Selective Cleavage of Proenkephalin-derived Peptides (~23,300 ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 25, Issue of September 5, pp. 12543-12553,1988 Printed in U.S.A.

Selective Cleavage of Proenkephalin-derived Peptides(~23,300 Daltons) by Plasma Kallikrein* (Received for publication, December 15, 1987)

Kathleen M. Metterstg, Jean RossierS, Joanne Paquinll, Michel Chretienv 11, and NabilG. Seidahn From the SLaboratoire de Physiologie Nerveuse du Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France and the llJ. A. de Seve Laboratories of Biochemical and Molecular Neuroendocrinology, ClinicalResearch Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canuda H2 W 1 R7

The ability of human plasmakallikrein to hydrolyze human plasma form of IRCM-SP1 hasbeen achieved (6). The several proenkephalin-derived peptides has been studhuman plasma enzyme also cleaves prohormone-derived pepied, including the synthetic peptides BAM 12P and tides exclusively at dibasic and single basic residues (6). peptides E,F, and B as well as synenkephalin-contain- Further characterization of purified human plasma IRCMing peptides (8.6, 18.2, and 23.3 kDa) purified from SP1 by protein sequence analysis showed that the first 25 bovine adrenal medulla chromaffin granules. All the residues of both the regulatory and catalytic subunits of this identified cleavages occurred either COOH-terminal to or between pairs of basic amino acids, with plasma enzyme (6) are identical to those reported for human plasma kallikrein recognizing Lys-Lys, Lys-Arg, and Arg-Argkallikrein (7). In addition the human plasma enzyme purified as processing signals. Moreover, plasmakallikrein was asIRCM-SP1 was immunoprecipitable by an antiserum raised againsthuman plasma prekallikrein (6). Therefore, found tocleave at the COOH terminus of the basic pairs human plasma IRCM-SP1 is indistinguishable from human of amino acids preceding enkephalin sequences thereby plasma kallikrein on the basis of the structural and immureleasing the biologically active form of the peptide with the free NHz-terminal Tyr needed for receptor nological parameters so far evaluated. In view of these data, recognition. the plasma enzyme used during the studies described in this paper and originally purified as plasma IRCM-SP1 will be referred to as plasma kallikrein throughout. Plasma kallikrein is already known to play a role in biologBiologically active peptides are synthesized as partof large ical events involving precursor maturation at both single and inactive precursors and are subsequently generated by selec- pairs of basic amino acids. This enzyme is responsible for the tive proteolytic cleavages. Elucidation of the amino acid se- formation of bradykinin, by cleavage of high molecular weight quences for many precursors has shown that biologically kininogen between a Lys-Arg bond and COOH-terminal to a active peptides are typically found bracketed between pairs of Phe-Arg sequence, during the series of proteolytic cleavages basic amino acids within these molecules and that these involved in coagulation (8).This enzyme also hydrolyzes prodibasic residues provide the most common signal for process- renin to renin, again by cleavageat a Lys-Arg bond (9). Since ing (1, 2). IRCM-serine protease 1 (IRCM-SPl),’ originally plasma kallikrein can selectively cleave i n vitro several biopurified from porcine pituitary (3) and rat heart (4), was logically active peptide precursors at sites supposed to be proposed as a candidate maturation enzyme on the basis of cleaved i n vivo, it is plausible that this enzyme could play a its ability to selectively cleave proopiomelanocortin-derived role in the maturation of a wider variety of propeptides. In peptides (5) and proatrial natriuretic factor (4) in vitro at the case of circulating plasma kallikrein there may be an both pairs of basic amino acids and at single Arg residues involvement in the extracellular processing of precursors. known to be cleaved in vivo. Recently, the purification of the This is supported by the selective cleavage of proatrial natriuretic factor, since this precursor is thought to be processed * This investigation was supported by the J. A. De SBve Foundation, by a research grant from the Medical Research Council of either duringor immediately following secretion (10-13). This Canada, grants from the United States National Institutes of Health, type of model could also apply to the maturation of proenand by a grant from the “Association pour la Recherche sur le Cancer kephalin-derived peptides since enkephalin precursors have de Villejuif.” The costs of publication of this article were defrayed in been shown to be released from perfused adrenal glands (14part by the payment of page charges. This article must therefore be 17) and are also present in the circulation (18, 21). hereby marked “advertisement” in accordance with 18U.S.C. Section In this study the ability of human plasma kallikrein to 1734 solelyto indicate this fact. hydrolyze several enkephalin-containing peptides has been § Supported by a grant from the Foundation Simone et Cino del Duca. Present address: J. A. De Seve Laboratory of Biochemical and investigated to establish whether this enzyme also displays Molecular Neuroendocrinology. strict selectivity toward cleavage sites within proenkephalin. 11 To whom all reprint requests should be addressed. The proenkephalin-derived peptides used as substrates inThe abbreviations used are: IRCM-SP1, IRCM-serineprotease 1; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonicacid; RIA, clude the synthetic peptides BAM 12P andpeptides E, F, and radioimmunoassay; SYN, synenkephalin (proenkephalin-1-70); ME, B, as well as synenkephalin (SYN)-containing peptides (8.6, [Met’lenkephalin; LE, [Leu’lenkephalin; SDS-PAGE, sodium dode- 18.2, and 23.3 kDa) purified from bovine adrenal medulla cy1 sulfate-polyacrylamide gel electrophoresis; HPLC, high perform- chromaffin granules (22). The SYN-containing peptides inance liquid chromatography; ( T y P ) SYN-octapeptide (63-70), clude the largest naturally occurring putative intermediates ( T y P ) synenkephalin-octapeptide (63-70); TPCK, L-l-tosylamido2-phenylethyl chloromethyl ketone; HEPES, 4-(2-hydroxyethyl)-l- in the processing of proenkephalin and are therefore of parpiperazineethanesulfonic acid; EESHLLA,SYN-heptapeptide (64- ticular interest as substrates for candidate maturation en70). zymes.

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12544

Cleavage of Proenkephalin-derived Peptidesat Pairsof Basic Amino Acids EXPERIMENTAL PROCEDURES~

RESULTS

Plasma Kallikrein Hydrolysis of Synthetic Enkephalin-containing Peptides-The ability of human plasma kallikrein to hydrolyze a series of proenkephalin-derived peptides has been studied. Initial experiments were performed using the synthetic bovine enkephalin-containing peptides known as peptide B (34),peptide F (35),peptide E (36),and BAM 12P (37). In all cases, the enzyme was incubated with the appropriate synthetic peptide and the reaction medium analyzed by reverse-phase HPLC as described under “Experimental Procedures.” The peptide peaks were collected and characterized by amino acid analysis (Fig. 1). Peptide B contains one copy of the enkephalin sequence which is in the form of the heptapeptide ME-RF andis located at the COOH terminus of the peptide. Plasma kallikrein cleaves peptide B following the Lys-Arg basic pair which precedes the heptapeptide, liberating ME-RF and the complementary peptide peptide B1-24(Fig. L4 and Fig. 2). These were the only observed cleavage products with approximately 35% hydrolysis of peptide B under the experimental conditions used as based on amino acid analysis of digestion products recovered by reverse-phase HPLC (Table MI). In particular there was no evidence of cleavage at theArgG position within the heptapeptide. Plasma kallikrein hydrolysis of peptide B was found to be linear with respect to time with a K,,, of 83 f 24 X M anda kc, of25.3 s-l (23). Peptides E and F show essentially the same pattern of cleavage by plasma kallikrein. Both peptides contain two enkephalin sequences, located at the NH2 and the COOH termini. The sequence occurs as ME except at the COOH terminus of peptide E where it is in the form of LE. The ME at the NH2 terminus is followedbyArg-Arg and Lys-Lys within peptides E and F, respectively, with plasma kallikrein cleaving between the basic pair in both cases (Fig. 1,B and C, and Fig. 2). Only with peptide F as substratewas a secondary cleavage observed, occurring after the Lys-Lys bond and accounting for 33% of the hydrolysis around this basic pair (Table M3). Incontrastthe enkephalin sequence atthe COOH terminus is preceded by Lys-Arg in bothpeptides, and cleavage byplasma kallikrein occurs predominantly after the basic pair to liberate authentic enkephalin,with a secondary cleavage between the Lys-Arg residues. The major cleavage is responsible for 65 and 84% of the COOH-terminal enkephalin generated from peptides E and F, respectively (Tables M2 and M3). BAM 12P, which has the sequence of peptide E1-12, was also incubated with plasma kallikrein and demonstrated the same cleavage pattern as thelarger peptide (Fig. 2). Plasma Kallikrein Hydrolysis of Synenkephulin-containing Peptides-Having established that plasma kallikrein is capable of hydrolyzing small enkephalin-containing peptides, the ability of this enzyme to cleave larger proenkephalin-derived peptides (8.6,18.2, and 23.3 kDa) was investigated. These peptides were purified from bovine adrenal medulla chromaffin granule lysate (22) and all contain SYN (proenkephalin1-70) at the NH2 terminus (Fig. 4). Following incubation of the individual peptides with the plasma enzyme, the reaction medium was separated by reverse-phase HPLC as described under “Experimental Procedures.” The eluant fractions were analyzed by RIA in order to characterize the peptide products Portions of this paper (including “Experimental Procedures,”Fig. 1, and Tables Ml-M6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

Peptide R

10

.\rll~-PhcAlaGluProLeuProSerGluGluGluGlyGlu

Ser TYr

20

9 GlyC1yTyrArgl,ysGluhlctGluProValCluLysSer Phe 30 MctArgPhe-COOH

Peptide E 10

NH2-TvrGlyGlyPheMetArgArgValGlyt~rgProGlu 9

TrP TrP Met

Peptitlc F 10

9.) NHZ-TyrGlyClyPheMetLysLysMetAspGluLeuTyr Pro Leu GluClyClyAsnAlaGluGluCluValGlu 20

Va 1 Leu G~yLy~r$~rClyClyPheMet-COOH

HAM-1 2P 10

NIIZ-TyrGlyClyPheMetArgArgValGlyArgProGlu-COOIf 9 FIG. 2. Sequences of the bovine synthetic peptidesB,E, F, and BAM 12P showing sites of cleavageby plasmakallikrein. Human plasma kallikrein was incubated with the appropriate synthetic peptide and the peptide products identified by amino acid analysis following separation by reverse-phase HPLC as described in Fig. 1. Bolder arrows indicate preferred cleavage.

with respect to theirME, LE, ME-RGL, SYN, and EESHLLA content. The SYN antiserum detects all peptides containing proenkephalin-1-70 by direct RIA.However, the COOHterminally directed ME, LE, ME-RGL and EESHLLA antisera show little or no cross-reactivity in RIA with NH2- and COOH-terminal extensions of these peptides and no crossreactivity when these sequences occur internally. Therefore, samples were further digested in order to liberate immunoreactive forms of ME, LE, ME-RGL, and EESHLLA which could then be measured by RIA. Digestion by trypsin releases COOH-terminally located ME-,LE-,andEESHLLA-IR, while digestion by trypsin followed by carboxypeptidase B liberates these sequences where they occur internally. Similarly, digestion by Staphylococcus aureus protease alone, or followed bycarboxypeptidase B, releases ME-RGL-IR where this is located COOH terminally, or internally. Finally, any small ME-, LE-, ME-RGL-, or EESHLLA-containing peptides liberated from precursors by plasma kallikrein hydrolysis at paired basic residues should still contain COOH-terminal basic amino acid(s). Therefore, digestion of samples with carboxypeptidase B alone was used to remove the remaining basic residues and unmask the immunoreactive peptides. The major peptide products separated by HPLC were also characterized by either aminoacid analysis, protein sequencing, or SDS-PAGE inconjunction with immunoblots. Hydrolysis of the 23.3-kDa Peptide-Plasma kallikrein has been shown to cleave either between or following pairs of

Cleavage of Proenkephalin-derived Peptides at Pairs basic amino acids and also at certain single basic residues. Since the 23.3-kDa peptide contains 10 pairs of basic amino acids, in addition to 11 singlebasic residues, this species provides many potential cleavage sites for plasma kallikrein, with the capacity for generating a variety of different peptides. In view of the possible complexity of the reaction products using this substrate, an initial incubation of the 23.3-kDa peptide with plasma kallikrein was analyzed by specific RIA and also by SDS-PAGE followed byimmunoblotting,to assess the degree of hydrolysis under these conditions. RIAwas performed both before and after additional enzymatic digestion of the incubation medium by trypsin and carboxypeptidase B in order to liberate immunoreactive peptides (Table I). When the incubation medium was assayed for LE-IR, comparison of the values obtained before digestion, which represents COOH-terminal LE-IR generated by plasma kallikrein, and after digestion with trypsin and carboxypeptidase B, which represents the total LE-IR content of the substrate, indicated that approximately 31% of the 23.3-kDa peptide washydrolyzed at the COOH-terminal Lys-Arg bond by plasma kallikrein, under these experimental conditions (Table I). As expected, neither ME- or ME-RGL-IR were detectable by direct RIA since these internal sequences would not be liberated by plasma kallikrein alone.However, subsequent digestion of the incubation medium by carboxypeptidase B liberated both immunoreactivities, showing that small MEand ME-RGL-containing peptides were present. In the case of ME-IR, this represented only 8% of the total ME-IR content of the 23.3-kDa peptide, indicating that themajority of ME was still in the form of large peptides. A similar pattern was observed forEESHLLA-IR. As previously stated EESHLLA constitutes the COOH-terminal 7 residues of SYN and is followed by Lys-Lys within the 23.3kDa peptide. Plasma kallikrein hydrolysis at thisdibasic pair would liberate EESHLLA-containing peptides with residual COOH-terminal basic amino acid(s), which can then be removed by carboxypeptidaseB, in order to reveal EESHLLAIR. Digestion of the incubation medium with carboxypeptidase B increased radioimmunoassayable EESHLLA %fold, indicating that this peptide was indeed present in a form containing COOH-terminal basic residuesdue to plasma kallikrein cleavage at theLys-Lys bond. Comparison of the value for EESHLLA-IR obtained following carboxypeptidase B TABLE, I Hydrolysis of the 23.3-kDa SYN-containingpeptide by plasma kallikrein: analysis of the incubation medium by radioimmunoassay for ME, LE, ME-RGL, and EESHLLA Plasma kallikrein was incubated with the 23.3 kDa SYN-containing peptide as outlined in Fig. 3. Prior to SDS-PAGE a sample (3 pl) of the incubation medium was removed and diluted 500-fold in 50 mM Tris-HC1,pH 8.0 at 37 “C, containing 2 mM CaC12.Aliquots were taken and assayed for ME-, LE-, ME-RGL-, and EESHLLA-IR, both before and after enzymatic digestion with either trypsin and/or carboxypeptidase B, as indicated. Under these conditions the electrophoresis sample buffer had no effect on either the enzymatic digestion or RIA. ND = not detectable. ME-RGL-IR was not measured following trypsin and carboxypeptidase B treatment since trypsin cleaves at the Arp“ position within this sequence. LE-IR was not measured following carboxypeptidase B alone since LE occurs at the extreme COOH terminus without a carboxyl basic amino acid extension. Total pmol IR in the incubation medium Enzyme treatment ME-RGL EESHLLA LE ME 31 None ND 484 ND Carboxypeptidase B 608 125 99 Trypsin and carboxy7660 1573 1011 peptidase B

of Basic Amino Acids

12545

digestion, with the value for total EESHLLA-IR estimated followingtrypsin and carboxypeptidaseB treatment suggested that approximately 7% of thesubstrate washydrolyzed around this basic pair. In this case a minor amount of immunoreactivity was detectable by direct assay, reflecting the cross-reactivity of the antiserum with larger peptides such as SYN and the8.6-kDa peptide. These RIA results indicated that the 23.3-kDa substrate was partially hydrolyzed by plasma kallikrein to yield small enkephalin-containing peptides, as well as larger intermediates. This was confirmedby immunoblotting following SDSPAGE of the incubation medium (Fig.3). The immunoblot of the hydrolysate was developed using SYN (Fig. 3, lane C), ME-RGL (Fig. 3, lane D),and ME (Fig. 3, lane E ) primary antisera. Visualization of resolved hydrolysate proteins by SYN antiserum revealed fourimmunoreactivebands, migrating with apparent M, values of 29,700, 23,000, 21,700, and 10,700 ( l a n e C). Except for the band at 18.4 kDa all these species contained ME-IR with no additional bands revealed by the ME antiserum ( l a n e E). In contrast only the SYN immunoreactive bands at 23 and 21.7 kDa were strongly stained with the ME-RGL antiserum, while additional bands of ME-RGL-IR were observed with apparent M, of27,200 and 14,400 ( l a n e D).The pattern of SYN immunoreactive bands obtained following incubation with plasma kallikrein was compared with that observed for both the purified 23.3kDa substrate ( l a n e E ) and also for the bovine adrenal medulla

SYN CON JAlNlNG -~ PEP TIDES

A B C D E

kdalton

23.3 4 18.2

ne adrenal medulla chromaffin granule I y s a I ~by affinity chromatography and TSX g e l irltrarlon HPLC 8 s described by llefferr and Rorrler (22).

Enzyme incubaflonr were performed in a final volume of 100 irl of 200 nH BE8 pH ronfalnlng 2 m H EUTA, 1.2~7.0U of purified human plasma kallikreln and elther I S nnol of synfhefic bovine pepcide (peptides E . P , B and BAM 12P) or I nnol of purified 5111c o n r a > n l n g pepcide (8.6, 18.2 and 23.3 kdalron). All incubaflonr " e r e conducfed a t room ~ r m p e r a l u r e f o r 2 - 5 h and the reaction terminated by the addition of SO rcl of glacial r r e r r c acid. The incubation medium vae then analyzed by reverse-phase HPLC over s Vydac 2IITP5L P ~ o f e i n C1 column ( 0 . 4 6 x 25 cn) eluted V1Ih a 120 m l n linear gradient of 8 - 3 6 ) . hepfafluorobutyric acid (HPBA) a7 a flov rate o f 1 ( u i r l acelonlrrile I n 0.l1Z (u,u) i.mlinin. I n :he case 01 t h e 18.2 and 21.3 kdalton peptides the incubation niedium "8s diluted "5th 1110 t.1 o f 6 H gilanidln~-HCl prior to analysis by HPLC. Reaction products "ere nonlrored by absorbance a t 230 and 280 nm. Colunn fraelionr (1-3 ml) "ere collected al~romdf~cally except f o r peptide peaks vhich Yere collected manually and Ehacacterlzed by e i l h r i amino acld antilysis. prorein sequencing o r sodium dadecyl sulphate polyacLyiamide urI electrophorerls (SDS-PAGE) in conjunction vith lmmunobloffing. Co wm fractions vere specific radloinmunoarsay (RIA) for [Lcu']enkephrlln (LE), also, analyzed by IHcl-)enkephalin (HE), HE-RGL, S Y N and EESHLLA, the C-terminal region Of SW. R.5,

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Y ~ E conducted In gradlent (12-16X acrylamlde) slab gelr as modified from Laemli (28). P r o t e i n hands were transferred electrophoretically ( 5 V l c m for 16 h a t IS'C) on t o nlfrocellulare (29) using a Blo-Rad transblot cell. The nlirocellulosc vas then developed by irnnunohlorllng as descrlbed by Pztcy e t il. (30) using the folloving primary anrlrera: HE pastfeur 4165 (311 and *E-RGL AC 2227 (12). both a t L flnel dilufion of 1:iOOO and SYN SVii ( 3 3 ) a t a final dilutlon of 1:SOO. The HE-Pasteur 4165 antirerun vas used only far the 1mmonabloI and is not spedfically directed towards the C-terminus of HE. The inrnunareacrive bands were viruallled ufing horreradlrh peroxidase labelled anU-rabbll gamma globulin as secondary antiserum. The SYN-containing peptldespresent in bovlne adrenal medulla chromaffin granules have been vel1 characterized by l h l S cornblnation Of electrophoresis and Irnmunablol 122.10). Therefore, a preparation of rhese peptides. purlfled by SYH afflnily chromatography (22). was included during SDS-PACE as a standard. In addition Lo ,he pwlider used as svbstrster in this study. t h l r affinity purlfled preparation rovflnely conlnlns a 1 2 . 6 kdalfon peplide (proenkCphalln 1-84) and a higher molecular "eight form of the 18.2 kdalfon peptide (Plg. 3 ) .

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Cleavage of Proenkephalin-derived Peptides at Pairs of Basic Amino Acids

TABLE MI. AMINO ACID COMPOSI~IONS OF THE P E P r t o E PRODUCTS FOLLOWING T H E INCUBATION OF tl. PLASMA KALLIKREIN WITHPEPTIDE

Numbers in p a r e n t h e s i sr e p r e s e n tt h ec a l c u l a t e d number of amino a c i d s basedon the anmo a c i d sequence, f i g . 5.

Cleavage of Proenkephalin-derived Peptides at Pairsof Basic Amino Acids I'BLE

TABLE '16

M5.

T

T

t 1 1.1

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