Carboxypeptidase inhibitors from Ascaris suum: The primary structure

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 270, No. 1, April, pp. 153-161,1989

Carboxypeptidase

Inhibitors from Ascaris sum:

GENE A. HOMANDBERG,*t3 ROBERT

The Primary Structure’*2

ROBERT D. LITWILLER,t J. PEANASKYS

AND

*Department of Biochemists, Rush-Presb&&an-St. Luke’s Medical Center, Chicago, Illinois TMayo Clinic, Rode&e-r, Minnesota 55905; and $Department of Biochemistry, University of South Dakota, VermiUion, South Dakota 57069 Received

July

28,1988,

and in revised

form

October

60612;

27,1988

The carboxypeptidase A inhibitor from Ascaris suum was isolated from aqueous extracts by affinity chromatography toward immobilized carboxypeptidase A. The amino acid sequence is DQVRKCLSDT”‘DCTNGEKCV~KICSTIVE301QRCEKEHF’T?IPCKSNNDCQr”‘VWAHEKICNK’?LPWGL65. The carboxypeptidase A inhibitor is not homologous with the chymotrypsin/elastase or trypsin inhibitors from Ascaris, but shows homology in a g-residue internal sequence with the 37/39-residue carboxypeptidase inhibitors from tomato and potato. The carboxy-terminal 5 (4) residues in the three inhibitors are similar, suggesting a common mechanism of inhibition. o 1989 Academic press, I~~.

Comparisons between the sequence of the Ascaris carboxypeptidase inhibitor with those isolated from potato (19-22) and tomato (23, 24), the other characterized carboxypeptidase inhibitors, would allow the identification of any similarity that would involve interactions between the catalytic site of the enzyme and the reactive site of the inhibitor. Modifications of residues in the catalytic site of carboxypeptidase A reduce binding of the enzyme with the Ascaris inhibitor (12), suggesting that the inhibitor binds in proximity to these catalytic residues. The interaction between potato inhibitor and carboxypeptidase A was shown by x-ray analysis to be at the catalytic site of the enzyme (25). The carboxypeptidase inhibitor from potato has 39 residues, terminates with the sequence Gly-Pro-Tyr-Val-Gly3’ (22), and binds one molecule of enzyme per molecule of inhibitor (21,22). The Ascaris inhibitor contains 65 or 66 residues, terminates in a leucyl residue, and interacts in a stoichiometry of one molecule of enzyme with two molecules of inhibitor (11). This atypical stoichiometry was based on the observation that a molecular mass of 16,000 D of

Ascaris suum are intestinal parasites that produce inhibitors to pepsins of the host stomach (l-3) and inhibitors of chymotrypsin and elastase (1,4-6), trypsin (1, ‘7-lo), and carboxypeptidases A and B (1, 11-13) of the host intestine. The chymotrypsin inhibitors which also inhibit elastase have been separated into five isoinhibitors (14) and the sequence (15) and the reactive site of each determined (16). The sequences are homologous to that of the Ascaris trypsin inhibitor (9, 17), but no other trypsin/chymotrypsin inhibitor. It has been proposed from the sequences of the Ascaris chymotrypsin/elastase and trypsin inhibitors that they represent a separate family of inhibitors of serine endopeptidases (18). It was of interest to us to determine whether the carboxypeptidase inhibitor is a member of the Ascaris trypsin/chymotrypsin inhibitor family. 1 This work was supported in part by National Institutes of Health Grant HL-28444 (G.A.H.) and National Institute of Allergy and Infectious Diseases Grant AI-10992 (R.J.P.). a A preliminary report of some of this work has appeared (17). ’ To whom correspondence should be addressed. 153

0003-9861/89 Copyright All rights

$3.00

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

154

HOMANDBERG,

LITWILLER,

inhibitor was required to inactivate one gram mole of enzyme. The apparent molecular mass of one mole of fully active pure inhibitor was around 8000 D. This molecular weight estimation is based on minimal amino acid composition, SDS4-polyacrylamide gel electrophoresis, and gel filtration. Since this stoichiometry is atypical and the methods of size analysis are prone to error, the primary sequence of the inhibitor will be an important verification of the relative mass of the inhibitor and the stoichiometry of the reaction. MATERIALS

