Immunochemical Studies and Complete Amino Acid Sequence of the ...

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Nov 1, 1991 - The complete amino acid sequence of the streptokinase (SKase) of Streptococcus pyogenes M type 12 strain. A374, isolated from a patient with ...
INFECTION AND IMMUNITY, Jan. 1992,

p.

Vol. 60, No. 1

278-283

0019-9567/92/010278-06$02.00/0 Copyright C 1992, American Society for Microbiology

Immunochemical Studies and Complete Amino Acid Sequence of the Streptokinase from Streptococcus pyogenes (Group A) M Type 12 Strain A374 HISASHI OHKUNI,1* YUKO TODOME,1 HIROSHI SUZUKI,' MANABU MIZUSE,l NAOTO KOTANI,2 KAZUHIKO HORIUCHI,2 NORIKO SHIKAMA,2 AKIRA TSUGITA,2 AND KENNETH H. JOHNSTON3 Division of Immunology, Institute of Gerontology, Nippon Medical School, 1-396, Kosugi-cho, Nakahara-ku, Kawasaki 211, Kanagawa,' and Research Institute for Biosciences, Science University of Tokyo, Noda 278, Chiba,2 Japan, and Department of Microbiology, Immunology and Parasitology, Louisiana State University Medical Center, New Orleans, Louisiana 701123 Received 17 June 1991/Accepted 1 November 1991

The complete amino acid sequence of the streptokinase (SKase) of Streptococcus pyogenes M type 12 strain A374, isolated from a patient with poststreptococcal glomerulonephritis (PSGN), was determined. The epitope domain for the monoclonal antibody N-59, which cross-reacts with SKases of both the PSGN-associated strain and S. equisimilis H46A (a non-PSGN-associated strain), was predicted to be localized in residues 370 to 374. The epitope domain specific for monoclonal antibody RU-1, which reacts only with the PSGN-associated SKase, was localized to residues 164 to 236.

were removed by centrifugation, and the supernatant fluid was concentrated to 1/20 of its original volume with PM 10 (Amicon Corp., Lexington, Mass.). The concentrated solution was brought to 60% saturation with ammonium sulfate and allowed to stand at 4°C overnight. After centrifugation, the pellet was dissolved in a small volume of 0.01 M ammonium bicarbonate, dialyzed against the same solution, and lyophilized. A374 SKase was purified essentially as reported by Johnston and Zabriskie (11) and Ohkuni et al. (16). Briefly, lyophilized material was dissolved in 50 mM Tris-HCI buffer, pH 7.5, and applied to a column of DEAESepharose CL-6B (Sigma Chemical Co., St. Louis, Mo.) equilibrated with 50 mM Tris-HCI buffer. The column was developed with an NaCl gradient (0 to 0.5 M) in 50 mM Tris-HCI buffer. Fractions containing SKase eluted from a DEAE-Sepharose CL-6B ion-exchange column were applied to a Sephadex G-100 (Pharmacia Fine Chemicals, Uppsala, Sweden) column and eluted with 0.1 M phosphate buffer, pH 7.5, containing 0.2 M NaCl. SKase-containing fractions were dialyzed against 25 mM piperazine (Sigma Chemical Co.), pH 6.0, and applied to a chromatofocusing (PBE 94; Pharmacia) column. The column was developed with polybuffer 74 (Pharmacia), diluted 1:10, and titrated to pH 4.0 with 1 N HCI. SKase was identified by sodium

Streptokinase (SKase), a 46-kDa extracellular protein produced by the majority of group A, C, and G streptococci, interacts with human plasminogen to form SKase-plasmin complexes (1). It has been proposed that these complexes facilitate the spread of streptococci in the development of focal infection (17); however, it has recently been proposed that certain SKase variants are intimately involved in the initiation of poststreptococcal glomerulonephritis (PSGN) (5-7). Genetic (8-10, 14, 23, 24), chemical (3, 4), and immunological (2, 11, 25) analyses of SKases from different streptococcal groups and strains have suggested that as a group, SKases are quite heterogeneous. We have previously reported (16) that a monoclonal antibody (MAb), N-59, prepared against SKase from a group A, M type 12 streptococcal isolate (strain A374) from a patient diagnosed as having had PSGN recognized an epitope also present in SKase from a group C streptococcus (Streptococcus equisimilis H46A) which is not a PSGN isolate. However, another MAb, RU-1, prepared against SKase from strain A374 reacted only with the homologous antigen and not with SKase from strain H46A. In this report, we present data to delineate specific domains of the A374 SKase molecule which represent epitopes recognized by MAb RU-1 and MAb N-59. Definition of these domains was accomplished by partial fragmentation and Western blot (immunoblot) analysis; the specific amino acid sequence of these epitopes was determined by the use of conventional protein sequencing methods which resulted in the determination of the complete amino acid sequence of A374 SKase.

dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (13, 15) and immunodot (11) and Western blot (11, 18) analyses using MAbs against A374 SKase. As ampholytes used in chromatofocusing interfered with subsequent amino acid analyses and proteolytic digestions, samples were desalted by gel permeation chromatography with a Biogel P-10 (Bio-Rad Laboratories, Richmond, Calif.) column (1.0 by 90 cm) equilibrated with 0.1 M pyridine-acetate buffer adjusted to pH 8.0 with collidine. Production of MAb to A374 SKase. Murine MAb N-59 was prepared against SKase isolated from strain A374 (16). MAb RU-1, prepared by J. Friedman, was kindly supplied by J. B. Zabriskie, Rockefeller University, New York, N.Y. SDS-PAGE. Electrophoretic separation of cyanogen bromide (CNBr)- and staphylococcal V8 protease-generated

MATERIALS AND METHODS Purification of SKase. Streptococcus pyogenes (group A) M type 12 strain A374, isolated from a patient diagnosed as having had PSGN, was grown in a chemically defined medium (22) with constant pH control at 37°C for 18 h. Cells * Corresponding author. 278

_

EPITOPE DOMAIN AND COMPLETE SEQUENCE OF STREPTOKINASE

VOL. 60, 1992

279

I 10 20 30 40 60 60 70 H-IAOPWLLDiIP$YNNSQLVYSYAOTVBGTNQEI SLXFFEI DLTSRPAHCGCTEQCLSPXSXPFATNSSAI _m Im

CNBr Vs LEP Trp

_

_

b

o! C

71 100 80 90 110 120 130 140 PHKLEKADLLXAIQEQLI ANYHSNDOYFVY I DJASDAT I TDINOXYYFADRDDSYTLPTQPYQEFLLSOH

CNBr V8 LEP

_~~~~~~~~

Trp 150

141

~

160

180

170

I

190

200

210

VRYRPYQPXAVHNSAERVNVt4YEYS7YSETONLDPTPSLXEnYHLTTLAVODSLSSQBLAAI AQFI LSXB

CNBr Vs LEP

-

=

Trp 211

220 230 240 260 260 270 280 HPDYI I TXRD5S I YTHDND I FIT I LPI4DQEFTYIi I KDREQAYXANSXTO I VETNNTDLI SEXYTYLXXI

CNBr Vs LEP Trp

1__J

281

290

' A z - -E N

-1

300

310

320

330

340

350

BEPYDPFDRSHLXLFT IXYYDYDT4ELLXSEQLLTAS3ERNLDIRDLYDPRDKAXLLYNNLDAFO I NDYTL

CNEr Vs

LEP Trp

__~~I

m

= 360 351 370 380 390 400 410 TGKYEDNI4HDtRI ITVYNOK1WKCOHIASYHLAYDXDRYTHEZREYYSYLMrOtPI PDtXDX-OH

CNBr V8 LEP

-~~~~~~~~~~~~~~~~~-I*

Trp FIG. 1. Amino acid sequence of A374 SKase. The amino acid sequence of the A374 SKase peptide was derived by sequencing peptides obtained from treatment with CNBr, V8 protease (V8), lysyl endopeptidase (LEP), and TPCK-treated trypsin (Trp). Bars represent isolated fragments. The shaded and closed bars represent those regions which were determined by sequencing. The CNBr cleavage fragments with molecular masses of 35, 29, and 14 kDa were isolated by SDS-PAGE, and parts of their sequences were determined (shaded bars). These fragments corresponded to residues 71 to 369, 1 to 237, and 236 to 369, respectively. Other CNBr cleavage peptides, which consisted of residues 1 to 70 and 370 to 414, were isolated by HPLC, and the former peptide was further digested with Asp-N-protease. V8 protease digestion of the native SKase resulted in the generation of three fragments. These fragments, which had molecular masses of 20, 14, and 11 kDa, were isolated by SDS-PAGE, and parts of their sequences were determined (shaded bars). These fragments corresponded to residues 1 to 163, 273 to 414, and 273 to 374, respectively. V8 protease digestion was also carried out with the heat-denatured SKase, and lysyl endopeptidase digestion was carried out with the native SKase. Both digests were isolated separately by reverse-phase HPLC. Tryptic peptides were chromatographed on Biogel P-10 column. The sequences of these peptide fragments were determined (closed bars).

