Vol. 59, No. 5

INFECTION AND IMMUNITY, May 1991, p. 1818-1822

0019-9567/91/051818-05$02.00/0 Copyright © 1991, American Society for Microbiology

Synthetic Peptide Substrates for the Immunoglobulin Al Protease from Neisseria gonorrhoeae (Type 2) STEPHEN G. WOODt AND JAMES BURTON* Evans Memorial Department of Clinical Research, University Hospital, Boston University Medical Center, Boston, Massachusetts 02118 Received 7 December 1990/Accepted 18 February 1991

Neisseria gonorrhoeae secretes proteases which inactivate human immunoglobulin Al (IgAl) by cleavage of specific peptide bonds in the hinge region. The type 2 IgAl protease (EC 3.4.24.13) is secreted as a 169-kDa precursor which undergoes autoproteolysis at three sites (A, B, and C) to release the 106-kDa active form of the enzyme (J. Pohlner, R. Halter, K. Beyreuther, and T. F. Meyer. Nature [London] 325:458-462, 1987). Synthetic decapeptides consisting of five residues on each side of the three autoproteolytic cleavage sites and their potential pentapeptide catabolites were prepared by solid-phase synthesis. Cleavage of the decapeptides by the type 2 IgAl protease from N. gonorrhoeae was monitored by high-performance liquid chromatography. Peptides homologous with the amino acid sequences around the B and C sites are cleaved by the IgAl protease. Amino acid analysis and Edman degradation show that the cleavage products have both the composition and amino acid sequence which would be expected from cleavage at the predicted sites. Km values of 1.35 mM and 3.43 mM and k,.t values of 280 pmol/h/U and 439 pmollh/U for the site B and site C peptides, respectively, were determined. The catalytic efficiency (kcat/Km) for the synthetic substrates is about 10% of that reported for intact IgAl. Cleavage of the peptides is inhibited by IgAl protease inhibitors such as the tetrapeptide substrate analog inhibitor HRP-48, human colostrum, and a peptide-boronate transition state inhibitor. An extract from an N. gonorrhoeae construct lacking active IgAl protease failed to cleave the synthetic substrate, while an extract from the control construct which secretes active enzyme completely hydrolyzed the synthetic peptide. Neither the site A peptide nor synthetic decapeptides encompassing cleavage sites in the hinge region of IgAl are hydrolyzed by IgAl protease. These are the first synthetic substrates to be reported for any IgAl protease.

The human pathogen Neisseria gonorrhoeae secretes proteases which inactivate human secretory immunoglobulin Al (IgAl) by cleavage on the C-terminal side of specific prolyl residues in the hinge region (11, 13). The proteases are highly specific and do not cleave synthetic peptides which are homologous with the IgAl hinge region (3). The lack of synthetic substrates has limited both detailed molecular studies of the mechanism of action of the enzyme and development of a simple assay. Currently employed assays are based on cleavage of the natural substrate (IgAl)

in the hinge region of human IgAl were prepared and tested as substrates for the type 2 IgAl protease. MATERIALS AND METHODS

Peptide synthesis. Techniques for the chemical synthesis, purification, and characterization of the peptide substrates and fragments have been described previously (4). Details of these syntheses will be presented elsewhere. The five decapeptides tested as substrates were labeled at two specific prolyl residues on either side of the putative cleavage site by incorporation of a tritiated amino acid during the course of chemical synthesis. Locations of the label and specific activity are given in Table 1. Peptides which would constitute fragments from cleavage were deliberately synthesized without a label. All peptides are homogeneous and have been fully characterized by amino acid analysis, high-performance liquid chromatography (HPLC), thin-layer chromatography, molar rotation, specific activity, and E. The sequences, specific activities, and HPLC retention times of the peptides are listed in Table 1. HPLC of peptides. Peptide digests were subjected to HPLC on a model 336 gradient system (Beckman Instruments, Palo Alto, Calif.) employing either a semipreparative (Beckman, 10 by 250 mm, C-18, 5,u) or an analytical (Beckman, 2.5 by 250 mm, C-18, 5,u) column, eluted at flow rates of 2 and 1 ml/min, respectively, with solution A. A gradient from 0 to 100% solution B over 20 min was used. Solution A contains 0.2% CF3COOH-H20, and solution B contains 0.2% CF3COOH-CH3CN. A fraction collector (Gilson, Middleton, Wis.) was used to collect fractions from the HPLC. Each fraction was col-