AND

METHODS

Bovine carboxypeptidase A (EC 3.4.17.1), hippuryl@-phenyllactate, cyanogen bromide, Sepharose 4B, Sephadex G-50, TPCK-treated trypsin, and DMAA buffer were from Sigma Chemical Company (St. Louis, MO). Submaxillaris protease (EC 3.4.21.40), dansyl chloride, phenylisothiocyanate, and TLC polyamide plates were from Pierce Chemical Company (Rockford, IL). Isolution of cnrboxgpeptiduse inhibitor. The carboxypeptidase inhibitor was isolated in our initial studies by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography (11). For these studies the inhibitor was isolated from the dissolved ammonium sulfate precipitate by affinity chromatography using carboxypeptidase A bound to Sepharose 4B. To prepare the affinity gel, 1 to 2 mg/ml of carboxypeptidase A in 0.2 M NaCV0.2 M sodium bicarbonate buffer, pH 9.5, was added to cyanogen bromide-activated Sepharose 4B prepared by the method of March et al. (26). Coupling proceeded for 24 h at 22°C. Up to 60% of protein typically coupled and about half of the bound enzyme was active in assays toward the ester substrate hippuryl-@-phenyllactate (27). Carboxypeptidase inhibitor was prepared from aqueous extracts of body walls of Ascaris swum that were centrifuged at 57,OOOg and then adjusted to pH 2 and incubated at 37°C for 75 min. When the pH was raised to 5.5, the precipitate that formed was removed and the supernatant solution was adjusted to 80% saturation with ammonium sulfate at pH 8.9. The pellet was dissolved in water, the amount of in-

4 Abbreviations used: DMAA, 0.4 M NJV-dimethylN-allylamine/trifluoroacetic acid in pyridine/water (3/2), pH 9.5; PITC, phenylisothiocyanate; PTH, phenylthiohydantoin; SDS, sodium dodecyl sulfate; TPCK, L-l-p-tosylamino-2-phenylethyl ehloromethyl ketone.

AND

PEANASKY

hibitor determined (28), and enough carboxypeptidase A-Sepharose to completely remove the inhibitor was stirred with the extract for 1 h at pH 8. This time is adequate to reach equilibrium since the reaction between pure inhibitor and enzyme occurs in less than 10 s at 4,22, and 37°C. When the inhibitor was bound to the affinity gel, the insoluble complex was transferred to a sintered-glass funnel and washed with several volumes of water, 0.5 M NaCl, and finally 20 mM Tris buffer, pH 7.4, until the absorbance at 280 nm of the washes reached baseline. The gel was then washed with 3 vol of water to remove salt and the inhibitor was released by suspending the gel in 1% formic acid. The supernatant (containing inhibitor) was recovered by filtration, and the gel was washed with 3 to 4 vol of 1% formic acid until the absorbance of the filtrate reached baseline. The eluants were combined, lyophilized to dryness, and then redissolved in a minimum volume of 1% formic acid. An insoluble fraction was removed by centrifugation and the supernatant was applied to a Sephadex G-50 column (1.5 X 90 cm) equilibrated in 1% formic acid. The chromatograms showed three peaks, the first and third peaks contained a total of 20% of the material applied, but this material did not inhibit carboxypeptidase A. The middle peak inhibited hydrolysis of hippuryl-@-phenyllactate by carboxypeptidase A (50 nM) when assayed as described (11). The middle peak, when examined on 20% polyacrylamide-SDS gels (29), showed a smeared band that migrated at about 8000 Da. Three different bands were evident by disc gel electrophoresis (30, 31) as observed for inhibitor purified by our original method (11). All three isoforms had the same amino acid compositions. We earlier reported the similarities in amino acid compositions and common amino-terminal sequences, carboxyl-terminal amino acids, and molecular weights. A Ferguson plot showed that the three isoforms were charge isomers. The inhibitor was lyophilized for storage. Inhibitor (40 mg) was obtained from 4.3 kg of A. suum body walls. A solution of inhibitor containing 1 mg/ml had an absorbance at 280 nm of 1.46 through a l-cm light path (11). A value of 6.42 X 10’ Me1 cm-’ (32) and a M, of 34,400 (33) were used to determine enzyme concentrations.

