Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity

Peptides, Vol. 7, pp. 87-90, 1986. © Ankho International Inc. Printed in the U.S.A. 0196-9781/86 $3.00 + .00

Opioid and Other Peptides as Inhibitors of Leumorphin

(Dynorphin B-29) Converting Activity

L A K S H M I D E V I * A N D A V R A M G O L D S T E I N * t 1

*Addiction Research Foundation and tStanford University Palo Alto, CA 94304

R e c e i v e d 3 S e p t e m b e r 1985

DEVI, L. AND A. GOLDSTEIN. Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity. PEPTIDES 7(1) 87-90, 1986.--A thiolprotease from rat brain membranes was shown to convert synthetic dynorphin B-29 (Dyn B-29, "leumorphin") to the tridecapeptide dynorphin B (Dyn B, "rimorphin"). This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. The dynorphin converting activity displayed typical Michaelis-Menten kinetics with an apparent Km for the substrate of 0.58/xM. Surprisingly, a synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site, did not inhibit the activity. Dyn A inhibited the activity competitively with an apparent K~ of 3.7/xM. The converting activity was also inhibited by Dyn A-(6--17) but not by Dyn A-(8-17), suggesting a role of Arge-Arg r in the inhibition of converting activity. Bovine adrenal medulla Peptide E inhibited the converting activity substantially whereas metorphamide did not, suggesting the importance of COOH-terminal residues in recognition./3-Endorphin was an effective inhibitor of converting activity, and [a-N-acetyl]/3-endorphin was not, indicating a crucial role of the free NH2-terminus in recognition by the enzyme. ACTH inhibited the activity competitively with an apparent K~ of 39 nM. The converting activity was also inhibited substantially by ACTH-(1-13) but not by a-MSH, again indicating a requirement of the free NHz-terminus for recognition. The above results suggest that the converting enzyme recognizes peptides of the three known opioid gene families.

Leumorphin Opioid Neuropeptide Processing Protease Substrate specificity

PROCESSING of many neuropeptides and hormones occurs at pairs of basic residues, especially Lys-Arg, with a tryptic cleavage to the right (i.e., on the COOH-terminal side) of arginine, then carboxypeptidase B-like activity leading to sequential removal of arginine followed by lysine [6]. Apart from this, a number of processing mechanisms occur that involve either a single arginine residue or no basic residues [10]. A single-arginine processing is required for the formation of Dynorphin A-( l -8) from Dynorphin A, neurophysin II from propressophysin, and Dynorphin B (Dyn B) from Dynorphin B-29 (Dyn B-29, also called " ieumorphin" [12], the COOH-terminal 29-residue peptide of prodynorphin [9]).

There have been reports concerning single-arginine processing enzymes [13,15]. We reported [4] that a thiolprotease is responsible for the conversion of Dyn B-29 to Dyn B. We also reported that this enzyme cleaves the Thr~3-Arg 14 bond in a single step, thereby converting Dyn B-29 to Dyn B [5]. This enzyme has not yet been purified to homogeneity, and therefore its substrate specificity cannot be determined. However, inhibition of activity can be studied in a crude enzyme preparation, in order to obtain information about recognition of various peptides. In this paper we present an analysis of the inhibitory potencies of peptides derived from the three opioid gene families. Relevant sequences are shown below:

Dyn B-29 (porcine): (H)YGGFLRRQFgKVVT~3RJiSQEDPNAY2ZYEELFDV29(OH)

Dyn A (porcine): (H)YGGFLRRIaRPKLKWDNQ~r(OH)

BAM Peptide E (bovine): (H)YGGFMRRVSGR'°PEWWMDYQKRYGGFLZS(OH)

fl-Endorphin (human): (H)YGGFMTSEKgSQTPLVTLFKNAIIKNAYKKGE31(OH)

ACTH (human): ( H)SYSMEH FRWGKPVGI4K~SKRRPVKVYPNGAEDESAEAFPLEF39(OH)