peptide fragments was performed by SDS-PAGE (13) with a 15% polyacrylamide separating gel and a 5% polyacrylamide stacking gel. Duplicate gels (20) were prepared to permit simultaneous Western blot analysis and protein chemical

analysis. For protein chemical analysis, one plate was stained with Coomassie brilliant blue (Sigma), and individual bands were extracted with 70% formic acid (19). Western blot analysis. Peptide fragments resolved by SDS-

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INFECT. IMMUN.

TABLE 1. Amino acid composition of A374 SKase

No. of residues as determined froma: Sequence Analysis 59 59 27 30 29 30 44 44 20 18 25 24 23 26 3 4 18 22 36 37 16 22 15 16 28 29 11 12 21 20 19 20 1 414

Amino

acid

Asx Thr Ser Glx Gly

Ala Val Met Ile Leu Tyr Phe Lys His Arg Pro Trp Total a Values were calculated on the basis that the number of Glx residues = 44.

PAGE were transferred to a nitrocellulose membrane (SS BA85; Schleicher & Schuell Co., Dassel, Germany) with a constant current (1 A) for 1 h according to the method of Towbin et al. (18). After nonspecific membrane protein binding sites were blocked with 10 mM Tris-HCl buffer (pH 8.2) containing 0.5% (vol/vol) Tween 20, 0.5 M NaCl, and 0.04% NaN3 for 1 h at room temperature, the membranes were probed with either MAb N-59 or MAb RU-1. After

A

B

1

2

3ter..

14K11 K_

FIG. 2. SDS-PAGE pattern (A) and Western blot analysis (B) of fragments of A374 SKase partially digested with V8 protease. (A) After SDS-PAGE, the gel was stained with Coomassie blue. (B) After SDS-PAGE, the peptide fragments were transferred to a nitrocellulose membrane, and the membrane was probed with either MAb N-59 (lane 1) or MAb RU-1 (lane 2). Molecular weights are indicated in thousands.

1

2

3

35K _ 29 K

_

-14K

-4.5K

FIG. 3. Staining pattern and Western blot analysis of partial CNBr-cleaved fragments. After SDS-PAGE, the peptide fragments were transferred to a nitrocellulose membrane. Lane 1 was stained with India ink, lane 2 was reacted with MAb N-59, and lane 3 was reacted with MAb RU-1. Molecular weights are indicated in thousands on the right.

incubation for 2 h, the membranes were developed according to the method of Johnston and Zabriskie (11) using alkaline phosphatase-labelled goat anti-mouse immunoglobulin G antibody (Cappel Laboratories, Cochranville, Pa.). Amino acid analysis. Hydrolysis of the protein or peptides was performed at 158°C for 22.5 and/or 45 min in an acid-vapor mixture, HCl-trifluoroacetic acid-water (6:1:3 vol/vol/vol) containing 0.1% phenol (21). Amino acid analysis was carried out with an amino acid analyzer (Irica A-500; Irica Co., Kyoto, Japan). N-terminal sequence analysis. Automatic Edman degradation was performed with a pulse-liquid sequencer (ABI 477A; Applied Biosystems, Foster City, Calif.), and phenylthiohydantoin amino acids were analyzed with an AP-302

reverse-phase high-pressure liquid chromatography (HPLC) column (S-6 300A ODS; Yamamura Chemicals Laboratory, Kyoto, Japan) connected to the sequencer. Succinylation. Before trypsin digestion, protein samples were succinylated with succinic anhydride (12) and subsequently chromatographed on Biogel P-10 equilibrated with 70% formic acid. Enzyme digestion. Partial digestion with staphylococcal V8 protease (Miles Laboratories, Inc., Naperville, Ill.) was carried out at 37°C for 6 h in 0.1 M pyridine-acetate buffer, pH 6.5, containing 0.1 mM CaCl2. Peptides were separated by SDS-PAGE (15% polyacrylamide). Digestion with V8 protease was also carried out at 37°C for 16 h after the sample was denatured for 10 min at 100°C. Achromobacter lyticus lysyl endopeptidase (Wako Pure Chemicals Co., Osaka, Japan) digestion was performed at 37°C for 6 h in 0.1 M pyridine-acetate-collidine buffer, pH 8.2. L-1-p-tOsylamino-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biochemicals, Freehold, N.J.) digestion was performed at 37°C for 16 h in 0.1 M pyridine-