Frwmentation

of carbaqpeptidase

inhibitor

with

submaxilluris protease. Inhibitor (6 mg) was reduced and carboxymethylated with iodoacetic acid (34) in the presence of 8 M urea and 0.5% (w/v) SDS. The reduced/alkylated product(s) was dialyzed against 100 mM iV-ethylmorpholine/acetate, pH 8, and submaxillaris protease (60 pg) was added. Digestion was monitored by examining samples by electrophoresis in 20% SDS-gels (34). The samples were reacted with fluorescamine (35) prior to electrophoresis to allow visualization under uv light. Staining with Coomassie

PRIMARY

STRUCTURE

OF

CARBOXYPEPTIDASE

blue and destaining in methanol/acetic acid/water solutions extracted the fragments from the gel. The submaxillaris protease digest fragments were separated on a 4.6 X 250-mm widepore C-18 HPLC column from J. T. Baker, Inc. (Phillipsburg, NJ). Volumes (500 ~1) containing up to 250 pg of fragments were applied. Tryptic peptides. Tryptic peptides were prepared from fractions that were obtained by first digesting reduced alkylated intact inhibitor with submaxillaris protease. Fractions (100 nmol) from this digest were combined, lyophilized, and redissolved in 100 mM Nethylmorpholine/acetate, pH 8. TPCK treated trypsin (1 mol%) was added and the digestion continued for 24 h at 37°C. The solution was lyophilized to dryness and the fragments were separated on a C-18 HPLC column. Gas-phase sequencing. One to twelve nanomoles of sample was subjected to sequencing on an Applied Bio-Systems (Foster City, CA) Model 470A protein sequenator using the protocol of Hewick et al. (36). Manual Edwmn sequencing. The carboxy-terminal tryptic peptide, residues 61-65, presented difficulties in gas-phase sequencing and was sequenced by a modified Edman procedure (37). The peptide (20 nmol) was dissolved in 100 ~1 of 75% pryidine/25% water and 10 pl PITC added. After 1 h at 45OC, the solution was lyophilized to dryness. Trifluoroacetic acid (100 ~1) was added and the anilinothiazolinone amino acid was released from the peptide after 15 min at 45°C. The solvent was removed by lyophilizing. A portion was dissolved in 10% (v/v) trifluoroacetic acid and incubated at 30°C for 10 min to convert the anilinothiazolinone amino acid to the PTH derivative. The remainder of the dried sample was dissolved in 100 ~1 of 75% pyridine/25% water and the cycle repeated. The PTH derivatives were identified by the HPLC method of Tarr (38) and of Bhown et al (39). In this procedure the PTH derivatives always contained the PTH amino acids from earlier cycles. Some PTH amino acids were hydrolyzed by incubation in 0.1% (w/v) SnClz/6 N HCl for 4 h at 140°C and the free amino acids identified after reaction with o-phthalaldehyde (35). The carboxy-terminal tryptic peptide was also examined by the dansyl-Edman procedure (37). To avoid losses of peptide the method was modified as follows: the acetone precipitation normally used after coupling of protein and PITC was deleted, the solution was dried and cleaved with trifluoroacetic acid. For dansylation, aliquots (1 ~1) were withdrawn, dried, and dissolved in 10 ~1 of DMAA buffer. Dansyl chloride solution (5 pl) was added. After 1 h at 37°C the solution was dried, the residue hydrolyzed in 6 N HCl, and the dansyl amino acid identified by TLC (37) or by HPLC (40). Carboxy-terminal analysis Reduced and carboxymethylated inhibitor or fragments and carboxypepti-