METHOD

Reagents

Except as otherwise noted, all peptides were obtained from Peninsula Laboratories Inc. (Belmont, CA). Peptide E was from Bachem (Torrance, CA). Dyn B-29-(9-22) was from Vega Biochemicals (Tucson, AZ). Metorphamide was a gift of Dr. E. Weber (Standford Univ.). Purity of Dyn B, Dyn B-29, Dyn A and Dyn B-29-(9-22) was verified by reverse- phase analytical HPLC on a Waters Associates (Milford, MA) system with a C18/zBondapak column. Elution was by a linear gradient from 20-50% acetonitrile in 5 mM tri- fluoroacetic acid (30 min, 1.5 ml/min). The sequences of the synthetic peptides Dyn B-29 and Dyn-B-29-(9-22) were verified in the gas-phase sequenator by Dr. Martha Bond (DNAX Research Institute, Palo Alto, CA). All peptides

'Requests for reprints should be addressed to Avram Goldstein, Addiction Research Foundation, 701 Welch Road, Suite 325, Palo Alto, CA 94304.

87

88 DEVI AND GOLDSTEIN

TABLE 1

POTENCIES OF PEPT1DES AS 1NHIBITORS OF CONVERTING ACTIVITY

IC~o (/zM)

Dynorphin-related peptides Dyn B-29-(9-22) > 1,000 Dyn A 3.8 _+ 0.8 (N=9) Dyn A-(l-8) 340 _+ 19 Dyn A-(6-17) 44 _+ 6 Dyn A-(8--17) >2,300 Gly-Gly-Gly-Dyn A-(8-17) >2,300

Peptides derived from pro-enkephalin BAM Peptide E 0.19 + 0.4 Metorphamide 290 _+ 29 [Met]enkephalin-ArgG-Phe 7 > 1,000

Peptides derived from pro-opiomelanocortin /3-Endorphin (human) 19 _+ 5 [c~-N-acetyl]/3-Endorphin > 570

ACTH (human) 0.040 _+ 0.007 ACTH-(1-13) 3.3 _+ 1.6 ACTH-(1-13) amide 7.2 + 0.8 [a-N-acetyl]ACTH-(1-13) amide (a-MSH) 380 _+ 62

The reaction was carried out as described under the Method section except that various peptides were used as inhibitors to obtain esti- mates of 50% inhibitory concentrations (IC~u). Unless otherwise indicated, data are means _+ SEM based on four or five independent experiments with each peptide.

were at least 90% pure by TLC, electrophoresis and amino acid analysis furnished by the supplier.

Preparation of Membranes and Assay of Con vetting Activity

A male Sprague-Dawley rat was killed by decapitation. The brain was homogenized using a Tissumizer (Tekmar, Cincinnati, OH) in 10 vol. per g wet weight of ice cold 50 mM sodium phosphate buffer at pH 7.4 (buffer A). The homoge- nate was centrifuged (26,000 x g, 20 min, 4°C) and the pellet was resuspended in the original volume of buffer A and homogenized in a Teflon Dounce homogenizer using pestle B. Following centrifugation as above, the pellet was washed again. For solubilization, the pellet was incubated in 10 vol. of buffer A containing Triton X-100 (0.1%) for 30 min at 4°C and centrifuged as above. The supernatant solution was used in the assay. The converting activity remained in the supernatant after centrifugation at 105,000 × g for 1 hr.

The assay mixture consisted of 15 txl of 250 nM Dyn B-29 in buffer B (buffer A containing 0.1% Triton X-100), 15/xl of 1 mM Dyn B-29-(9-22) (except when Dyn B-29-(9-22) was the test peptide, 15/xl of 1 mM Dyn A-(l-8) was used), 15/xl of a peptide to be tested for inhibitory activity, 50 gl of 150 mM sodium phosphate buffer (pH 7.4), 50/xl (5-10/xg of protein) of enzyme preparation, and distilled water to a final volume of 150/xl. Dyn B-29-(9-22) was used to saturate nonspecific peptidases [5]. The reaction was carried out at 37°C for 10 rain and was terminated by boiling for 3 min. There was no measurable enzyme activity in boiled membranes. Radioim- munoassay (RIA) and preparation of radiolabelled tracers were as described elsewhere [81. The antiserum 17S used in

