Synthesis, monitoring and structure-function studies on some ...

PEPTIDE A N D PROTEIN GROUP approximately equal batches by treating the peptide resin with thioanisolc ( 1 ml; S%), v/v). ethylmethylsulphide ( 1 ml; S ' L . v/v) and anisole ( 1 ml; S%, v/v) for 20 min followed by the addition of 95% ( v / v ) aqueous TFA (20 ml) at room temperature for 3 h. , After the TFA was removed iri V ~ C I I OP-mercaptoethanol (2%, v/v) in diethyl ether (SO ml) was slowly added to the gently stirred residue t o remove the organic scavengers. The diethyl ether was removed by decantation and the procedure was repeated twice. The white solid obtained was immediately dissolved in N,-saturated urea buffer [SO mMNH,OAc/8 M-urea/lO mwdithiothreitol (DTT), pH 4.51 and left t o equilibrate at 4°C overnight with gentle stirring, and at room temperature for a few hours before it was applied t o a high resolution Sephadex G-SO column (2.6 cm x 136 cm), previously equilibrated with urea buffer. The fractions obtained were sequentially dialysed against decreasing urea concentration: N,-saturated SO mMNH,OAc/4 M urea/lO mM-DTT pH 4.5 ( 2 I, 24 h); N,snturated 50 m ~ - N H , o A c / 2M urea/lO mM-DTT pH 4.5 ( 2 1, 24 h); N,-saturated SO mM-NH,OAc/lO mM-DTT pH 4.5 ( 2 I. 24 h); N,-saturated 5 0 mM-NH,OAc pH 4.5 ( 4 x 2 I. 24 h). Thcse fractions were assayed by isoelcctric focusing (i.c.f.) o n Phast gel pl range 3-9 and reverse phase ( r p )h.p.1.c. The desired fractions wcre further purified by cation-exchange chromatography on a CM-Sepharose CL6B (Pharmacia) column ( 1.6 cm X 40 cm) eluting with SO mM-NH,OAc pH 4.5 ( I bed volume) followed by sequential pH and salt gradients in the order: (1) SO mM-NH,OAc pH 4.5-50 mMNH,OAc pH 5.5; ( i i ) 5 0 mM-NH,OAc pH 5.5-0.3 MNH,OAc pH S.S. The desired material obtained (assayed by Phast i.e.f. and r.p. h.p.1.c.) was purified further by anion-exchange chromatography o n DEAE-Sepharose (Pharmacia) column ( 1.6 cm x 40 cm) eluting with SO mM-NH,HCO, pH 9.3 ( I bed volume) followed by salt gradient elution up to 0.3 MNH,HCO, pH Y.3. Two major peaks werc obtained which assayed similarly by Phast i.e.f. and r.p. h.p.1.c. These peaks. designated A and H. were judged t o be > Y 0 " h and > 80% purc. respectively. Semi-preparative r.p. h.p.1.c. purification gave synthctic ubiquitin A (40mg) and ubiquitin B ( 5 0 mg) in 4.2% overall

