α-Enolase, a Novel Strong Plasmin(ogen) Binding Protein on the Surface of Pathogenic Streptococci

The plasmin(ogen) binding property of group A streptococci is incriminated in tissue invasion processes. We have characterized a novel 45-kDa protein displaying strong plasmin(ogen) binding activity from the streptococcal surface. Based on its biochemical properties, we confirmed the identity of this protein as α-enolase, a key glycolytic enzyme. Dose-dependent α-enolase activity, immune electron microscopy of whole streptococci using specific antibodies, and the opsonic nature of polyclonal and monoclonal antibodies concluded the presence of this protein on the streptococcal surface. We, henceforth, termed the 45-kDa protein, SEN (streptococcal surface enolase). SEN is found ubiquitously on the surface of most streptococcal groups and serotypes and showed significantly greater plasmin(ogen) binding affinity compared with previously reported streptococcal plasminogen binding proteins. Both the C-terminal lysine residue of SEN and a region N-terminal to it play a critical role in plasminogen binding. Results from competitive plasminogen binding inhibition assays and cross-linking studies with intact streptococci indicate that SEN contributes significantly to the overall streptococcal ability to bind plasmin(ogen). Our findings, showing both the protected protease activity of SEN-bound plasmin and SEN-specific immune responses, provide evidence for an important role of SEN in the disease process and post-streptococcal autoimmune diseases. The plasmin(ogen) binding property of group A streptococci is incriminated in tissue invasion processes. We have characterized a novel 45-kDa protein displaying strong plasmin(ogen) binding activity from the streptococcal surface. Based on its biochemical properties, we confirmed the identity of this protein as α-enolase, a key glycolytic enzyme. Dose-dependent α-enolase activity, immune electron microscopy of whole streptococci using specific antibodies, and the opsonic nature of polyclonal and monoclonal antibodies concluded the presence of this protein on the streptococcal surface. We, henceforth, termed the 45-kDa protein, SEN (streptococcal surface enolase). SEN is found ubiquitously on the surface of most streptococcal groups and serotypes and showed significantly greater plasmin(ogen) binding affinity compared with previously reported streptococcal plasminogen binding proteins. Both the C-terminal lysine residue of SEN and a region N-terminal to it play a critical role in plasminogen binding. Results from competitive plasminogen binding inhibition assays and cross-linking studies with intact streptococci indicate that SEN contributes significantly to the overall streptococcal ability to bind plasmin(ogen). Our findings, showing both the protected protease activity of SEN-bound plasmin and SEN-specific immune responses, provide evidence for an important role of SEN in the disease process and post-streptococcal autoimmune diseases. Streptococcus pyogenes is responsible for a wide variety of human diseases that range from suppurative infections of the throat (pharyngitis), skin (impetigo), and underlying tissues (necrotizing fasciitis), to an often fatal toxic shock syndrome, and the post-streptococcal sequelae, rheumatic fever, and acute glomerulonephritis. Bacterial surface proteins play a major role in these disease processes by exhibiting a wide range of functions. As data have become available, it is clear that most surface proteins found on Gram-positive bacteria, particularly those on group A streptococci, have a great deal of structural similarities (1Fischetti V.A. Pancholi V. Schneewind O. Dunney G.M. Cleary P.P. McKay L.L. Genetics and Molecular Biology of Streptococci, Lactococci, and Enterococci. American Society for Microbiology, Washington, D. C.1991: 290-294Google Scholar, 2Fischetti V.A. Pancholi V. Schneewind O. Orefici G. New Perspectives on Streptococci and Streptococcal Infections. Gustav Fischer Verlag Jena Gmbh, Jena, Germany1992: 165-167Google Scholar). Proteins for which the function(s) has been defined have been found to be multifunctional, whereas in others a function has only been attributed to one of two or more domains (2Fischetti V.A. Pancholi V. Schneewind O. Orefici G. New Perspectives on Streptococci and Streptococcal Infections. Gustav Fischer Verlag Jena Gmbh, Jena, Germany1992: 165-167Google Scholar, 3Kehoe M.A. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishing Co., Inc., New York1994: 217-261Google Scholar). Thus, the multifunctional characteristics of these surface proteins increase the complexity of the Gram-positive surface beyond what has been previously imagined. We recently described one such multifunctional protein, streptococcal surface dehydrogenase (SDH), 1The abbreviations used are: SDH, streptococcal surface dehydrogenase; EACA, ε-aminocaproic acid; MES, 2-[N-morpholino]ethanesulfonic acid; Plr, plasmin receptor; plasmin(ogen), plasminogen and plasmin; SEN, streptococcal enolase; PAGE, polyacrylamide gel electrophoresis; PEP, phosphoenolpyruvate; 2-PGE, 2-phosphoglycerate; PVDF, polyvinylidene difluoride; PMSF, phenylmethylsulfonyl fluoride; tPA, tissue-type plasminogen activator; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BSA, bovine serum albumin; cfu, colony-forming units; ASD, azido-salicylamido]ethyl-1–3′-dithiopropionate. 1The abbreviations used are: SDH, streptococcal surface dehydrogenase; EACA, ε-aminocaproic acid; MES, 2-[N-morpholino]ethanesulfonic acid; Plr, plasmin receptor; plasmin(ogen), plasminogen and plasmin; SEN, streptococcal enolase; PAGE, polyacrylamide gel electrophoresis; PEP, phosphoenolpyruvate; 2-PGE, 2-phosphoglycerate; PVDF, polyvinylidene difluoride; PMSF, phenylmethylsulfonyl fluoride; tPA, tissue-type plasminogen activator; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BSA, bovine serum albumin; cfu, colony-forming units; ASD, azido-salicylamido]ethyl-1–3′-dithiopropionate.as a major surface protein on group A streptococci and other streptococcal groups which is structurally and functionally related to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar). SDH also has an ADP-ribosylating activity (5Pancholi V. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8154-8158Crossref PubMed Scopus (155) Google Scholar) and exhibits multiple binding activities to several mammalian proteins such as fibronectin and cytoskeletal proteins (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar). A structurally and enzymatically similar streptococcal protein, Plr, was also identified on group A streptococci, based on its ability to bind plasmin (6Lottenberg R. Broder C.C. Boyle M.D.P. Kain S.J. Schroeder B.L. Curtiss III, R. J. Bacteriol. 1992; 174: 5204-5210Crossref PubMed Google Scholar). SDH, however, is a weak plasmin-binding protein (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar). During our studies characterizing the SDH molecule, we reported that a 45-kDa protein was also found in high amounts on the surface of group A streptococci (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar). While determining the relative plasmin binding activity of SDH with respect to other streptococcal surface proteins, we found that the 45-kDa protein had in fact strong plasmin binding activity. 2V. Pancholi and V. A. Fischetti, unpublished data. 2V. Pancholi and V. A. Fischetti, unpublished data. The plasmin(ogen) system displays a unique role in the host defense by dissolving fibrin clots and serving as an essential component to maintain homeostasis and vascular potency (7Plow E.F. Felez J. Miles L.A. Thromb. Haemostasis. 1991; 66: 32-36Crossref PubMed Scopus (124) Google Scholar, 8Plow E.F. Herren T. Redlitz A. Miles L.A. Hoover-Plow J.L. FASEB J. 1995; 9: 939-945Crossref PubMed Scopus (381) Google Scholar, 9Saksela O. Rifkin D.B. Annu. Rev. Cell Biol. 1988; 4: 93-126Crossref PubMed Scopus (717) Google Scholar). Studies on the ability of Gram-positive bacteria to subvert the fibrinolytic activity of human plasmin(ogen) to their own advantage for tissue invasion have been largely focused on pathogenic streptococci and were described first as early as 1933 by Tillet and Garner (10Tillet W.S. Garner R.L. J. Exp. Med. 1933; 58: 485-502Crossref PubMed Scopus (317) Google Scholar). This property was subsequently attributed to the plasmin(ogen) activator, streptokinase (11Christensen L.R. J. Gen. Physiol. 