EPITOPE DOMAIN AND COMPLETE SEQUENCE OF STREPTOKINASE

VOL. 60, 1992

A374-SKase

1

164

236

374

370

NH2- C

281

414 --I -COOH

B

A PV 2

PV 1

PV 3

CNBr 2

CNBr 3 .I g III11111101;009WAV.*AVff0OA

111,

I

CNBr 4 1=

I

CNBr 1

FIG. 4. Epitope domain of A374 SKase. The open bars represent the regions which did not react with either MAb N-59 or MAb RU-1. The solid bars represent the MAb N-59-reacting regions. The stippled bars represent the MAb RU-1-reacting regions. Three V8 protease-digested fragments having molecular masses of 20, 14, and 11 kDa were designated PV 1, PV 2, and PV 3, respectively. CNBr cleavage of A374 SKase resulted in the generation of four fragments with molecular masses of 35, 29, 14, and 4.5 kDa, which were designated CNBr 1, CNBr 2, CNBr 3, and CNBr 4, respectively. Numbers above the map are the residue numbers of the A374 SKase, which were predicted from the sequences of amino acid compositions, molecular masses, and cleavage specificities. A, specific epitope domain against MAb RU-1; B, common epitope domain against MAb N-59.

acetate-collidine buffer. The resulting peptides were separated by reverse-phase HPLC with an AP-302 column with a linear gradient of 0 to 80% acetonitrile in 0.1% trifluoroacetic acid, by using a 600E Multisolvent Delivery System (Waters, Div., Millipore Corp., Milford, Mass.). Digestion of Asp-Nendoproteinase from Pseudomonas fragi (Boehringer GmbH, Mannheim, Germany) was carried out with a CNBr peptide at 37°C for 1 h in 0.1 M pyridine-acetate-collidine buffer, pH 8.0. CNBr cleavage. CNBr cleavage was carried out in 70% formic acid with the addition of CNBr (molar excess of methionine residues = 50) at room temperature for 48 h, and the resulting peptide mixture was fractionated by SDSPAGE (15% polyacrylamide). RESULTS AND DISCUSSION Complete amino acid sequence of A374 SKase protein. Although the A374 SKase contains four methionine residues, the mild CNBr cleavage reaction resulted in four fragments: CNBr 1 (residues 71 to 369), CNBr 2 (residues 1 to 237), CNBr 3 (residues 238 to 369), and CNBr 4 (residues 370 to 414). Three (CNBr 1, CNBr 2, and CNBr 3) of these fragments were isolated by SDS-PAGE, and parts of their sequences were determined. Cleavage at Met-346 was not observed. The partial N-terminal sequence of fragment CNBr 4, which was isolated by HPLC, was determined twice to confirm the sequence. The proteolytic V8 digestion of the native SKase resulted in the generation of three fragments: PV 1 (residues 1 to 163), PV 2 (residues 273 to 414) and PV 3 (residues 273 to 374). These fragments were isolated by SDS-PAGE, and parts of their sequences were determined. Both V8 protease digestion of the heat-denatured sample and lysyl endopeptidase digestion of the native sample were also carried out, and the digests were separated by reverse-phase HPLC. These peptides covered 93% of the entire sequence with the following regions not covered: residues 74 and 75, 1% to 198, 250 to 262, 333 to 353, and 372 to 386. The sequences of the peptide obtained by CNBr cleavage covered residues 74, 75, 250,