(8, 9, 14). The type 2 protease secreted by N. gonorrhoeae (EC 3.4.24.13) is synthesized as a 169-kDa precursor which undergoes autoproteolysis at three sites (A, B, and C) to release the 106-kDa form of the enzyme (15). (IgAl proteases are classed as zinc proteases on the basis of research by Plaut et al. [14] with the IgAl protease isolated from Streptococcus sanguis. The type 2 IgAl protease isolated from N. gonorrhoeae appears to be a serine protease [1] and should be classed with these enzymes.) The amino acid sequences around the autoproteolytic sites are somewhat different from that of the IgAl cleavage site (Fig. 1). This offers the possibility that synthetic peptides which are homologous with the amino acid sequences around the autoproteolytic sites may be substrates for the type 2 IgAl protease. To this end, synthetic decapeptides which are homologous with the amino acid sequence around the A, B, and C cleavage sites as well as the type 1 and type 2 cleavage sites *

Corresponding author.

t Present address: Procter & Gamble Company, Miami River Valley Laboratories, P.O. Box 398707, Cincinnati, OH 45239. 1818

VOL. 59, 1991

SYNTHETIC PEPTIDE SUBSTRATES FOR IgAl PROTEASE

N. gonorrhoeae IgA 1 Protease

1819

70006000-

Autoproteolytic Sites P2

P1

PI' P2'

5000-

Site A: -Val94" -Lys-Pro-AIa-ProjSer-Pro-Ala-Ala-AsnSite B: -Val" -Val-Ala-Pro-ProlSer-Pro-Gln-Aba-AsnSite C: -Leu1'° -Pro-Arg-Pro-Pro AIa-Pro-Val-Phe-Ser-

40003000-

cpm appled

(cpm recovered

=

2000-

IgA 1 Cleavage Sites

-

168)

0.5008

r2=

0.996

1000-

Type 1: -Thr225 -Pro-Pro-Thr-Pro Ser-Pro-Ser-Cys-CysType 2: -Pro22' -Ser-Thr-Pro-Prc Thr-Pro-Ser-Pro-Ser-

, 0

T

I

I

I

I

I

I

2000 4000 6000 8000 10000 12000 14000 cpm

Applied

FIG. 1. Amino acid sequences around the cleavage sites of human IgAl and pre-prolgAl protease from N. gonorrhoeae by IgAl protease.

FIG. 2. Recovery of acetyl-Leu-Pro-Arg-Pro-Pro-Ser-Pro-GlnAla-Asn-NH2 (HRP-93) from HPLC. cpm, counts per minute.

lected for 15 s (0.5 ml), and aliquots of the fractions were analyzed. The quantity of labeled material recovered from HPLC was determined from a standard curve constructed by chromatographing 50-pul aliquots of five concentrations of the intact substrate and measuring the amount of radioactivity in the various fractions which eluted from the column. The appropriate blanks and 3H standards were also counted. Recovery of both labeled substrates from the HPLC column was approximately 50%. Equation 1 was developed from data collected for the site C peptide (HRP-93) to allow the amount of material in the sample chromatographed to be calculated from the number of counts recovered (Fig. 2). Recovery of the site B peptide (HRP-92) from HPLC was, within experimental error, the same as for the site C peptide (HRP-93).