1945; 28: 363-383Crossref PubMed Scopus (99) Google Scholar), an extracellular 48-kDa protein secreted in culture supernatants (12Castellino F.J. Sodetz J.M. Brockway W.J. Siefring G.E. Methods Enzymol. 1996; 45: 244-257Crossref Scopus (89) Google Scholar). The role of pathogenic bacteria in tissue invasion utilizing this system has recently been reviewed (13Lottenberg R. Minning-Wenz D. Boyle M.D.P. Trends Microbiol. 1994; 2: 20-24Abstract Full Text PDF PubMed Scopus (146) Google Scholar). In the present communication, we describe purification and characterization of the 45-kDa protein and show that it is the major plasmin(ogen) binding molecule on the surface of streptococci. We also show that this protein has significant sequence similarity with one of the important glycolytic enzymes, α-enolase, found generally in the cytoplasm. While bound on the surface of group A streptococci, this 45-kDa protein is found to retain its α-enolase activity, hence we named it SEN (streptococcal surface enolase). It is distinct from the 48-kDa streptokinase (12Castellino F.J. Sodetz J.M. Brockway W.J. Siefring G.E. Methods Enzymol. 1996; 45: 244-257Crossref Scopus (89) Google Scholar, 14Huang T.T. Ferretti J.J. Mol. Microbiol. 1989; 3: 197-205Crossref PubMed Scopus (48) Google Scholar), the 35.8-kDa SDH (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar), the 41-kDa Plr (6Lottenberg R. Broder C.C. Boyle M.D.P. Kain S.J. Schroeder B.L. Curtiss III, R. J. Bacteriol. 1992; 174: 5204-5210Crossref PubMed Google Scholar), or the 45-kDa plasminogen-binding protein, PAM (15Berge A. Sjobring U. J. Biol. Chem. 1993; 268: 25417-25424Abstract Full Text PDF PubMed Google Scholar), all of which have been reported to bind plasmin to varying degrees. α-Enolase has not been previously identified on the surface of bacteria; however, it has been shown to be expressed on the surface of neuronal (16Nakajima K. Hamanoue M. Takemoto N. Hattori T. Kato K. Kohsaka S. J. Neurochem. 1994; 63: 2048-2057Crossref PubMed Scopus (110) Google Scholar), cancer (17Lopez-Alemany R. Correc P. Camoin L. Burtin P. Thromb. Res. 1994; 75: 371-381Abstract Full Text PDF PubMed Scopus (43) Google Scholar), and some hematopoietic cells (18Redlitz A. Fowler B.J. Plow E.F. Miles L.A. Eur. J. Biochem. 1995; 227: 407-415Crossref PubMed Scopus (208) Google Scholar, 19Miles L.A. Dahlberg C.M. Plescia J. Felez J. Kato K. Plow E.F. Biochemistry. 1991; 30: 1682-1691Crossref PubMed Scopus (485) Google Scholar) as a novel plasmin(ogen) receptor. Here, in addition to the structural and functional characterization of SEN, we also describe the biological activity of SEN, the functional consequence of plasmin(ogen) binding to SEN, and the enzymatic activity of SEN-bound plasmin. Group A β-hemolytic streptococcal strains of various M types and standard strains used for streptococcal grouping were from The Rockefeller University Culture Collection (New York, NY) and are listed as follows: M2(D626), M4(F694), M6(D471), M9(F690), M11(F743), M14(T14/46), M15(D176A), M22(D943), M25(B554), M35(C171), M40(C270), M44(C757), M49(B910), M51(A291), M58(D632), M60(D630), M61(D336), M62(D458), M63(D459), M66(D794), group B (0902), group C (C74), group D (D76), group E (K131), group F (F68C), group G (D166B), group H (F90A), group L (D167B), and group N (C559). These strains were grown overnight in Todd-Hewitt broth (Difco) and washed once with 50 mm ammonium bicarbonate followed by two washes in 50 mm phosphate buffer, pH 6.1, to eliminate the presence of any soluble streptokinase, which may interfere with analysis. Type M6 (D471) streptococci (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar) were used for the isolation of SEN, whereas the other strains were used to study the prevalence of the plasmin-binding 45-kDa related proteins in different streptococcal groups and group A serotypes. Purified human plasminogen and plasmin (lysine-plasmin) were purchased commercially (Sigma). Plasmin was also generated from plasminogen by incubation with urokinase (20 units/ml, Sigma) in HBS gel buffer (50 mmHEPES/NaOH, pH 7.4, containing 1 mm MgCl2, 0.15 mm CaCl2, and 0.1% of gelatin) containing 40 mm lysine. Conversion of plasminogen to plasmin was maximal after 1 h at 37 °C. This method consistently converted more than 95% of the single chain zymogen molecule plasminogen to the heavy and the light chain of the plasmin molecule as reported earlier (20Lottenberg R. Broder C.C. Boyle M.D.P. Infect. Immun. 