and 372 to 386, but the sequences between residues 196 and 198, 251 and 262, and 333 and 353 remained undetermined. To fill in the unresolved residues, a third digestion technique was carried out. The native SKase was succinylated and digested with trypsin. Trypsin is specific for lysyl and arginyl peptide bonds and digests arginyl peptide bond only after succinylation of lysine residues. The sequences of the tryptic peptides covered all of the remaining peptide sequences with the exception of the overlapping region at residues 59 and 60. The peptide fragment CNBr 5 (residues 1 to 70), obtained by cleavage for 72 h and isolated by HPLC, was digested with Asp-N-protease, and one of the resulting peptides (residues 41 to 70) was sequenced. These results overlapped residues 59 and 60. C-terminal analysis of protein by carboxypeptidase B digestion resulted in a lysine residue which coincided with the C terminus of the SKase sequence described above. In this manner, the complete amino acid sequence of A374 SKase was determined (Fig. 1). The amino acid composition of the A374 SKase from the sequence coincided with that from the amino acid analysis shown in Table 1. The A374 SKase consists of 414 residues in a single polypeptide chain having a molecular weight of 47,169. Localization of epitope domains in A374 SKase. MAb RU-1, which reacts only with SKases secreted by group A streptococci associated with PSGN (16), and MAb N-59, which reacts with SKases secreted by both PSGN- and non-PSGNassociated group A streptococci, were analyzed for immunological reactivity to peptide fragments of A374 SKase. Partial digestion with V8 protease generated three major peptide fragments, PV 1, PV 2, and PV 3 having molecular masses of 20, 14, and 11 kDa, respectively (Fig. 2A). Western blot analysis with MAb N-59 indicated reactivity with fragments PV 2 and PV 3 but not with fragment PV 1 (Fig. 2B, lane 1); however, MAb RU-1 did not react with any of the PV fragments (Fig. 2B, lane 2). The heavy bands which were digested insufficiently with V8 protease are visible in Fig. 2, and these bands had epitopes reacting with both MAb N-59 and MAb RU-1. CNBr cleavage of the

282

INFECT. IMMUN.

OHKUNI ET AL.

A374 NZ131 USA275 SF130/13 F665 H46A G19908

30 40 50 60 70 10 20 1 H-IAGPEWLLDRPSVNNSQLVVSVAGTVBGTNQEISLKFFEIDLTSRPAHGGKTEQGLSPKSKPFATNSSAM 2 2 22 2:: 2 222 2 2 2 22 2 22 2 2 2S S:sDKGs 2 S S 22S 2 22 2 2 22 222 22 HH-2s2YG::P:2:PI:222222:M: :I:: :DKKVFIN:2::::22:Q:22222222222s:::222DNG:2 H-

2 H-: 2 tY: 2222222ss 222

H-: 2

2 222

71 A374 NZ131 USA275 SF130/13 F665 H46A G19908

A374 NZi31 USA275 SF130/13 P665 H46A G19908

NZ131 USA275 SF130/13 P665 H46A G19908 A374 NZ131 USA275

2

2 22 2

2

2 2

2

22

2

2:D: 2 2 2

2

2 22 22 222 2

2

2

2 2

2

2

L2 2 D:G: 2

80

120 130 140 90 100 110 PHKLEKADLLKAIQEQLIANVHSNDGYFEVIDFASDATITDRNGKVYFADRDDSVTLPTQPVOEFLLSGH R:

2

:: :2 ::2 222:::22 24 2 222 2160 21702222 190 VK:21 180 200 22::R 222::2P22:KS2D2R2222::D:222222:2KsV: :22222222:K:GI:::tQ2t:S22K::2 tsJ ttDsE:T:QsTssNP:DssR:G:KT::s::X:It:TIT:::sLss::SttNsT SsssR:ssEstPt2 141 210 180 190 200 150 160 170

Ss3t::KEl:|:PIss:::X:DsEsTDQ::::NP::s:G:sDTKsK:s:I:sTsIs|Tl:::s:sst:NsN 2 2 Kz 22222222222222:t2 222 222D2 22 2D 22 2L2RNQ2 22222222222222222222222 22K 2:::as:tsEsPitOsKStD:Est:T:QD:LNsD:s3:As:DT:X:ss::ltsKTIT:::I::Q:tSs:NKlN 2 R2 2 2 22222222222 2 22 2 2 2 2 2 N: 2 2 SaKER: 222222 A2 222 22222222 22222R :iGR:TsKE:PQtQ:KS:D2ETQsTPLNPD:RVG:KN::KL::K:S2212:TIT22I2LQ2252:::N:T :PIQTP:KS :DIRT:Q:TPLNPD:K::V:NKDTKLKR:2:SNY:TLNI:L:Q:::S:::NET 22T:RsKE VRVRYIITKAVHNSAERVNVNYI OFYIDRQYAS7IETNTLSKYLK 22222 :KE:PIQ:Q:KS:D:E:T:Q:TPLNP:D: 2R2G2 DTKL2K2sitIt,TIT: 22 LiQi iS:sNiN 22222 2KE2P2Q2Q2KS2D2E2T2Q2TPLNP :D: :R:A: DTKL.2K2 :1:ItTIT: 22 L2Q2 :5: N:N