Determination of enzyme kinetic parameters. The type 2 IgAl protease from N. gonorrhoeae was prepared by previously described techniques (7). The enzyme preparation did not hydrolyze "4C-labeled hemoglobin and thus did not appear to contain nonspecific proteases. The synthetic peptides HRP-91 through HRP-95 (Table 1) were tested for cleavage by dissolving the purified material in Tris (50 mM, pH 10.0), adjusting the pH of the solution to 7.66 with 1 N HCI, and diluting with buffer. Subsequently, a 100-,u aliquot of the neisserial IgAl protease (GC740, 2.2 U/,l) was added so that the final concentration of substrate was 1.00 mM, and the solution was incubated at 37°C. Samples of the digest (100 pul) were subjected to HPLC analysis at 1-h intervals. HRP-92 and HRP-93 (Fig. 3) were cleaved while other peptides remained intact when incubated with the type 2 IgAl protease produced by N. gonorrhoeae. Kinetic parameters (K, kcat) of HRP-92 and HRP-93 were measured by dissolving the peptide substrates in Tris (50 mM, pH 7.66, 475 pul) at concentrations of 2.0, 1.5, 1.0, 0.50, and 0.10 mM. A 25-pA volume of GC740 (2.2 U/pl) was then

cpm applied

where

cpm

is counts

=

per

(cpm recovered . 0.50) - 170 (r2 = 0.99)

(1)

minute.

TABLE 1. Amino acid sequence, HPLC retention times, and specific activites for synthetic peptides

HfRP

Site

91 92 93 94 95 105 106 107 108 109 112 113 114 115 116

A B C 1 2

Sequenceb

Ac-Val-Lys-Pro-Ala-[3H]Pro-Ser-[3H]Pro-Ala-Ala-Asn-NH2 Ac-Val-Val-Ala-Pro-[3H]Pro-Ser-[3H]Pro-Gln-Ala-Asn-NH2 Ac-Leu-Pro-Arg-[3H]Pro-Pro-Ala-[3H]Pro-Val-Phe-Ser-NH2 Ac-Thr-Pro-Pro-Thr-[3H]Pro-Pro-Ser-[3H]Pro-Ser-Cys-Cys-NH2 Ac-Pro-Ser-Thr-Pro-[3H]Pro-Thr-[3H]Pro-Ser-Pro-Ser-NH2 Ser-Pro-Ala-Ala-Asn-NH2 Ser-Pro-Gln-Ala-Asn-NH2 Ala-Pro-Val-Ser-Phe-NH2 Ser-Pro-Ser-Cys-Cys-NH2 Thr-Pro-Ser-Pro-Ser-NH2 Ac-Val-Lys-Pro-Ala-Pro Ac-Val-Val-Ala-Pro-Pro Ac-Leu-Pro-Arg-Pro-Pro Ac-Thr-Pro-Pro-Thr-Pro Ac-Pro-Ser-Thr-Pro-Pro

Retention time

Specific activity

15.8c 16.8c 18.1c 16.3c 16.8c 11.5d 11.2c 13.3c

0.048 0.045 0.013 0.067 0.032

14.8d 16 0d

12.4d 14.2c 16.2c

12.3d 15.2d

a Laboratory identification number. b Ac, acetyl. c Altex C-18 (10 by 250 mm) column, 2.0 ml/min. Solvent A, 0.2% trifluoroacetic acid in water; Solvent B, 0.2% trifluoroacetic acid in acetonitrile, gradient 0

to 100%o over 20 min. d Altex C-18 (4.6 by 250 mm) column, 1.0 ml/min. Solvent A, 0.2% trifluoroacetic acid in water; Solvent B, 0 to 100% over 20 min.

0.2% trifluoroacetic acid in acetonitrile, gradient

1820

INFECT. IMMUN.

WOOD AND BURTON

1.50

0

"4 S 0 a

0.70

.0 44

-0.10 I 0

VI 200

400

I

I

600

500

J

1000

'

I

1200

1400

1600

Time (3.) FIG. 3. HPLC of the mixture from the partial hydrolysis of the site C peptide acetyl-Leu-Pro-Arg-Pro-Pro-Ser-Pro-Gln-Ala-Asn-NH2 (HRP-93) by the type 2 IgAl protease from N. gonorrhoeae. HPLC conditions were as described in Table 1, footnote c.

