/1263-O.
References TSUBOKAWA, M. KISHIDA, and B.I. BAKER, KAWAZOE, M. H. KAWAUCHI, I. Nature 305, 321-323 (1983). 2. M. ENAMI, Science 121, 36-37 (1955). SHERBROOKE, A.M.L. CASTRUCCI and M.E. 3. ~~,EYw1~~~c."*,";4,H~~LlIW;3C<1984). 4. B.C. GILKES, V.J. HRUBY, A.M.L. CASTRUCCI, W.C. SHERBROOKE and M.E. 613-619 (1984). HADLEY, Biophys. Res. Commun. Biochem. 122, 5. E. KAISER, R.L. COLESCOTT, C.C. BOSSINGER and P.I. COOK, Anal. Biochem. 2, 594-598 (1970). GISIN, Helv. 1476-1482 (1973). Chim. Acta, 6. B.F. 56, 248-249 (1974). GISIN, Anal. Chim. Acta, 7. B.F. 58, 26-34 (1982). Int. J. Peptide Protein Res., 20, 8. G.R. MATSUEDA, 9. B.C. WILKES, T.K. SAWYER, V.J. HRUBY and M.E. HADLEY, Int. J. Pept. Protein Res. 2, 313-324 (1983). 553-560 10. K. SHIZUME, A.B. LERNER and T.B. FITZPATRICK, Endocrinology, 2, (1954). 11. A.H.L. CASTRUCCI, M.E. HADLEY and V.J. HRUBY, Gen. Comp. Endocr1nol. 55, 104-111 (1984). 486-490 12. H. IDE, I. KAWAZOE and H. KAWAUCHI, Gen. Comp. Endocrinol. 2, (1985). 67-79 (1968). 13. J.T. BAGNARA, M.E. HADLEY and J.D. TAYLOR, Cell Biol. 2, 14. N. OSHIMA, H. KASUKAWA, R. FUJII, B.C. WILKES, V.J. HRUBY, A.M.L. CASTRUCCI Zool. 235, 175-180 (1985). and M.E. HADLEY, J. Exp. Y. NAKAI, H. KAWAUCHI and Y. HAYASHI, Cell Tissue Res. 242, 15. 4"i-4"sAI;pp6s5). 16. N. ZAMIR, G. SKOFITSCH, M.J. BANNON and D.M. JACOBOWITZ, Proc. Natl. Acad. Sci., 1J.S.A.g, 1528-1531 (1986). 17. B.I. BAKER, D.J. BIRD and J.C. BUCKINGHAM, J. Endocrinol. 106, R5-R8 (1985). 1.