270 280 250 260 230 240 K EFTYHIKDREQAYKANS TAGIVEKTNNTDLISEKYYVLKKG EEPYDPQ

211

A374

2 2

220

2 2G2T2YE2 222222 222 2222 22 2 2 2 T:YE: 22 2 2

2222222

V:N: 222 EI:P2 22 K: 2

2222:sV:

222222

Q2

2 :G:T:YE: 22222:2::::: 22222222 2 2:RV:N: 222 R12K2S21.N2E1 222222 22222 222 2 :G:T:YE: 22 2 2 2222222 2222 2252S222S : V:N: :2: RI :K:S:LN:EI 2222222222222222 320 330 340 350 281 290 300 310 EEPYDPFDRSHLKLFTIKYVDVDTNELLKSEQLLTASERNLDFRDLYDPRDKAKLLYNNLDAFGIMDYTL

SF130/13 P665

H46A G19908

222222222222222222222222222222tss 390 400 410

21(22222222222222222222N22222222222222222

360

351

370

A374

TGKVEDNHNDTNRIITVY?.

NZ131

2 2

2

222222

380

~EN&SYHLAYDKDRYTEEEREVYSYLRYTGTPIPDNPKDK-OH 22222 2 222 222222222222 D2t-OH

222 222 22222222222

USA275 SF130/13

2s2 s22

222222 :sKsAKG 222s222s2 L2s 222 DKN1(sW:::

P665 H46A G19908

2D2 2222 22

iDaD222222

-OH KAs2 2222s22222222 iN: i-OH -OH

22 22222 2 222 2 222222222222222222222222N 2-OH 2 2222 2 222 2 222222 2 22222222222222222222tN:t-OH

FIG. 5. Comparison of amino acid sequences of SKases from group A, C, and G streptococci. Strains: A374, S. pyogenes M type 12; NZ131, S. pyogenes M type 49 (8); USA275, S. pyogenes T type 3 (10); SF130/13, S. pyogenes M type 1 (23); F665, S. pyogenes M type 33 (10); H46A, S. equisimilis (group C) (14); G19908, a group G Streptococcus strain (24). A374, NZ131, and USA275 are PSGN-associated strains. F665, SF130/13, H46A, and G19908 are strains not associated with PSGN. Boxed sequences indicate domains specific for MAbs (see Results and Discussion).

native molecule generated four fragments, CNBr 1, CNBr 2, CNBr 3, and CNBr 4, having molecular masses of 35, 29, 14, and 4.5 kDa, respectively (Fig. 3, lane 1). MAb RU-1 reacted with fragments CNBr 1 and CNBr 2 but not with fragments CNBr 3 and CNBr 4 (Fig. 3, lane 3). MAb N-59 did not react with any of the CNBr fragments (Fig. 3, lane 2). Although heavy bands were also observed for the CNBr cleavage fragments, these bands were not subjected to amino acid sequencing. Figure 4 schematically represents the immunological reactivities of MAbs N-59 and RU-1 with these peptides. These immunological assays demonstrated that A374 SKase has at least two epitope domains. By the process of subtraction, it may be proposed that MAb RU-1 has specificity for the domain consisting of amino acids 164 to 236 and MAb N-59 has specificity for the domain defined by amino

acids 370 to 374 (as shown by the boxes in Fig. 5). The inability of MAb N-59 to recognize fragment CNBr 4 may be explained by the disruption of the conformation of the epitope defined by residues 370 to 374 by the absence of amino acid residues preceding this domain. Sequence similarity between SKases. Previous investigations done in our laboratory (16) have demonstrated that MAb RU-1 is specific for SKases secreted by group A streptococci associated with PSGN. In this study, 16 isolates, of which 14 secreted an SKase which was recognized by MAb RU-1, were studied. All SKase molecules studied to date have the same number of amino acids (414) but exhibit amino acid heterogeneity (Fig. 5). However, this heterogeneity is localized primarily within an internal variable domain (amino acids 146 to 218). Johnston et al. (9, 10) demonstrated that this internal domain could be classified

VOL. 60, 1992

EPITOPE DOMAIN AND COMPLETE SEQUENCE OF STREPTOKINASE

into six classes, three of which (I, II, and VI) exhibited >95% homology with each other and were associated with SKases from PSGN-associated group A streptococci. The other classes (III, IV, and V) demonstrated