1323 yield and in >9SnL1 purity. The synthetic material s o obtained exhibited identical h.p.1.c. and i.e.f. behaviour in comparison with authentic bovine ubiquitin. Amino acid analysis figures agree well with thcorctical values. Elcctrospray mass analysis o f this material showed the expected molecular ion 8564.8. The primary sequence of the synthetic ubiquitin was confirmed by automated Edman degradation. Tryptic digestion of oxidized ubiquitins A and B and bovine ubiquitin gave a very interesting picture which led u s t o speculate that peak B corresponds to a material which may represent an intermediate state of folding. The current hypothe5is is that protein folding takes place in a few discreet steps 17, 81. Isolation of materials at intcrmediate stages in the process opens the way to kinetic and thermodynamic investigation of the development o f tertiary structure. The tryptic digest was found to be most variable in the C-terminal region and one could spcculatc that the fitting o f this region into the final tertiary structure occurs at a late stage. Clearly the ncxt step is direct physical investigation of the materials, which we are carrying out by spectroscopic and electrophoretic methods. We thank the S.E.K.C.. Applied l3iosystems Inc. and Merck. Sharp & Dohmc f o r financial support. We are grateful t o B. Whigham and K. Shaw for their invaluable technical support. Brian Green ( V C Instruments) for the mass analysis. Dr L. A . FothergillCilmore and Ms L. Kerr o f the University o f Edinburgh sequencing unit, WELMET. lor the sequencing o f these samples. Green. J. & Kamage. K.( 1987) Tc~irctheclror~ I.c,tr. 28. 2287 Merrifield. K. B. ( 1963)J . Am. C'hcJtn..Yoc,. 85, 2 147 Merrifield. K.13. ( 1985) S c i e n w 232. 341 -347 Hriand. J.-P., Muller. S.. Raboy. 13. & Van Dorsselaer. A . ( I 989,) /",pi. Hes. 2. 38 1-388 5. Armarego, W. L. F., Perrin, D. I). & Perrin, D. K.( 1980) I'uriJicw riot? oj'luhorcrio~y('lic~rniccrh. 2nd cdn.. Pergamon Press. Oxford 6 . Green, J., Ogunjobi, 0. M. & Kamage. K. ( 1 9 8 9 ) Terrrrheclrorr 1,rrr. 3 0 . 2 149-2 I 5 2 7. Anfinsen, C . 13. & Schcraga. H. A . ( 1075) A d i . . I'roi. (‘hem. 29, 205-300 ( I98 I ) Ad,'. I'tW. C'hc,tt/. 34. 167-339

I. 2. 3. 4.

Received 1 May I990

Synthesis, monitoring and structure-function studies on some neurokinin A analogues DAVID J. S. GUTHRIE.* AHMED A. ABU SHANAB,* JAMES M. ALLEN.1- G. BRENT IRVINE.* NEIL V. McFERRAN* and BRIAN WALKER* * Ilitisiori of' Hiochernistty. The School of' HioloD arid Hiochernistry, The Qireeri j. Universiy of' Relfast, Belfast R I ' V 7HL. N. Irelrnd, U.K . and t Riornerlical Sciences Kiwrrrch C'entre. Uriiver.sityof' Ulster, Ncwtowriuhhey H7'37 OQB, N . Irchrid, U.K . Neurokinin A (NKA) is one o f three tachykinins found in mammals, the others being substance P (SP)and neurokinin B (NKB) (Table I ) . Thcse peptides arc widely distributed Abbreviations t-hutoxycarbonyl; substance P; Aib. carhoxylic acid; receptor.

Vol. 18

used: Fmoc. fluorenylmethoxycarbonyl; Roc. NKA. neurokinin A; NKH. neurokinin 13; SP. a-aminobutyric acid; Ach. I -aminocyclohexanef.a.h.. fast atom bombardment; NK2, NKA-

throughout the nervous system and show a variety of activities [ 1 I. Three types of receptor with specificitics matching the thrcc tachykinins ( N K I for SP, NK2 for NKA and NK3 for NKB) have been characterized. The relationships between these peptides and their receptors are very complex. Receptor-stimulating activity resides in the very similar C-terminal regions and a degree o f cross-reactivity has been postulated [ 1 I. Some cells express mainly one receptor type, but many cells express more than one. Furthermore, SP and NKA are synthesized together and arc probably co-released from peripheral neurons. We wanted t o investigate the possible rolc o f tachykinins in conditions such as asthma, where it has been suggested that abnormal release of SP or NKA could be involved in bronchoconstriction [ 2, 31. More recently, it has been shown that in the airways NKA is more potent in contracting smooth muscle, while SP is a more potent stimulant o f mucous hypersecretion. vasodilation and resulting oedema

BIOCHEMICAL SOCIETY TRANSACTIONS

1324

Table 1. Tachykinins and analogues 0

1

2

3

4

5

6

Pro His

Gln

Gln

Asp

Phe Ser

Phe Phe Phe Phe Phe

Pro

Lys

Asp

Met

His

Lys

Peptide I Peptide 2 Peptide 3

His

Lys Lys

Thr Asp Asp Ala Thr Asp Ala T h r Asp Ala

Peptide 4

His

Lys

Thr

Substance P Neurokinin B

Neurokinin A

Arg

Asp

[3,4]. Investigations of the roles of SP and NKA were hampered by the lack of good antagonists of NKA and we set out to remedy this deficiency. In attempting to design and synthesize antagonists of NKA we were guided by the many previous studies on SP [S] and by the ideas of Schwyzer 161. The latter had analysed the activities of several families of peptides in terms of a 'message domain' and an 'address domain'. The message domain of the tachykinins obviously lies in the similar C-terminal sequences, while he assigned the address domain, which would guide an individual class member to its specific receptor, to the N-terminus. Thus, the full size tachykinin should have maximum specificity. Various authors, e.g. Cotrait & Hospital [7], had concluded that the C-terminal region of active analogues of SP was involved in some type of bend or partial helix. We chose to synthesize analogues of NKA containing aaminoisobutyric acid (Aib) and l-aminocyclohexanecarboxylic acid (Ach)at positions near the C-terminus, since these residues favour folded conformations [8, 91 (Table 1). In addition, we replaced the C-terminal methionine with leucine or norleucine to remove complications owing to oxidation of the thioether function of methionine. This is a conservative change that has often been used in SP analogues IS]. We also decided to replace Ser-S with Ala because the Asp-Ser combination can give problems in solid-phase peptide synthesis with strong acid cleavage [lo]. This was assumed to be a neutral replacement, later justified by the results of Rovero et al., who showed that (Ala5]NKA is slightly more potent than NKA itself [ 111. Some shortened analogues, lacking N-terminal residues, were also synthesized t o test the importance of the full NKA sequence. Synthesis

The NKA analogues, peptides 1-4, were initially synthesized on methylbenzhydrylamine resin using N-Boc protected amino acids and di-isopropylcarbodi-imidewith final deprotection and cleavage by the 'low-high' H F method of Tam et a/. [ 121. The extent of reaction was monitored using the Kaiser ninhydrin test [ 13). Unfortunately, Aib and Ach d o not give strong colours with this test and, in the case of coupling to these amino acids, the strategy of a double coupling followed by capping with acetic anhydride was adopted. The peptides were analysed and purified by h.p.1.c. on C- 18 Bondapak columns. After purification, the peptides gave satisfactory amino acids analyses and showed the expected molecular ions in fast-atom bombardment (f.a.b.) mass spectrometry. Subsequently, peptides 3 and 4 have been resynthesized using conductance to monitor the extent of reaction. An Applied Biosystems 4 3 1A automatic peptide synthesizer was modified so that during synthesis, supernatant was periodically removed from the reaction vessel. passed to a conductance cell and then returned to the reaction vessel to continue reacting. The syntheses were carried out on a Rink

Ala

7

8

Phe

Gly

Leu

MetNH,

Gly Gly

Leu Leu

MetNH,

Aib

Leu

Aib Aib Gly

Leu

Val

Val

Phe

Val Val Val

Phe

Val

9

I0

MetNH?

Leu

LeuNH, LeuNH: LeuNH?

Ach

NleNH,

amide resin using N-methylpyrrolidone containing 0.5% (v/v) di-isopropylethylamine as solvent. Peptide 3 was synthesized using Fmoc amino acid pentafluorophenyl esters (OPfp) except for Aib which was coupled as the 1-hydroxybenzotriazole ester (HOBt) formed in the instrument's prereaction vessel. Peptide 4 was synthesized entirely from Fmoc 1-hydroxybenzotriazole esters formed similarly. Fig. 1 shows a conductance trace for the addition of three residues in the synthesis of peptide 3. The points A , R and C gave the conductance in the reaction mixture immediately before contact with the resin so that the changes A-A', B - B' - B" and C - C' represent the increase in conductance on coupling. Coupling of FmocAibOBt and FmocPheOPfp esters show a rapid increase in conductance which was essentially complete by the time that the first conductance measurement was made. Reaction was allowed to proceed for 40 min, but no significant change in conductance occurred. However, the coupling of FmocValOPfp gave a quite different profile. Little increase in Conductance was apparent after 1 h and the reaction mixture was allowed to remain in contact with the resin overnight without further sampling. When readings were restarted, the conductance of the solution in the reaction vessel had risen to R". Two additional coupling reactions were carried out at this position using slightly different sets of conditions: FmocValOPfp + 0.1 equiv. HOBt for 1 h and then FmocValOPfp + 10% (v/v)-butanol (to disrupt any clumping of peptidc chains) for 2 h. At this stage, the total increase in conductance for this step was approximately equal to that for previous steps and reaction was moved on to the next residue (Phe).Subsequent residues were coupled without any problems. The small spike A" is due to the carbamic acid salt formation during Fmoc deprotection by piperidine. Reverse phase h.p.1.c. indicated that the crude peptide consisted of one main component with one minor contaminant, unlike the previous synthesis which had produced significant levels of several h.p.1.c.-resolvable components. The purified peptide was identical to that obtained previously, as judged by amino acid analysis, f.a.h. mass spectrometry and n.m.r. spectroscopy. In the synthesis of peptide 4, all couplings werc complete within 1 h, as indicated by the conductance trace, but the coupling of FmocGly to the Ach was noticeably slower than the other couplings. Biological testing The peptides were tested for their ability t o contract guinea-pig tracheal rings and to antagonize contractions produced by SP, NKA and NKB. The experimental method was as described earlier [ 161. Carbachol at lo-' M was used to contract the trachea, which was then allowed to relax. Subsequent contractions due to peptides were expressed as a percentage of the carbachol-induced contraction. Peptides were examained at several concentrations from l o - " to lo-" M.

1325

PEPTIDE AND PROTEIN GROUP

Aib (OBt ester)

I

Val

(OPfp ester)

& then FrnocValOPFP. 10% t-BuOH 2 h

I

Phe

e 50

300

400 overnight

60

Time (min)

Fig. 1. Conductimetric monitoring of the progress of coupling reactions for addirioti of residues, 8, 7arid 6 ofpeptide 3 (Ilk-Lys-Thr-Asp-Ala-Phe-Val-Aih-Leu-Leu-NH,) Pentafluorophenyl esters were used in 2-fold molar excess, 1-hydroxybenzotriazole ester in 4-fold molar excess.

Kesltlls

NKA. SP and NKB all caused contractions of guinea-pig tracheal rings over the concentration range tested. NKA was most potent, and with it contractions reached a maximum within the concentration range. Peptide 3 ([Ah', A h x , Lcu"']NKA) was without agonist activity at all concentrations used ( I W Y - l W hM). In the presence of peptide 3, contractions due to NKA were reduced, but the antagonism was not o f a simple competitive nature, since the maximum contraction achieved was reduced in a dose-dependent manner. Peptide 3 did not produce any reduction in the contractions produced by SP, NKB or carbachol. Peptide 2 ([Ah5, Aibx, Leu1"]NKA(2-10)}showed no ability to contract the trachea and also antagonized contractions due to NKA, but, in this case, the antagonism appeared to be competitive in nature as the same maximum contraction was eventually achieved at all antagonist concentrations. Analysis by Schild plot yielded a straight line, but with slope - 0.6 rather than the theoretical value of - 1 [ 141. A similar deviation from unity has been reported for another NK2 antagonist [ IS]. Peptide 2 did not antagonize contractions produced by SP or NKB, but it did slightly reduce contractions produced by carbachol. Pcptidc 1 {[Ala?, Aibx, Leul"INKA(4- 10)) did cause contractions of tracheal rings at concentrations above 10V' M. At concentrations below l o - ' M, it antagonized contractions caused by NKA and SP but not those caused by NKB. Peptide 4 {[Ala', Achy, Nle"']NKA(4-10)} behaved in a similar manner to peptide 3. It failed to produce contractions itself, but did antagonize those produced by NKA, again with a reduction in the maximum contraction achieved. It did not affcct contractions produced by SP or NKB. The reduction in the magnitude o f contractions due to l o - ' M-NKA caused by the four peptides, also at l o - ' M concentration, were as follows: peptide 1, 53%; peptide 2, 22%; peptide 3, 19%; and peptide 4, 27%. T h u s it has been possible to produce specific antagonists of NKA-induced contractions of guinea-pig trachea by incorporation of amino acids such as Aib and Ach which restrict backbone conformational freedom. The use of an online monitoring system has alerted us to coupling difficulties with these residues, allowing remedial treatment which produced a significant increase in purity of the crude peptides.