• 2 r

1/v

l Dyn A J

f / J -

: / j / ' "

1 J J ~

I CONTROL

2 4 6 - - -~ 1/S

FIG. I. Lineweaver-Burk plot for inhibition by Dyn A. The reaction was carried out as described in the Method section except that the reaction mixture contained various concentrations of Dyn B-29 in the presence and absence of 20/zM Dyn A. Units of S are/~M. In order to normalize results in different experiments, v, the rate of reaction (pmol Dyn B formed x mg protein ~ x min 1) was computed as percent of control Vm,x. Shown are means and SEM from five independent experiments fitted by least-squares lines.

RIA does not crossreact with Dyn B-29-(9-22) or Dyn B-14 and crossreacts poorly with Dyn B-12 [5]. Protein estima- tion was by the method of Bradford [2] with BSA as stand- ard.

The integrity of peptide inhibitors after incubation was tested by HPLC analysis of the reaction mixture as described above. Absorbance at 228 and 280 nm was moni- tored, The reaction mixture with enzyme preparation boiled for 3 rain was taken as control. Under our incubation condi- tion there was no measurable breakdown of inhibitory peptides except for ACTH. In that case, there was an additional absorbance peak eluting at 35.5% CH:~CN (ACTH elutes at 35.0%). The formation of this new peak could be blocked completely by 100/~M Dyn B-29, suggesting that the breakdown of ACTH may be specifically due to the dynorphin converting activity.

RESULTS

The results are shown in Table 1. Dyn B-29-(9-22), a synthetic peptide that contains the cleavage site Thr~Z-Arg 14 did not inhibit at the highest concentration available. Thus, recognition by the converting enzyme requries more than the central portion of the substrate that includes the cleavage site for inhibition.

From the double-reciprocal plot (Fig. 1), the Km for Dyn B-29 as substrate was computed to be 0.58 /zM. Dyn A inhibited the activity competitively, with an apparent Ki of 3.7/zM. A K~ value for Dyn A of 3.7/xM was also obtained from the IC5, value in competition experiments (Table 1 ), by means of the Cheng-Prusoff equation [3]. Dyn A-(I-8), which occurs naturally, was much less potent, demonstrating the importance of COOH-terminal residues for recognition of the larger peptide, Dyn A, by the enzyme. The importance of the NH2-terminal enkephalin sequence is shown by the reduced potency of Dyn A-(6--17) as compared with Dyn

SPECIFICITY OF DYNORPHIN B-29 CONVERTING ACTIVITY 89

.15

FIG. 2. L ineweaver-Burk plot for inhibition by AC T H. The proce- dure was as descr ibed in Fig. 1 legend except that the incubation was carried out in the presence and absence o f 100 nM ACTH.

A. A critical role for arginine-6 and/or arginine-7 is indicated by the total loss of inhibitory activity in Dyn A-(8-17); even with the NH2-terminai extension Gly-Gly-Gly, this peptide is not recognized.

BAM Peptide E is a potent inhibitor. Metorphamide, a naturally occurring amidated peptide that contains the NH2- terminal eight amino acids of Peptide E, was 1500-fold less potent, showing that the COOH-terminal sequence plays an important role in recognition. [Met]enkephalin-Argn-Phe 7 did not inhibit.

/3-Endorphin, which contains both an NHe-terminal enkephalin sequence and a basic residue at position 9, was a moderately potent inhibitor. However, acetylation at the NH2-terminal caused a potency loss of more than 30-fold, showing that the free NH2-terminal tyrosine plays a crucial role in the inhibition of converting activity. ACTH, inter- estingly, was a highly potent competitive inhibitor with apparent K~ of 39 nM (Fig. 2 and Table 1). ACTH-(1-13) had 82-fold lower potency than ACTH itself, indicating a signifi- cant role of some of the CODH-terminal 26 amino acids in the inhibition. ACTH-(1-13) amide had only two-fold lower potency than ACTH-(1-13). However, [ct-N-acetyl]ACTH- (I-13) amide (a-MSH) was 53-fold less potent than ACTH- (1-13) amide, demonstrating the importance of free NH2- terminal serine for recognition by the enzyme.