added to the various solutions of substrate. At 10-min intervals, 100-pAl aliquots were taken from the digests for HPLC analysis. Fractions from the HPLC containing the decapeptide were collected and counted, and the amounts of substrate remaining in the reaction mixture at various times were calculated by using equation 1. The rate of the cleavage reaction (-dc/dt) remained constant over the 1-h period used to determine the kinetic parameters. The reciprocal of the rate of cleavage (l/v) was plotted against the reciprocal of the concentration of the synthetic peptides (1/S) (LineweaverBurk plot) to yield Km and kcat (6). The Lineweaver-Burk plot used to determine Km and kcat for the site B peptide (HRP-92) is shown in Fig. 4. Amino acid sequence determination. Edman degradation was used to determine the sites of cleavage of the peptide substrates. This was done with an Applied Biosystems sequencer (Applied Biosystems, Culver City, Calif.). A solution of the site C peptide (HRP-93) was treated with 30 Km = 1.35 mM kcat = 280 pmol hr-'u-' r' = 0.999

-1000 0

5,000 1/S (M-')

10,000

.B

FIG. 4. Lineweaver-Burk plot for cleavage of the site peptide acetyl-Val-Val-Ala-Pro-Pro-Ser-Pro-Gln-Ala-Asn-NH2 (HIRP-92) by the type 2 IgAl protease from N. gonorrhoeae.

IgAl protease until approximately 40% of the peptide chains were cleaved (Fig. 3). The solution was then deproteinized with an equal volume of 10% trichloroacetic acid solution, extracted with Et2O, and the aqueous phase was lyophilized. The lyophilizate was then subjected to gas-phase Edman degradation as previously described (5). Amino acid analysis. The amino acid compositions of various fractions from HPLC were determined by pooling and lyophilizing fractions containing radioactive label. The lyophilizate was hydrolyzed at 110HC for 24 h under vacuum in 6 N HCl (2). Amino acid analyses of the hydrolysate were performed on a Beckman D-6000 analyzer (Table 2). Enzyme specificity studies. In order to determine whether the hydrolysis of the synthetic peptides was due to IgAl protease and not some contaminating enzyme in the preparation, studies were done both with relatively specific inhibitors of IgAl protease (1, 7, 17) and with extracts from bacteria which had been engineered so that they did not secrete IgAl protease (10). IgAl protease inhibitors. (i) Substrate analog. The site C decapeptide (HRP-93) was dissolved in 475 ,ul of 50 mM Tris to make a 1 mM solution. Enough tetrapeptide IgAl protease inhibitor (HRP-48; 50% inhibitory concentration, 5.3 ,uM) to

TABLE 2. Elution times and compositions of cleavage fragments of HRP-92 and HRP-93 Peptide and Amino elution timea acids (min) HRP-92 11.2 .... Ser, 0.85; Pro, 0.96; Glx, 1.03; Ala, 1.04; Asx, 1.12 14.2 .... Val, 1.67; Ala, 1.15; Pro, 2.18 HRP-93 13.3 .... Ala, 1.08; Pro, 1.02; Val, 0.83; Ser, 1.08; Phe, 0.99 16.2 ........... Leu, 1.01; Pro, 2.99; Arg, 0.99 a HPLC conditions were as described in Table 1, footnote c.

VOL. 59, 1991

SYNTHETIC PEPTIDE SUBSTRATES FOR IgAl PROTEASE

yield a 1 mM solution was added, and the pH of the mixture was adjusted to 7.66. Then 25 .l1 of the standard neisserial IgAl protease solution was added, and the digest was incubated at 37°C for 6 h. HPLC analysis using the conditions shown in Table 1 showed that nearly all label eluted at the position of the intact decapeptide substrate (18 min). Less than 5% of the counts eluted at the position of the C-terminal cleavage product (Ala-Pro-Val-Phe-Ser-NH2, HRP-107, 12.1 min). Under identical conditions in which HRP-48 was omitted from the digestion mixture, complete hydrolysis of the synthetic decapeptide substrate occurred. (ii) Human colostrum. HRP-93 was exposed to the IgAl protease as described above except that human colostrum (7) rather than HRP-49 was used as an IgAl protease inhibitor. After 24 h of incubation, HPLC analysis showed