INHIBITOR

FROM

Ascuris

155

dase Y were dissolved separately in 0.1 M N-ethylmorpholine/acetate buffer, pH 5, and dialyzed against this same buffer to remove endogenous amino acids. Carboxypeptidase Y and inhibitor were mixed in the ratio 0.05 mol enzyme per mole of inhibitor (fragment) and digestion was continued for 24 h at 37°C. The digest was lyophilized to dryness and dissolved in 10 pl DMAA buffer. Free amino acids were determined by dansylation and identification by TLC (37). In some experiments the digest was derivatized with o-phthaldehyde (37) and separated on HPLC. Amino abd composition Amino acids in peptides and in the inhibitor were determined by either the Pica-Tag system (41) or the hydrolysate was derivatized with o-phthalaldehyde and separated on an HPLC column (35). RESULTS

Isolatim of inhibitor. The Ascati inhibitor used in these experiments was removed from the dissolved ammonium sulfate fraction by affinity chromatography using carboxypeptidase A liganded to Sepharose 4-B. This procedure could conceivably release the carboxy-terminal amino acid if this residue is cleaved when enzyme and inhibitor interact, as predicted by the classical inhibitor/protease mechanism for serine endoproteases. When inhibitor, isolated by affinity chromatography, was digested with carboxypeptidase Y, 0.9 mol of leucine and lesser amounts of glycine per mole of inhibitor were released after a 24h digestion. In earlier experiments, when this carboxypeptidase inhibitor was prepared without the affinity chromatography step, these were still the only two amino acids detected by hydrazinolysis. Therefore, affinity chromatography used in this way apparently does not release carboxyterminal residue(s). The inhibitor sample used for these studies showed three differently charged forms on polyacrylamide gel electrophoresis at pH 8.6. This is the same electrophoresis pattern seen when inhibitor was isolated without affinity chromatography. These isoforms were shown by a Ferguson plot to be charge isomers with similar amino acid compositions (11). These isoforms were not separated prior to fragmentation for sequencing although this did complicate HPLC chromatography of the submaxillaris protease digests. More

156

HOMANDBERG,

LITWILLER,

than one HPLC peak yielded the same amino acid composition/sequence. Sequence analysis of native inhibitor. Carboxypeptidase inhibitor (12 nmol) was subjected to 39 cycles of sequencing. The yields are shown in Table I. The amount of material applied overloaded the support, prevented optimal exposure to the sequencing agents, and led to a background of some nonsynchronized sequences. The gaps at positions 6,12,18,25, and 34 were assigned as Cys. Sequence analysis of reduced and alkylated inhibitor residues 533 confirmed assignment of residues 6,12, and 18. Residue 34 was confirmed by sequencing the carboxy-terminal fragment, and residue 25 was assigned from the amino acid composition of fragment 5-33. Sequence analysis of fragmnts obtained

by submaxillaris nyl residues.

protease cleavage at argi-

Amino acid composition showed the presence of two arginyl residues. These were located at positions 4 and 33 in the sequence analysis of intact inhibitor and provided the overlap strategy needed to align a carboxy-terminal fragment that would begin with residue 34. Carboxypeptidase inhibitor was reduced and alkylated in 1% (w/v) SDS which was necessary to maintain solubility of the derivative. This reduced and alkylated inhibitor required 7 days incubation with submaxillaris protease (which cleaves at the carboxyl of arginine) before SDS-gel electrophoresis showed that digestion was complete. The digest was chromatographed on an HPLC wide-pore C-18 column (Fig. 1). Nine regions were combined and the samples lyophilized, and then each was rechromatographed using the same gradient. Examination of amino-terminal residues and amino acid compositions suggested that the same major peptide was present in regions 2, 3, and 4; each began with Lys and had an amino acid composition predicted for residues 5-33. The amino acid composition of the major peptide in region 4 is shown in Table II. The same major peptide was present in regions 5, 6, ‘7, and 8 as determined by amino acid composition and the amino-terminal residue which was CM-Cys. Region 9 contained intact inhibitor. The amino acid

AND

PEANASKY

TABLE I SEQUENCE ANALYSIS OF NATIVE Amwis CARBOXYPEPTIDASE INHIBITOR’”

Cycle

Amino

acid D

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Q V R K C L S D T D C T N G E K C V

Q K N K I C S T V E h R C E K E H F

Yield bmol)

Background (pmol)

11600 11600 12800 3300 7627

G G I K N

656 353 237 214 230

8453 295 4162 2138 1986

K K L L

940 413 848 257

K D

295 881

D G G K K K

707 1037 638 977 908 722

Q

9= 253 764 194

874 1034 1469 1766 1877 1420 1275 1529 820 1795 1161

K N K

538 622 901 799 626 904 288 276 433 642 468 196 197

a Over 12 nmol was applied to increase sequencing duration. Background is due to this overload and inefficient coupling. ‘Gaps in the sequence were assigned Cys as expected for unreduced inhibitor. Assignments were confirmed by separate runs of reduced and alkylated inhibitor and by amino acid composition of fragments (see text).

PRIMARY

STRUCTURE

OF CARBOXYPEPTIDASE

INHIBITOR

FROM Ascaris

157

Search for the ultimate-carboxy terminal peptide. The ultimate carboxy-terminal peptide ought to be released by tryptic cleavage at Lye?‘. This peptide should contain tryptophan and be readily identified. Since peptides 7 and 8 (Fig. 1) contain this peptide, lOO-nmol samples of each peptide were combined and digested with trypsin. Separation on an HPLC C-18 column FIG. 1. Separation of fragments obtained from a showed 11 major peaks (Fig. 2). The aminosubmaxillaris protease digest of reduced and alkylterminal residue, amino acid composition, ated inhibitor on a wide-pore C-18 HPLC column (4.6 and absorbance at 280 nm of each peak x 250 mm) equilibrated with 0.1% trifluoroacetic acid were determined. Peptide 1 was residues in HzO. Flow rate was 1.0 ml/min. The gradient was 34, 35, and 36; peptides 2-4, residues 57 to increased linearly from 0.1% trifluoroacetic acid to 60; peptides 5 and 6, residues 37 to 44; and 18% acetonitrile/O.l% trifluoroacetic acid between 0 peptide 7, residues 45 to 56. A solution (2 and 10 min, then linearly to 30% acetonitrile/O.l% recovered from peptrifluoroacetic acid between 10 and 20 min. The col- ml) of the material umn was run isocratically in 30% acetonitrile/O.l% tides 7 and 8 had an absorbance at 280 nm trifluoroacetic acid until 30 min and then increased of 0.1 through a l-cm light path, while all linearly to 60% acetonitrile/O.l% trifluoroacetic acid other peaks had negligible absorbance at between 30 and 60 min. All of the fractions were col- that wavelength. The tryptophan in peplected, lyophilized, and rechromatographed under the tide 7 was assigned to position 52 (see Tasame conditions for additional purification (not ble III). Peptides 8 and 9 had a near-inteshown). ger composition of 2 Leu and 1 Gly and began with Leu. No Asp was found. Since Pro cannot be detected by the o-phthalaldecomposition of the major peptide in region 7 is shown in Table II. Table III shows the hyde method, an amino acid hydrolysate of sequence and yield of PTH amino acids in peak 8 was dansylated and found to contain Pro. Peptides 10 and 11 were similar, peptide 7. Cycles 1 to 6 of the sequence, began with CM-Cys, and resembled nearly shown in Table III, correspond to positions 34 to 39 of the native inhibitor (Table I). intact inhibitor. A portion of peptide 8 (5 nmol) was seThe data in Tables I and III provide the proceamino acid sequence through residue 60 of quenced by a manual dansyl-Edman dure. The sequence obtained was Leu-Prothe inhibitor. The secondary residues in (Ser,Gly,Ala) was reTable III agree with the sequence of resi- (Ser,Gly,Ala)-Gly. leased at the third cycle. Identification of a dues 5 to 32 shown in Table I. This shadow sequence is probably due to incomplete re- fifth residue was not possible because the accumulation of salt prevented assignduction of disulfides. ment of that residue by TLC. Since the The major peptide in region 8 was subjected to 25 cycles of sequencing. The se- three PTH derivatives found at the third quence was identical to that of peptide 7. cycle could have been formed by acid destruction of dansyl-Trp, peptide 8 (20 From the difference between the amino acid composition of native inhibitor and nmol) was sequenced by the Edman the sequence of residues 1 to 60 (Tables I method and the PTH amino acids were identified directly. The sequence observed and III), residues remaining after residue A sample of pep60 should consist of a Gly, Pro, Trp, Asp, was Leu-Pro-Trp-Gly. tide 8 was treated with carboxypeptiand two Leu. However, from the amino acid composition of the three fragments in dase Y and leucine and glycine were the Table II, there may be one less Asp in the only amino acids found by HPLC and by sequence than predicted and residues 61- TLC following dansylation of the digest. 65 now likely consist of Gly, Pro, Trp, and Reduced and alkylated intact inhibitor two Leu residues. treated in parallel gave identical results.

158

HOMANDBERG,

LITWILLER,

AND

TABLE AMINO

ACID COMPOSITION DIGEST

PEANASKY

II

OF NATIVE INHIBITOR AND SELECTED PEPTIDES FROM SUBMAXILLARIS OF REDUCED AND ALK~ATED CARBOXYPEPTIDASE INHIBITOR

PROTEASE

Amino acid

Native inhibitor

Peptide 4, residues 5-33

Peptide 7, residues 34-65

Peptide A, residues l-4

Asx Glx Ser GUY His Arg Thr Ala Pro

lO(9)C 9 3 2 2 2 4 1 2

3.9 (4) 3.7 (4) 1.8 (2) 0.8 (1) 0.1(O) 0.9 (1) 2.8 (3) 0.0 (0) 0.1(O)

4.4 (4) 4.1(4) 0.8 (1) 1.0 (1) 1.8 (2) 0.1(O) 0.7 (1) 1.2 (1) 1.9 (2)

(1) (1) 0 0 (b 0 0 0

1.8 (2) 0.0 (0) 0.0 (0) 2.9 (3) 0.9 (1) 0.1(O) (0) 3.9 (4) 3.6 (4)

0.8 0.0 (1) (0) 0.0 (0) 1.8 (2) 1.9 (2) 0.9 (1)

A 0 0 0 0

Mb

0

4.0 (4) 3.5 (4)

0 0

29

32

4

Vai 5r Met Ile Leu Phe ‘b LYS

CYS Total

40 0 5 3 1 2 8 8 66 (65)’

0 This peptide was not isolated but consists of Asp, Gln, Val, and Arg as shown in Table I. b Trp content was based on sequence data. c The compositions of peptide 4 and peptide 7, as well as the sequence of the amino-terminal tryptic peptide, suggest that the Asx composition of the native inhibitor should be corrected from 10 to 9 residues and the total number of residues from 66 to 65.

Figure 3 shows the complete sequence of the Ascaris carboxypeptidase inhibitor and the sequence of the carboxypeptidase inhibitors from potatoes (22) and tomatoes (24) for comparison. Note the homology in the sequences between residues 42 to 50 of the Ascuris inhibitor and residues 11 to 19 of the potato inhibitor and 10 to 18 of the tomato inhibitor and the similarities between the last 5 (4) residues of all three inhibitors. DISCUSSION

We had shown earlier that the three isoforms of the inhibitors had similar amino acid compositions and identical molecular weights, specific activities, carboxyl-terminal residues, and mobilities on nondenaturing gels that were dependent only on charge (11). Other unpublished data

showed that one charge form could be converted to the others on long-term storage. Therefore, these forms were not separated prior to protease digestion and the resultant sequence would be a consensus sequence of the three forms. The individual sequences would differ only in amide assignment. In our original report on the carboxypeptidase inhibitor from Axa& (11) we reported an amino acid composition of 66 residues including 10 Asx residues. The amino acid sequence of the inhibitor presented in this paper accounts for only 9 Asx residues; and this should be corrected in the original report (11). The other 65 amino acid residues reported are accounted for in this sequence. Our earlier work with the Ascaris carboxypeptidase inhibitor suggested that it

PRIMARY

STRUCTURE

OF CARBOXYPEPTIDASE

INHIBITOR

159

FROM Ascaris

TABLE III SEQUENCE ANALYSIS OF PEPTIDE 7 OBTAINED FROM SUBMAXILLARIS PROTEASE DIGEST OF REDUCED ALKYLATED Asemis CARBOXYPEPTIDASE INHIBITOR Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Amino acid

Yield (pm4

CMC E K E H F T I P CMC K S N N D CMC

4006 3206 2811 2697 1761 2127 2134 1945 1138 1082 1327 856 678 741 692 872 691 530

Q

V w A H E K I CMC N K

NQ* 509 337 392 210 200 249 157 98

Background” (pmol) K CMC L S D T D C T G E K

683 1505 986 345 478 156 478 617 534 167 236 400

V

188

Q

NQ

K N K I CMC S T V E I

176 155 181 130 206

NQ NQ 92 138 97

’ Note that the background sequence corresponds to residues 5-32. Incomplete reduction likely caused a small degree of contamination with fragment 5-33. *Not quantitated.

had a unique stoichiometry in that two molecules of inhibitor bound one molecule of enzyme (11). This stoichiometry was based on our determination of molecular weight by minimum amino acid composition, by gel filtration, and by SDS-gel electrophoresis. The sequence presented here verifies our molecular weight assignment. It could be argued that our inhibitor could consist of two identical sequences in tandem, but only one able to function. We feel this is unlikely since our inhibitor had mobilities on SDS-gels that were expected for fragments of a 65-residue protein. There-

FIG. 2. Separation of the tryptic fragments after peptides 7 and 8 of the submaxillaris protease digest of reduced and alkylated inhibitor (Fig. 1) were combined and treated with TPCK-treated trypsin for 24 h. Tryptic peptides were applied to a wide-pore C-18 HPLC column (4.6 X 250 mm) equilibrated with 0.1% trifluoroacetic acid in HaO. Flow rate was 1.0 ml/min. The gradient, from 0 to 60% acetonitrile/O.l% trifluoroacetic acid, over 60 min was operated as described in Fig. 1.

fore, our earlier assignment of an atypical stoichiometry cannot be explained by errors in estimation of the size of the inhibitor. Nonetheless we still have no mechanistic explanation for this stoichiometry. The sequence of the carboxypeptidase inhibitor bears no obvious homology with either the chymotrypsin/elastase or trypsin inhibitors from Ascaris. Some homology of the Ascaris inhibitor with a trypsin inhibitor from pumpkin (42), to factor X (43,44), to factor VIII (45) was observed in

&g.@.&

1 5 10 15 OQVRKCLSOTOCTNGEKCVQKNKIC

&a&

S T I V':

I P R C':

K E H F4:

20

25

I

potato Tomato

Ascarls

55 VVAHEKICNK

Potato

20 G A Y F C';

Tomato

60

rQi-----i< ILPYGLl I

135 A C H N3: A R T C; G P Y V3:/ I 38 I GGTFCQACURFAGTC:GPVV ;

FIG. 3. Primary structure peptidase inhibitor and the carboxypeptidase inhibitors mato (24). Solid box, region region of similarity.

of the Ascaris carboxyprimary structure of the from potato (22) and toof homology; dashed box,

160

HOMANDBERG,

LITWILLER,

homology searches in the NIH data base. We cannot offer an explanation of the significance of these homologies. One region of homology exists between the Ascwis and potato and tomato carboxypeptidase A inhibitors. This region may have a common function. Ascaris

42

P CKSNNDCZ 11 P CKTHDDC: PCSTQDDCS Tomato The homology between residues 42 and 50 of the Ascaris inhibitor and residues 11 to 19 and 10 to 18 of the other two carboxypeptidase inhibitors may suggest that this is a region of contact with carboxypeptidase A. However, the x-ray structure of the complex of potato inhibitor with enzyme (25) provides little suggestion of this. This region of the inhibitor is not in close contact with the enzyme. Although there was some contact between His15 of the potato inhibitor and the ring of Tyra8 of the enzyme and the phenyl ring of Phezl of the inhibitor and Phen9 of the enzyme, these residues are not common in the Ascaris inhibitor. Assuming this similar sequence has another important function, we should still note that it may not be homology between similar molecules but rather convergent evolution of the Ascaris and potato/tomato inhibitors to a more similar structure. Chou-Fasman predictions (46) of these sequences suggest that this is a pturn region with no propensities for formation of a-helical or ,&sheet structures. A second region of five amino acids at the carboxy terminus of these inhibitors shows some similarity which should also be interpreted with caution since it is limited. We present it here since this region would be expected to bind in the specificity pocket of the enzyme. 61 LPWG!? Ascaris 35 Potato GPYVE Tomato GPYV The common sequence appears to be N/APro-Aro-Gly-N/A, where N/A is Gly or Potato

AND

PEANASKY

an aliphatic residue like Leu in the Ascmis inhibitor, and Aro is an aromatic residue. This is consistent with the observation that at least five carboxy-terminal residues of a substrate may influence both the binding and the catalytic parameters of hydrolysis of a substrate by carboxypeptidase A (47). In contrast an x-ray crystallographic study of the complex between the potato-inhibitor and carboxypeptidase A at 2.5 A has shown that only the carboxyterminal three residues, Tyr37, Va138, and Glf19, appear to interact with the enzyme, perhaps because of the sharp turn in the peptide chain due to.Pro36. Further, the inhibitor binds like an extended substrate with the Va138-Gly39 peptide bond cleaved in the complex and the free Gly39 still trapped in the binding pocket of the enzyme (25). Since the inhibitor is small, perhaps resolution of the structure by NMR or x-ray analysis of the structure of its complex with carboxypeptidase A will help reveal the mechanism of action and allow a direct structural comparison of it with the potato inhibitor. ACKNOWLEDGMENTS We are grateful to Dr. David Fass of the Mayo Clinic for the use of his sequencing facility and for his critical review of the manuscript. We thank Professor M. Laskowski, Jr., from Purdue University who permitted some early experiments to be performed at his sequencing facilities. The efforts and contributions of Dr. I. Kato and W. H. Kohr from that laboratory are noted with gratitude. REFERENCES 1. PEANASKY, R. J., ABU-ERREISH, G. M., GAUSH, C. R., HOMANDBERG, G. A., O’HEERON, D., LINKENHEIL, R. K., KUCICIS, U., AND BABIN, D. R. (1974) in Bayer Symposium V, Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L. J., and Truscheit, E., Eds.), pp. 649-666, Springer-Verlag, New York. 2. ABU-ERREISH, G. M., AND PEANASKY, R. J. (1974) .I Bid Chem 249,1558-1565. 3. ABU-ERREISH, G. M., AND PEANASKY, R. J. (1974) J. Biol Chem. 249,1566-1571. 4. PEANASKY, R. J., AND LASKOWSKI, M. (1960) Bb chim Biophys. Acta 37,167-169. 5. RHODES, M. B., MARSH, C. L., AND KELLEY, G. W., JR. (1963) Exp. Parasitd 13,266-272.

PRIMARY 6. PEANASKY,

STRUCTURE

R. J., AND Szucs,

OF

CARBOXYPEPTIDASE

M. M. (1964)

J.

Bid

P., AND

FRAEFEL,

W. (1967)

Helv.

Chim Acta 50,2078-2087. 8. PUDLES,

J., ROLA,

F. H., AND MATIDA,

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