Neurotensin, Substance P, and bradykinin failed to inhibit even at 1 mM (data not shown).

DISCUSSION

We recently reported the presence of a dynorphin converting activity that is responsible for the formation ofDyn B from Dyn B-29. This activity was not inhibited by various peptides containing single or double arginine residues but was inhibited by Dyn A. The data presented here concern the relative potencies of various peptides and peptide frag- ments as inhibitors of the converting activity. The interpre- tations to follow are based on the finding that Dyn B is

formed, under our conditions, by the action of a single converting enzyme cleaving between threonine-13 and arginine-14 in a single step [5].

That the converting enzyme did not exhibit measurable affinity towards the synthetic peptide Dyn B-29-(9-22), which contains the cleavage site, is interesting. It supports the previous finding that the converting activity is not due to a nonspecific arginyl protease and shows that recognition by the enzyme requires more than the local sequence flanking the cleavage site.

Dyn A is a competitive inhibitor of Dyn B-29 converting activity, suggesting that the formation of Dyn A-(l-8) from Dyn A in the brain could be due to this same enzyme. In a recent study Wallace et al. [15] characterized an enzyme in extracts of atrial gland of Aplysia, which carries out that conversion. However, their enzyme differs from the one described here in that it is a soluble metallopeptidase, activated by cobalt chloride, with a pH optimum of 6.2.

The recent characterization of proenkephalin-converting activity in chromaffin granules prepared from bovine adrenal medulla is of particular interest because the authors reported the presence of a trypsin-like protease with affinity toward Peptide E [11,14] or BAM 12P [7]. These proenkephalin converting activities cleaved at paired basic residues, whereas the converting activity described here (which is strongly inhibited by Peptide E) is directed toward a cleavage site in Dyn B-29 that contains a single arginine residue. The formation of metorphamide from Peptide E requires cleavage at Glyg-Arg ~° followed by amidation.

ACTH is a potent inhibitor of Dyn B-29 converting activity. In Dyn B-29, cleavage at the Thr~3-Arg TM bond leads to the formation of Dyn B. It is tempting to suggest that a similar cleavage at Gly~4-Lysl~ of ACTH would produce ACTH-(1-14), which, if followed by conversion ofglycine to a terminal amide [1], and a-N-acetylation, would yield a-MSH. The fact that in the incubation mixture containing ACTH, the formation of a new absorbance peak can be to- tally blocked by Dyn B-29 (data not shown) suggests that ACTH acts as a competitive substrate for the converting activity, and this is consistent with the Lineweaver-Burk analysis. Studies with pure enzyme are required to determine if ACTH is a substrate for this enzyme.

The results presented here indicate that the Dyn B-29 converting enzyme recognizes peptide products of the three known opioid gene families. It is puzzling that the common feature shared by the opioid peptides, namely, the NH2- terminal enkephalin portion, is not an absolute requirement for recognition, as shown by results with ACTH and Dyn A-(6-17). Comparison of Dyn A with the 100-fold less potent Dyn A-(l-8) indicates a role of the COOH-terminal extension, which contains several single basic residues. A similar conclusion can be drawn from the comparisons of BAM Peptide E and metorphamide, as well as ACTH and ACTH- (1-13). In each case the longer peptide could be a substrate of the converting enzyme.

In ACTH and related peptides, which exhibit high affinity towards the enzyme, the NH2-terminal serine must be free for good recognition; a-N-acetylation of ACTH-(1-13) amide leads to substantial reduction in potency. A similar conclusion can be drawn from the comparisons of/3-endorphin and [a-N-acetyl]/3-endorphin./3-Endorphin, which is an effective inhibitor, contains single and double lysine rather than arginine residues.