1987; 55: 1914-1928Crossref PubMed Google Scholar). Purified plasminogen and plasmin were radioiodinated with Na125I (17 Ci mg−1, NEN Life Science Products) by the chloramine-T method, using IODO-BEADs (Pierce) as described (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar). The labeled proteins were separated from free iodine by passage over a G-25 column (PD-10, Amersham Pharmacia Biotech) and collection in HBS gel. The labeled proteins were stored at −70 °C. Plasmin was also generated from the 125I-radiolabeled plasminogen by incubation with urokinase (20 units ml−1, Sigma) in HBS gel that contained 40 mm lysine (20Lottenberg R. Broder C.C. Boyle M.D.P. Infect. Immun. 1987; 55: 1914-1928Crossref PubMed Google Scholar). More than 95% of the radioactivity was found to be retained with the plasmin. Thus, the specific radioactivity of the labeled plasmin and plasminogen was found to be essentially the same. Furthermore, the specific radioactivity of commercially available purified plasmin (Sigma) and urokinase-generated plasmin was also the same. Typically, specific radioactivity of the 125I-labeled plasmin/plasminogen was achieved in a range of 1.2–2.0 × 106 cpm μg−1 protein. Proteins in the bacterial cell wall extracts were resolved by 12% SDS-PAGE gels and blotted electrophoretically onto a PVDF membrane as described (21Pancholi V. Fischetti V.A. J. Bacteriol. 1988; 170: 2618-2624Crossref PubMed Google Scholar,22Pancholi V. Fischetti V.A. J. Exp. Med. 1989; 170: 2119-2133Crossref PubMed Scopus (41) Google Scholar). Blots were incubated at room temperature for 3 h in a blocking HBST gel buffer (50 mm HEPES/NaOH, pH 7.4, containing 0.15 m NaCl, 1% acidified BSA, 0.5% gelatin, 0.5% Tween 20, 0.04% NaN3) and probed for 4 h at room temperature in the HBST gel buffer containing 2.0 mmPMSF and 125I-labeled human plasminogen or plasmin 3 × 105 cpm ml−1. The probed blots were washed several times with half-strength HBST gel buffer containing 0.35m NaCl, dried, and autoradiographed by exposure to Kodak X-OMAT AR film with an intensifying screen for 15 h at −70 °C. M6 strain D471 was grown to stationary phase at 37 °C for 18 h in 4–6-liter batches of Todd-Hewitt broth. Bacteria were pelleted by centrifugation, washed, and resuspended in 50 mm phosphate buffer (1/50th of the original culture volume) containing 30% raffinose and 5 mm dithiothreitol and 5 mm EDTA. Streptococcal cell wall extracts using lysin (an amidase) enzyme (128 units ml−1) was carried out as described (21Pancholi V. Fischetti V.A. J. Bacteriol. 1988; 170: 2618-2624Crossref PubMed Google Scholar) and was dialyzed against 50 mm Tris/HCl, pH 8.0, and concentrated 10-fold using Centriprep-10 concentrators (Amicon Inc., Beverly, MA). The muralytic enzyme, mutanolysin (20 μg ml−1, Sigma), was used to prepare cell wall extracts of each grouping strain suspended in 50 mm Tris/HCl buffer, pH 6.8, containing 30% raffinose as described (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar). The dialyzed and concentrated cell wall extracts were sequentially precipitated with ammonium sulfate at 40, 60, and 80% saturation. The precipitated proteins were then dialyzed against 50 mm Tris/HCl, pH 8.0, and concentrated to an appropriate volume. The proteins in the dialyzed preparations were resolved by SDS-PAGE, electroblotted onto a PVDF membrane, and probed with labeled plasmin(ogen). A strong plasmin(ogen) binding activity was found to be mainly associated with a 45-kDa protein of the sequentially fractionated cell wall extract with 40–60% saturation of ammonium sulfate (Fig.1, A and B). For further purification, 40–60% ammonium sulfate precipitates were used as starting material. The dialyzed precipitate was concentrated (Centriprep-10, Amicon) and stored at −70 °C until further use. The concentrated sample was applied to a Mono Q FPLC column (HR10/10, Amersham Pharmacia Biotech) pre-equilibrated with 50 mmTris/HCl buffer, pH 8.0. After washing with 5-column volumes of this buffer, bound proteins were eluted with a 70-ml linear NaCl gradient from 0 to 700 mm and then with a 20-ml linear NaCl gradient from 700 mm to 1 m. Protein elution profile in each fraction was determined by SDS-PAGE and by Coomassie stain. A duplicate gel was Western blotted and probed with125I-plasmin(ogen) as described above. The 45-kDa protein eluted at 630 mm NaCl. The pooled fractions containing the 45-kDa protein and exhibiting plasmin(ogen) binding activity were dialyzed against the starting buffer and re-chromatographed on the Mono Q column using the same conditions. The positive fractions were again pooled and concentrated to a volume of <1.0 ml, using Centriprep-30 and Centricon-30 concentrators (Amicon). The concentrated sample was applied to a Superose-12 FPLC column (Amersham Pharmacia Biotech) pre-equilibrated with 50 mm Tris/HCl, pH 8.0. Fractions containing both the 45-kDa protein and plasmin(ogen) binding activity were pooled. These fractions were then concentrated, mixed with an equal volume of 4 m(NH4)2SO4, and applied to a Poros BU/M hydrophobic column (Perspective Biosystems, Cambridge, MA) pre-equilibrated with 50 mm Tris/HCl buffer, pH 8.0, containing 2 m(NH4)2SO4. The proteins were eluted with a 20-ml decreasing linear gradient of (NH4)2SO4 from 2.0 to 0.0m. The 45-kDa protein was eluted in one fraction at 1.32m (NH4)2SO4. The eluted protein was then dialyzed and stored at a concentration of 250 μg/ml at −70 °C until further use. Protein concentration was determined by the BCA (bicinchoninic acid) method (Pierce). N-terminal amino acid sequence of the purified 45-kDa protein was determined as described (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar, 23Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). Briefly, the purified 45-kDa protein was resolved by SDS-PAGE and electroblotted onto a PVDF membrane. The protein was visualized by staining with 0.1% Ponceau S (Sigma) in 1% acetic acid. Plasminogen and plasmin binding activity was confirmed by autoradiography. The section of the membrane containing the protein band of interest was excised, destained with double distilled water, and subjected to automated Edman degradation. Each sample contained approximately 5 μg of the protein as determined by the BCA protein estimation method (Pierce). A duplicate sample of PVDF membrane was digested with lysine-specific endopeptidase (Lys-C, sequencing grade, Boehringer Mannheim), and the resulting peptide fragments were separated by capillary electrophoresis interphased with the matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Perspective Biosystems). N-terminal sequences of the two internal peptide fragments were then determined as described above. All microsequence analyses were performed at the Protein/Biotechnology Facility of the Rockefeller University. The strong N-terminal sequence homology of the 45-kDa protein with α-enolase prompted us to investigate whether this protein is enzymatically active. α-Enolase activity was measured essentially as described earlier by both the coupled assay (24Hunther F.-J. Psarros N. Duschner H. Infect. Immun. 1990; 58: 1043-1047Crossref PubMed Google Scholar) and the direct assay atA 240 (25Wold F. Ballou C.E. J. Biol. Chem. 1957; 227: 301-312Abstract Full Text PDF PubMed Google Scholar). In the coupled assay, α-enolase activity was determined by measuring the transformation of NADH·H+ to NAD+. The enzymatic reactions were performed at 37 °C in 100 mmHEPES buffer, pH 7.0, containing 3.3 mm MgSO4, 0.2 mm NADH, 0.3 mm 2-phosphoglycerate (2-PGE), 1.2 mm ADP, 10.3 IU of lactate dehydrogenase, and 2.7 IU of pyruvate kinase in a final reaction volume of 1.0 ml. The reaction was started by adding 100 μl of the test solution containing α-enolase. The α-enolase activity was measured in terms of the rate of reduction in the absorbance at 340 nm (i.e. increase in the production of NAD from NADH·H+). The decrease of the extinction at 340 nm was recorded as the change in (Δ)A 340 nm min−1, using a Spectronic 3000 spectrophotometer (Milton Roy, Rochester, NY). For kinetic studies, a single enzyme assay was used, involving only the transformation of 2-PGE to phosphoenolpyruvate (PEP) by α-enolase, thus avoiding interactions of the effectors with other enzymes. This reaction was performed at 37 °C in 100 mm HEPES buffer, pH 7.0, containing 10 mm MgSO4 and 7.7 mm KCl and different concentrations of 2-PGE (9–35 mm) in a final volume of 1.0 ml. Change in absorbance/min was monitored spectrophotometrically at 240 nm as described above. The results were recorded as the rate of PEP release at 5-s intervals for a period of 3 min at 240 nm. To determine the extinction coefficient of PEP, the absorbance of different concentrations of PEP (32.5–1040 μg ml−1) was measured at 240 nm in 1-cm cuvettes. By using the Lambert-Beer formula (A =a m cl), the extinction coefficient (a m) for PEP (disodium salt) was calculated as 1.164 × 10−3m−1. By using 5 μg of SEN and different concentrations of 2-PGE, the kinetic coefficients, K m andV max, were calculated from the values obtained for the intercepts and slopes of the double-reciprocal plots of Lineweaver-Burk (26Lineweaver H. Burk D. J. Am. Chem. Soc. 1934; 56: 658-666Crossref Scopus (7963) Google Scholar). To determine whether the 45-kDa protein is functionally active as an α-enolase on the streptococcal surface, an overnight culture of group A streptococci (D471) was washed (3 ×) with 100 mm HEPES/NaOH buffer, pH 7.0, and different concentrations of streptococci were incubated with and without 3 mm 2-PGE in 100 mm HEPES buffer, pH 7.0, containing 10 mm MgCl2 and 7.7 mm KCl as described above. The reaction was allowed to occur in a final volume of 1.0 ml for a period of 3 min at room temperature, after which the bacteria were removed by centrifugation (4000 × g for 10 min). The supernatants were analyzed by measuring absorbance at 240 nm as described above. For the remaining portion of the “Experimental Procedures,” the 45-kDa protein will be referred to as SEN (surface enolase). Polyclonal antibodies to SEN were prepared in New Zealand White rabbits immunized subcutaneously with 150 μg of purified SEN emulsified in complete Freund's adjuvant (1:1) at multiple sites. Rabbits were boosted twice, each time with 150 μg of the purified protein in incomplete Freund's adjuvant (1:1) at 3-week intervals. The rabbits were bled 10 days after the third immunization. All sera were filter-sterilized and stored at 4 °C. To prepare SEN-specific IgG, the polyclonal serum was subjected to sequential purification on protein-A Sepharose CL-4B (Amersham Pharmacia Biotech) and SEN affinity columns. The affinity column was made by covalently linking approximately 2 mg of purified SEN to 0.5 g of affinity matrix (Ultralink 3M-Carboxy beads, Pierce) with 200 μl of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide-HCl (Pierce, 120 mg ml−1), in a final volume of 2.0 ml of 100 mm MES buffer, pH 4.7, at room temperature for 3–4 h. The protein-coupled matrix was then washed, and finally the affinity column was equilibrated with 50 mm HEPES/NaOH buffer, pH 7.4. The polyclonal serum (2–3 ml) was first adsorbed to the protein A-Sepharose CL-4B column using 50 mm Tris/HCl, pH 8.0, as the initial buffer. After washing, the bound IgG was eluted with 200 mm glycine/HCl buffer, pH 2.5. Eluted IgG was dialyzed against the starting buffer, concentrated, and affinity purified on the SEN affinity column using a strategy similar to that as described above. SEN-specific IgG was then used for immunochemical analyses. BALB/c × SJL-F1 mice were subcutaneously immunized with 30 μg of purified SEN in complete Freund's adjuvant (1:1 v/v). After 3 weeks, mice were bled and tested for antibodies to SEN by enzyme-linked immunosorbent assay and Western blot analysis using a crude cell wall extract of group A M6 strain D471. Affinity purified rabbit polyclonal antibodies against SEN were used as a positive control. Mice with high antibody titers were given a second dose of antigen intraperitoneally in distilled water. Mouse spleens were excised 3–3.5 days after the last booster. The spleen cell fusion to P3-NS1/1AG4–1(NS-1) myeloma cells was performed as described (27Kohler G. Milstein C. Eur. J. Immunol. 1976; 6: 511-519Crossref PubMed Scopus (1386) Google Scholar,28Galfre G.S. Howe C. Milstein C. Butcher G.W. Howard J.C. Nature. 1977; 266: 550-552Crossref PubMed Scopus (1182) Google Scholar). Hybridomas cloned by limiting dilution were grown in 2-liter rolling tissue culture flasks. From these cultures, secreted monoclonal antibodies were precipitated at 50% ammonium sulfate saturation. The precipitates were then dialyzed and purified using a protein A-Sepharose affinity column. To determine the location of SEN in streptococcal cells, an overnight culture of strain D471 was subjected to lysine digestion in 30% raffinose buffer to extract streptococcal cell wall-associated proteins as described above. From the resulting protoplasts, the membrane and cytoplasmic fractions were separated as described previously (5Pancholi V. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8154-8158Crossref PubMed Scopus (155) Google Scholar, 22Pancholi V. Fischetti V.A. J. Exp. Med. 1989; 170: 2119-2133Crossref PubMed Scopus (41) Google Scholar). Proteins from each cellular fraction were resolved by SDS-PAGE and electroblotted onto a PVDF membrane. The presence of SEN in different cell fractions was monitored by affinity purified anti-SEN polyclonal (75.0 ng ml−1) and monoclonal antibodies (12 ng ml−1) as described (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar,5Pancholi V. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8154-8158Crossref PubMed Scopus (155) Google Scholar). Group A streptococci (D471) from the overnight TH broth cultures were harvested, washed, and adjusted to a concentration of 109 cfu/ml. An aliquot of 200 μl of the bacterial suspension was incubated with 4 μg of affinity purified anti-SEN(1A10) or anti-SDH (4F12) monoclonal antibodies for 4 h followed by a 2-h incubation with colloidal gold (5- and 10-nm sized beads for anti-SEN and anti-SDH labeled bacteria, respectively) anti-mouse IgG (Amersham Pharmacia Biotech, 1:25) at room temperature. The labeled bacteria were then fixed in 2.5% glutaraldehyde in 0.1m cacodylate buffer, pH 7.4, for 4 h at 4 °C. The fixed labeled bacteria were then processed for transmission electron microscopy as described (29Pancholi V. Fischetti V.A. J. Exp. Med. 1997; 186: 1633-1643Crossref PubMed Scopus (83) Google Scholar). Proteins from the cell wall extract of several M serotypes after lysin digestion and those of various streptococcal grouping strains (group A–H, L, and N) after mutanolysin digestion were resolved by SDS-PAGE and transferred to a PVDF membrane. The blots were blocked, probed with affinity purified anti-SEN rabbit polyclonal antibodies (75.0 ng ml−1) for 3–4 h, and the reactive protein bands were visualized as described (4Pancholi V. Fischetti V.A. J. Exp. Med. 1992; 176: 415-426Crossref PubMed Scopus (510) Google Scholar, 5Pancholi V. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8154-8158Crossref PubMed Scopus (155) Google Scholar). Purified SEN (10 μg) was treated with carboxypeptidase Y (Boehringer Mannheim) at a substrate to enzyme concentration ratio of 13.5:1 in 50 mm HEPES buffer, pH 7.0, at 37 °C for 6 h. Equal amounts of the enzyme-treated and untreated SEN were resolved by SDS-PAGE, and their plasminogen binding activity was determined by the blot overlay method as described above. In another set of similar experiments, plasmin(ogen) binding activity was measured in the presence of 0.1 m EACA or 0.1 mlysine. The ligand binding analysis was carried out using 96-well microtiter plates (C8 Maxi Break-apart, Nalge Nunc International, Naperville, IL). The plates were coated with 100 μl of purified SEN in 0.05 m carbonate buffer, pH 9.6 (5 μg ml−1), for 3 h at 37 °C and were then kept at 4 °C overnight. The plates were washed and blocked with HBST gel blocking buffer for 4 h at room temperature. A serial 2-fold dilution of aprotinin and PMSF-treated 125I-plasminogen (3.8 pmol) or 125I-plasmin (2.2 pmol) in a final volume of 100 μl of HBST gel buffer containing 2 mm PMSF was added to the SEN-coated wells and incubated for 4 h at room temperature on a shaker. The plates were then washed thre