VOl. 18

Maximum specificity has been achieved in peptides of the same length as NKA. When the 10-residue peptide 3 is shortened to the 7-residue peptide 1, specificity of antagonism is lost and, indeed, it behaves as an agonist at higher concentrations. Again peptide 4 is a s ecific anta onist of NKA, but a truncated analogue, [Ach)/hKA(4- 107 produced by Rovero et al., was essentially inactive as an agonist or antagonist on all three receptor types [ 1 11. This study was funded by a grant from the Northern Ireland Chest, Heart and Stroke Association and financial support from Applied Biosystems Inc. (Foster City, CA. U.S.A.). A.A.A.-S. thanks the Arab Student Aid International. New Jersey. U.S.A. for financial assistance. 1. Escher, E. & Regoli, D. ( 1 989) in I'epride Hormones us Prohormones (Martinez, J., ed.), pp. . . 26-52, Ellis Horwood, Chichester 2. Barnes. P. J. ( 1 986) Luncer i. 242-245 3. Joos, 6.F. (1'989)k'lin. Exp. Allergy 19 (Suppl. 1 ), 9- 13 4. Webber, S. E. ( 1989) Rr. J . I'harmuco/. 98. 1 197- 1206 5. Hakanson, R. & Sundler, F. (eds.) ( 1 98s) Tuchykinin An/ugonisrs, Elsevier Scientific, Amsterdam 6. Schwyzer, R. ( 1 987) E M B O J. 6.2255-2259 7. Cotrait, M. & Hospital, M. ( 1 982) Biochem. Biophys. HES. C'ommun. 109. 1 123- I 128 8. Patterson, Y.. Rumsey, S. M., Benedetti, E., Nemethy, G. 8( Scherage, H. ( 1981) J . Am. Chem.Soc. 103,2947-2955 9. Paul, P. K. C., Sukumar, M., Bardi, R., Piazzesi, A. M., Valle, (3.. Tonioli, C. & Balaram. P. (1986) J. Am. C'hern. Soc. 108. 6363-6370 10. Stewart, J. M. & Young. J. I>. ( 19x4) Solid Phase Peppride Synthesis, 2nd edn., pp. 22 and 44. Pierce Chemical Company, Rockford, Illinois, U.S.A. 1 1 . Rovero, P., Pestelhi, V., Rhaleb, N.-E., Dion, S., Rouissi, N., Tousimant. C.. Telemauue. S.. Ilrapeau. G. 8( Reaoli, D. ( 1989) Neuripeptides 13,263-'270 12. Tam. J. P.. Heath. W. F.& Merrifield. R. B. ( 1083) J . Am. ('hem. Soc. '105,'6442-6455 13. Kaiser, E., Colescott, R. L.. Hossinger, C. D. & Cook. P. I. ( 1 970) A n d . Biochem. 34,595-598 14. Kenakin, T. P. ( 1982) Cun. J . I%y.siol. I'hurmacd. 60, 249-265 15. Ireland. S. J.. Hagan. R. M.. Bailey, F., Jordan, C. C. & Stephens-Smith, M. L. ( 1990) Hr. J . I'hurmucol. 9 8 , 6 3 16. Abu Shanab, A. A., Allen, J. M., Guthrie. D. J. S., Irvine, G . B. & Murphy, R. F. ( 1989) Riochem. Soc. Trutis. 1 7 , 7 3 1-732

-

Received I May 1990