These peptides may share as yet unknown common fea- tures such as similarities in secondary or tertiary structures

90 DEV1 A N D G O L D S T E I N

tha t enab le s t h e m to be r ecogn ized by the c o n v e r t i n g en zyme . This s tudy has e n a b l e d us to de t e rmine the abil i ty of va r ious pep t ides to act as inh ib i to rs in c rude e n z y m e prep- a ra t ions , bu t s tudies wi th pu re e n z y m e are requi red to de- t e rmine the subs t r a t e specif ici t ies .

ACKNOWLEDGEMENTS

We are grateful to Dr. Martha Bond for verifying the authenticity of Dyn B-29 and Dyn B-29-(9-22) by sequence analysis. This inves- tigation was supported by grant BNS-84-16617 from the National Science Foundation.

R E F E R E N C E S

1. Bradbury, A. F., M. D. A. Finnie and D. G. Smyth. Mechanism of C-terminal amide formation by pituitary enzymes. Nature 298: 686-688, 1982.

2. Bradford, M. M. A rapid and sensitive method for the quantita- tion of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976.

3. Cheng, Y.-C. and W. H. Prusoff. Relationship between the inhibition constant (K0 and the concentration of inhibitor which causes 50 percent inhibition (I~0) of an enzymatic reaction. Bioehem Pharmacol 22: 309%3108, 1973.

4. Devi, L. and A. Goldstein. Dynorphin converting enzyme with unusual specificity from rat brain. Proc Natl Acad Sci USA 81: 1892-1896, 1984.

5. Devi, L. and A. Goldstein. Neuropeptide processing by single-step cleavage: Conversion of leumorphin (dynorphin B-29) to dynorphin B. Biochem Biophys Res Commun 130: 1168-1176, 1985.

6. Docherty, K. and D. F. Steiner. Post-translational proteolysis in polypeptide hormone biosynthesis. Annu Rev Physiol 44: 625- 638, 1982.

7. Evangelista, R., P. Ray and R. V. Lewis. A "trypsin-like" enzyme in adrenal chromaffin granules: A proenkephalin processing enzyme. Biochem Biophys Res Commun 106: 895-902, 1982.

8. Ghazarossian, V. E., C. Chavkin and A. Goldstein. A specific radioimmunoassay for the novel opioid peptide dynorphin. Lift, Sci 27: 75-86, 1980.

9. Kakidani, H., Y. Furutani, H. Takahashi, M. Noda, Y. Morimoto, T. Hirose, M. Asai, S. lnayama, S. Nakanishi and S. Numa. Cloning and sequence analysis of cDNA for porcine fl-neo-endorphin/dynorphin precursor. Nature 298: 245-249, 1982.

10. Lazure, C., N. G. Seidah, D. Pelaprat and M. Chretien. Proteases and posttranslational processing of prohormones: A review. Can J Biochem 61: 505-515, 1983.

11. Lindberg, I., H.-Y. T. Yang and E. Costa. Further characterization of an enkephalin-generating enzyme from adrenal medullary chromaffin granules. J Neurochem 42: 1411-1419, 1984.

12. Nakao, K., M. Suda, M. Sakamoto, T. Yoshimasa, N. Morii, Y. lkeda, C. Yanaihara, N. Yanaihara, S. Numa and H. Imura. Leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B. Bioehem Biophys Res Commun 117: 695- 701, 1983.

13. Straus, E., A. Malesci and R. S. Yalow. Characterization of a nontrypsin cholecystokinin converting enzyme in mammalian brain. Proc Nat! Acad Sci USA 75:5711-5714, 1978.

14. Troy, C. M. and J. M. Musacchio. Processing of enkephalin precursors by chromaffin granule enzymes. Lift, Sei 31: 1717- 1720, 1982.

15. Wallace, E. F., E. Weber, J. D. Barchas and C. J. Evans. A putative processing enzyme from Aplysia that cleaves dynorphin A at the single arginine residue. Biochem Biophys Res Commun 119: 415-422, 1984.

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Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity