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[43] Bacterial immunoglobulin A1 proteases
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[43] Bacterial Immunoglobulin A 1 Proteases B y MARTHA H . M U L K S a n d RUSSELL J. SHOBERG
Introduction The immunoglobulin A1 (IgA0 proteases are extracellular bacterial enzymes that specifically cleave human IgA of the IgA 1 subclass to yield intact Fabt~ and Fco~ fragments. 1-4 Bacteria which produce IgA1 proteases include the causative agent of gonorrhea, Neisseria gonorrhoeae; the three most common causative agents of bacterial meningitis, Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae; a variety of oral microorganisms including Streptococcus sanguis, S. mitis, S. oralis, Preootella (Bacteroides) sp., and Capnocytophaga sp.; as well as Ureaplasma urealyticum and Clostridium ramosum. Because many of the organisms that produce IgA 1 proteases are mucosal pathogens of humans and IgA 1 proteases specifically degrade the major class of immunoglobulin found in mucosal secretions, the production of IgA 1 proteases has been postulated to be a virulence factor for these bacteria. Each IgA l protease cleaves a single peptide bond within the hinge region of human IgA 1 (Fig. 1). Several of the species which produce IgA1 proteases, including N. gonorrhoeae, 5 N. meningitidis, 6 and H. influenzae, 7 produce at least two different types of IgA 1 proteases, as defined by the specific peptide bond cleaved, although, with very few exceptions, a given isolate produces only one type of protease. The genes encoding IgA 1 proteases from N. gonorrhoeae, 8 H. influenzae, 9 and S. sanguis 1°have been cloned and sequenced. Studies on nucleotide sequence homology and on inhibition of protease activity by specific antisera have shown that the IgA 1 proteases from Neisseria and Haemo1 M. Kilian, J. Mestecky, and M. W. Russell, Microbiol. Rev. 52, 296 (1988). 2 S. J. Kornfeld and A. G. Plaut, Rev. Infect. Dis. 3, 521 (1981). 3 M. H. Mulks, in "Bacterial Enzymes and Virulence" (I. A. Holder, ed.), p. 81. CRC Press, Boca Raton, Florida, 1985. 4 A. G. Plaut, Annu. Rev. Microbiol. 37, 603 (1983). 5 M. H. Mulks and J. S. Knapp, Infect. lmmun. 55, 931 (1987). 6 M. H. Mulks, A. G. Plaut, H. A. Feldman, and B. Frangione, J. Exp. Med. 152, 1442 (1980). 7 M. H. Mulks, S. J. Kornfeld, B. Frangione, and A. G. Plaut, J. Infect. Dis. 146, 266 (1982). 8 j. Pohlner, R. Halter, K. Beyreuther, and T. F. Meyer, Nature (London) 325, 458 (1987). 9 K. Poulsen, J. Brandt, J. P. Hjorth, H. C. ThCgersen, and M. Kilian, Infect. Immun. 57, 3097 (1989). 10 j. V. Gilbert, A. G. Plaut, and A. Wright, Infect. lmmun. 59, 7 (1991).
METHODSIN ENZYMOLOGY,VOL. 235
Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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ENZYME AND TOXIN ASSAYS
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A 220
225
I
230
J
235
I
I
, sTP sPscc,
IgAl
°""rumI,
240
I
| /
N, gonorrhoele 1 N, menl~gltldl$1
H. Influenzae 2 N. gonorrhoeae 2 N.meningitid|l 2 U. urealytlcum 5 H, aegyptlui H. Influenzae 1 4
s. ,,~g,,.
B. melaninogenicui C. ochracea 2
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l i - -
i--i--
U H
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i
i Fd
~
u
iu
i ,mm~"__
!
u
~
i L i
C
i
1
2
i
3
i
4
5
6
FZG. I. (A) Amino acid sequence of the human IgA] hinge region showing peptide bonds cleaved by bacterial IgAl proteases? -4 (B) Diagrammatic representation of IgAl protease digests of human IgA~ as seen on SDS-PAGE under reducing conditions, showing the relative mobilities of Fca and Fdc~fragments produced by IgA 1proteases of different specificities. Lane C, undigested control; lanes 1-6, cleavage patterns corresponding to digestion by proteases of the six different peptide bond specificities shown in (A). H, IgA 1 heavy chain; Fc, Fcc~ fragment; Fd, heavy chain portion of the Faba fragment; L, light chain. Horizontal dashed lines indicate where the gel would be cut for quantitative assays; section l (brace) contains intact heavy chain, whereas section 2 contains Fca and Fdct fragments.
philus species are closely related at both the gene and the protein level, whereas the IgA 1 protease from S. sanguis is distinctly different. ~°-~4 II R. Halter, J. Pohlner, and T. F. Meyer, E M B O J. 8, 2737 (1989). 12j. M. Koomey and S. Falkow, Infect. Immun. 43, 101 (1984). 13 K. Poulsen, J. P. Hjorth, and M. Kilian, Infect. Immun. 56, 987 (1988). 14 H. Lomholt, K. Poulsen, D. A. Caugant, and M. Kilian, I'roc. Natl. Acad. Sci. U.S.A. 89, 2120 (1992).
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Assay Procedures M a n y different methods of analyzing a samplc for the presence of IgAl protease have been developed, including immunoelectrophorcsis, 15 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), 7 enzyme-linked immunosorbent assay (ELISA), 16,17and high-performance liquid chromatography (HPLC).18 All have the following in common: the enzyme sample must be reacted with human IgA l as substrate and the results of that reaction analyzed for either degradation of intact IgA Ior appearance of IgA1 cleavage products. Synthetic peptide homologs of IgAl protease cleavage sites have also been tested and can serve as substrates for IgA~ proteases. ~9 However, thcsc peptide substrates are not available commercially or readily synthesized. Therefore, currently the only substrate used for the detection and quantitation of IgA~ protease activity is human IgA1. Preparation of Substrate
N u m e r o u s procedures have been described for the purification of IgA from normal human serum, from colostrum, and from plasma, serum, or other fluids of patients with IgA myeloma (see Ref. 20 for a review). The method outlined below is relatively simple and reliable, especially when used to purify myeloma IgAl from patient sera. Procedure
1. To 3 ml of IgA 1 myeloma serum, slowly add 2 ml of a saturated solution of ammonium sulfate in water, stirring constantly. Continue to stir slowly for 30 min at room temperature. Centrifuge for 20 min at 8000 g and 4 °. Discard the supernatant. Wash the precipitate once with 40% saturated ammonium sulfate and resuspend the washed precipitate in 3.0 ml o f 0.9% NaC1. Dialyze the sample extensively against 0.9% NaCI to r e m o v e all ammonium sulfate. 2. T o the dialyzed sample ( - 3 . 0 ml), add 6 ml of 60 m M sodium acetate buffer, p H 4.8. Dropwise, add 9.0 ml of caprylic acid, stirring constantly, at room temperature. Continue to stir slowly for 30 min. Centrifuge for 20 min at 8000 g and 4 °. Carefully remove the supernatant and discard the 15s. K. Mehta, A. G. Plant, N. J. Calvanico, and T. B. Tomasi, Jr., J. Irnmunol. 111, 1274 (1973). 16j. Reinholdt and M. Kilian, J. lmrnunol. Methods 63, 367 (1983). 17M. S. Blake and C. Eastby, J. Immunol. Methods 144, 215 (1991). 18S. B. Mortensen and M. Kilian, J. Chromatogr. 296, 257 (1984). 19S. G. Wood and J. Burton, Infect. Immun. 59, 1818 (1991). 2oj. Mestecky and M. Kilian, this series, Vol. 116, p. 37.
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ENZYME AND TOXIN ASSAYS
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precipitate. Dialyze the supernatant against 5 mM potassium phosphate buffer, pH 8.0. 3. Apply the sample to an anion-exchange column [e.g., Pharmacia (Piscataway, NJ) FPLC (fast protein liquid chromatography) Mono Q or Whatman (Clifton, NJ) DE-52] equilibrated with 15 mM Tris-phosphate buffer, pH 8.0. Elute the column with a linear buffered pH gradient, using 15 mM Tris-phosphate, pH 8.0, as the starting buffer and 0.3 M Tris-phosphate, pH 4.0, as the final buffer. 4. Screen each fraction by Ouchterlony double-diffusion analysis for IgG, IgA, IgM, and albumin, using appropriate antisera and controls. Pool fractions containing IgA, concentrate, and screen again by Ouchterlony to confirm the purity of the IgA. If necessary repeat the chromatography step to remove contaminating material. The major contaminant is usually albumin, which can also be removed by passage over an Affi-Gel blue (Bio-Rad, Richmond, CA) affinity column. An alternative procedure that is especially useful in the purification of IgA 1 from normal human serum is affinity chromatography over a jacalin-Sepharose column. Jacalin, or jack bean lectin, selectively binds human IgA 1 but not IgA2.21
Quantitative Assay Using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis The following assay technique is based on physical separation of the IgA~ cleavage products (Faba and Fco0 from the intact IgA~ molecule by electrophoresis. 7,22 Quantitative results are achieved by using trace amounts of radiolabeled IgA~ as part of the reaction mixture.
Reagents Pure human IgA1, at a concentration of 2.0 mg/ml 125I-labeled human IgA~, approximately 400/zCi/ml Assay buffer: 50 mM Tris-HC1, pH 7.5, containing 10 mM MgCI2, 10 mM CaCI2, and 0.05% bovine serum albumin (radioimmunoassay grade) SDS-PAGE sample buffer: 62.5 mM Tris-HCl, pH 6.8, containing 12.5% glycerol, 1.25% SDS, 1.25% 2-mercaptoethanol, and 0.006% bromphenol blue 21 D. L. Skea, P. Christopoulos, A. G. Plaut, and B. J. Underdown, Mol. lmmunol. 25, 1 (1988). 2z D. A. Simpson, R. P. Hausinger, and M. H. Mulks, J. Bacteriol. 170, 1866 (1988).
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Procedure
1. Mix 2.5/.d of the enzyme sample to be assayed, 2.5/.d of human IgA l (5/zg), 2.5/.d of [125I]IgA1 (--I /.~Ci), and 5/zl of assay buffer, and incubate the mixture in a 37° water bath. After incubation, add 50/zl of SDS-PAGE sample buffer and boil for 5 min to stop the reaction and reduce the IgA~ to polypeptide monomers. 2. Electrophorese the samples on a 9% polyacrylamide gel containing 0.1% SDS in a Laemmli buffer system 23 to separate the IgA 1 cleavage products. Note that we generally use 50 mM Tris, 384 mM glycine, 0.1% SDS as electrode buffer rather than the standard Laemmli recipe. 3. Stain the gel with 0.05% Coomassie Brilliant Blue in 35% methanol-10% acetic acid and destain in 35% methanol-10% acetic acid. Dry the stained gel onto Whatman 3MM paper and autoradiograph onto Kodak (Rochester, NY) XAR-5 film to localize the labeled IgA 1 bands. Using the autoradiograph as a cutting guide, excise the sections of each lane of the gel containing (1) the intact IgA1 heavy chain ( - 5 6 kDa) and (2) the F c a and Fda fragments ( - 2 5 - 3 5 kDa), as shown in Fig. lb. The Fdo~ is defined as the heavy chain portion of the Fabo~ fragment. Regions of the gel containing light chains ( - 2 2 kDa) or secretory component ( - 8 0 kDa) are discarded. The amount of radioactivity [counts per minute (cpm)] in each band is measured using a 3/counter. 4. The percent cleavage of the IgA1 in each sample is calculated as follows: cpm in Fd and Fc fragments = % fragments cpm in intact heavy chain + cpm in Fd and Fc fragments 5. The amount of IgA l cleaved is then calculated as % fragments in sample - % fragments in negative control % fragments in positive control ×/xg IgA l per digest =/~g IgA l cleaved The positive control is a sample in which complete cleavage of the available IgAl has occurred, and it provides a value for the maximum percentage of total radioactivity that is found in IgA1 fragments. This value is usually 75-80% rather than 100%. 6. Units of IgAl protease activity are expressed as micrograms IgA~ cleaved per minute. 23U. K. Laemmli, Nature (London) 227, 680 (1970).
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Example. An undiluted sample of supernatant from a late log phase broth culture of N. gonorrhoeae yields 25% fragments in a 20-min incubation. The percentage of fragments in the negative control is 5% and that in the positive control is 80%. The assay reaction mix included 2.5 /xl enzyme sample and 5/xg IgA~. For the calculation, (25% - 5%) x 5 ~g IgAl = 25/xg IgA1 cleaved/min/ml (80%) × 20 m i n x 0.0025 ml = 25 units/ml of sample
Comments. The SDS-PAGE procedure is the only method for the assay of IgAl protease activity which permits both quantitation of units of IgAl protease and determination of the type of cleavage, based on the size of the Fda and Fct~ fragments produced. The method can also be used for qualitative assays on culture supernatants or on suspensions of bacterial colonies. For qualitative assays, we recommend using only the radiolabeled substrate to yield increased sensitivity. The electrophoresis-based assays are the method of choice in use today by most laboratories studying IgA~ proteases. The technique allows for multiple samples to be processed simultaneously, offers consistency, is amenable to qualitative or quantitative studies, and is relatively rapid. The use of radiolabeled IgA~ limits the amount of IgA~ necessary for assays, but it can present a problem for laboratories that do not wish to employ radioisotopes. Detection of IgA1 bands by immunoblotting with antiserum against the a chain is an alternative, 24 although quantitation by densitometric scanning of developed blots is generally less sensitive and less reproducible than assay with radiolabeled substrate. Qualitative Plate Assay for Immunoglobulin A1 Protease Solid-phase plate assays can be used for screening large numbers of isolates simultaneously and are most often used to select an IgAl proteaseproducing isolate from a population of nonproducers. The first described method utilizes radiolabeled IgA1 immobilized on polyacrylamide beads via an anti-Fcot antibody. 25 The technique involves overlaying bacterial colonies on an agar plate with a layer of immobilized IgA~ in top agar or agarose. Secreted IgA~ protease from producing colonies will cleave the immobilized IgAl, liberating Fabot, which is diffusible. Diffused Faba fragments are blotted onto a nitrocellulose membrane, which is then autoradiographed. IgA~ protease-producing isolates are identified by a locationcorresponding radioactive signal. 24 T. Ahl and J. Reinholdt, Infect. lmmun. 59, 563 (1991). 25 j. V. Gilbert and A. G. Plaut, J. lmmunol. Methods 57, 247 (1983).
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Reagents 125I-Labeled human IgA1, approximately 400/zCi/ml Immunobead (Bio-Rad) reagent specific for human IgA Phosphate-buffered saline, pH 7.5 (PBS) 2% Agarose in PBS Nitrocellulose filters
Procedure 1. Reconstitute the lyophilized polyacrylamide beads at a concentration of 1 mg/ml in PBS. Let stand at least 30 rain at room temperature. 2. For each 100-mm petri plate to be assayed, mix 2 ml bead suspension plus 4-10 ~Ci [125I]IgA1 (10-20/zl). Incubate for 90 min at 37° with gentle shaking. Wash the beads three times with PBS to remove unbound IgA. Resuspend the beads to a concentration of I mg/ml in PBS and warm to 50° for 5 min. 3. For each plate to be assayed, melt 2 ml of sterile 2% agarose in PBS and cool to 50 °. 4. Rapidly mix 2 ml of the 125I-labeled bead suspension plus 2 ml agarose and pour onto the surface of the plate to be assayed. Swirl gently to distribute an even layer of agarose, being careful not to disturb the bacterial colonies. It is helpful to have the culture plates at 37° to prevent the agarose from solidifying too rapidly. Incubate at 37° in a moist chamber, such as a sealed box lined with damp paper towels. The incubation time will vary with the bacteria to be assayed: N. gonorrhoeae colonies need approximately 30 min, whereas Escherichia coil clones expressing iga genes may need 12-16 hr. 5. Lay a dry nitrocellulose filter over the agarose, being careful to avoid trapping any air bubbles, and mark the orientation of the filter. Incubate at 37° for 20 min in a moist chamber. Lift off the filter and wash twice in PBS to remove any adherent agarose or other debris. Air-dry the filter and autoradiograph onto Kodak XAR-5 film. 6. I g A 1 protease-producing colonies release radiolabeled IgAl fragments, which are picked up by the nitrocellulose. Therefore, exposed areas on the autoradiograph correspond to IgA~ protease-producing colonies.
Comments. It is necessary to adjust both incubation times and exposure times to achieve optimal sensitivity with minimal background. Strong IgA~ protease producers such as N. gonorrhoeae will need only 15-30 min of incubation for protease activity to be detectable, whereas E. coli clones, which are often very weak protease producers, may need 12-16 hr of incubation as well as increased exposure time for the autoradiograph. In any case, incubation with nitrocellulose filters should be for only 20-30
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ENZYME AND TOXIN ASSAYS
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min, or background levels may become too high for interpretation of results. It is possible to recover viable bacteria from under the overlay agarose if exposure to radiation is kept to a minimum. 26 Alternatively, bacteria or phage to be tested can be spotted in a grid pattern onto two plates, with one plate assayed by this method and the other held in reserve for the isolation of positive colonies. Two variations have arisen from this method. One involves prior immobilization of the radiolabeled IgA l on the nitrocellulose membrane and incubation on a bacteria-covered surface. 27 After allowing for diffusion of the liberated Fabot, IgA l protease producers can be detected by a decrease in the amount of radioactive signal. This method is not as sensitive as the original assay procedure, since it is much more difficult to detect a negative area than a positive one. A second variation incorporates t~-light chain antibodies bound to the nitrocellulose membrane to improve the efficiency of Fabo~ recovery. 28 Detection of bound Fca fragments with biotinylated antibodies to Fcct eliminates the need for using radioactive materials.
Immunoassay Procedure for Quantitation of lmmunoglobulin A 1 Protease Activity Several ELISAs for the quantitation of IgA l protease activity have been developed. 16,17These methods are not optimal for screening bacterial samples for IgA1 protease activity, since they do not permit identification of protease type nor do they distinguish between IgAi protease activity and nonspecific degradation ofimmunoglobulin Al. However, these assays generally are very sensitive and reproducible, and they have been especially useful in detection of monoclonal antibodies capable of inhibition of IgA~ protease activity. 17 Reinholdt and Kilian 16 developed an ELISA that utilizes antibody to human light chain to coat the wells and to bind IgAl by its Fab end. After digestion with IgA1 protease, horseradish peroxidaseconjugated antibody against Fco~ is used to quantitate the remaining uncleaved IgA~. In this assay, decreased absorbance correlates with increased protease activity. Blake and Eastby ~7developed a similar procedure that utilizes the IgAbinding protein from group B streptococci 29 to coat the wells and to bind 26 R. 27 R. 28 T. 29 G.
J. Shoberg, Ph.D. Thesis, Michigan State Univ., East Lansing (1991). Halter, J. Pohlner, and T. F. Meyer, EMBO J. 3, 1595 (1984). A. Brown and I. G. Leak, J. lmmunol. Methods 123, 241 (1989). J. Russell-Jones, E. C. Gotschlich, and M. S. Blake, J. Exp. Med. 160, 1467 (1984).
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IgAl substrate by its Fc end. After digestion with IgA~ protease, remaining Fabt~ is detected and quantitated using alkaline phosphatase-conjugated antibodies to human light chain. Decreased absorbance correlates with increased cleavage of IgAl and increased protease activity. Both of these ELISA techniques offer the ability to perform multiple assays rapidly and simultaneously, using very small amounts of human IgAl and no radioactivity, with very high sensitivity. However, the methods may not be useful for assay of IgA 1proteases from oral streptococci, which may cleave only one heavy chain rather than both and thus fail to release cleaved fragments.~6,z°
Enzyme Production and Isolation Several methods have been described for the production and partial purification of neisserial IgA l proteases.17,22'27'3° All rely on a combination of chromatographic procedures, including ion-exchange, chromatofocusing, molecular sieve, and hydrophobic interaction chromatography. The procedure we use to purify the gonococcal type 2 IgA~ protease is a modification of a previously published method 22 that gives a higher yield of active IgA 1 protease. Procedure
1. Inoculate brain-heart infusion (BHI) agar containing 10 ml/liter KeUogg's supplement 31 from a freezer stock of N . g o n o r r h o e a e stored at - 7 0 ° in 2% tryptone-20% glycerol. Grow overnight at 35 ° under 5% CO z. 2. Prepare brain-heart infusion broth. Filter through a 10,000 molecular weight cutoff Amicon (Danvers, MA) membrane. Collect and autoclave the filtrate; discard the retentate. Immediately before use, add 10 ml/liter Kellogg's supplement plus 10 ml/liter of 4.2% sodium bicarbonate (BHIK). 3. Inoculate 30 ml of this medium (BHIK) from the overnight plate culture. The starting OD520 should be approximately 0.05. Incubate the culture at 35° in a water bath shaker at 150 rpm for 3-4 hr, until the ODsz0 reaches 0.8-1.0. Use the broth to inoculate 500 ml of prewarmed BHIK. Incubate at 35 ° for 3-4 hr, to an OD520of 0.8-1.2 (late log phase). Centrifuge for 30 min at 6000 g and 4 ° to pellet the bacteria. 4. Carefully decant the supernatant into a clean, ice-cold flask. Discard the cell pellet. Add 0.5 M EDTA, pH 8.0, to a final concentration of 50 mM. Plaut, this series, Vol. 165, p. 117 (1988). 31D. S. Kellogg,Jr., W. L. Peacock, W. E. Deacon, L. Brown, and C. I. Pirkle, J. Bacteriol. 85, 1274(1963). 3o A . G .
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ENZYME AND TOXIN ASSAYS
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5. Add approximately 200 ml of DEAE-cellulose (Whatman DE-52) resin, equilibrated in 50 mM Tris-HC1, pH 7.5, to the supernatant. Stir slowly at 4° for at least 1 hr. Centrifuge for 30 min at 6000 rpm and 4 ° to pellet the resin. Decant and save the supernatant. Wash the pelleted resin with 50 mM Tris-HCl, pH 7.5, plus 5 mM EDTA; decant and save the supernatant. Pool the supernatants and discard the used resin. 6. Concentrate the pooled supernatant by positive pressure dialysis in an Amicon stirred cell using a 50,000 molecular weight cutoff membrane. If the material is to be stored, add 20% sterile, ultrapure glycerol (BRL, Gaithersburg, MD) to the concentrated sample. 7. Using an Amicon or similar membrane filtration device (not dialysis tubing), dialyze the sample into at least 2 volumes of distilled water, to dilute the salt concentration. Bring the sample to 47.5 ml with sterile distilled water. Add 2.0 ml of ampholytes (Pharmacia), pH 8.0-10.5 range, plus 0.5 ml of ampholytes, pH 5.0-8.0 range. Separate the sample in a Rotofor (Bio-Rad) isoelectric focusing cell, according to the manufacturer's instructions. 8. Pool the fractions with IgA 1 protease activity and exchange into 50 mM Tris-HC1, pH 7.5, plus 5 mM EDTA, 20% glycerol, and 0.2% Nonidet P-40 detergent. Chromatograph over a Pharmacia FPLC Superose 12 HR 10/30 column equilibrated with the same buffer. Screen fractions for IgAl protease activity by SDS-PAGE assay and for purity by Coomassie blue-silver staining. 32 Pool fractions containing IgA1 protease and exchange into 50 mM Tris-HCl, pH 7.5, plus 5 mM EDTA and 20% glycerol, and store at - 7 0 ° . Comments. We have successfully purified type 2 gonococcal IgA 1 protease to homogeneity by the above method. Type 1 gonococcal protease can be purified by similar methods, although it is much less stable. 22 Use of 50 mM Tris-acetate, pH 7.5, plus 5 mM EDTA, 1 mM dithiothreitol (DTT), and 20% glycerol as a stabilization buffer is necessary for purification of the type 1 gonococcal protease. The IgA 1proteases from N. meningitidis 6 and H. influenzae 7 have been partially purified by a combination of anion-exchange and molecular sieve chromatography. The method described here, with modifications such as optimization of appropriate stabilization buffers for each enzyme, may also be applicable to purification of IgA 1 proteases from these bacteria.
32 A. Gorg, W. Postel, J. Weser, H. W. Schivaragin, and W. M. Boesken, Sci. Tools (Sweden) 32, 5 (1985).
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TABLE I PROPERTIES OF BACTERIALIMMUNOGLOBULINA1 PROTEASES Species
Enzyme classa
Mr (× 10-3)
pl
Kmb (/,~mol)
Refs.
Bacteroides melaninogenicus Capnocytophaga spp. Haemophilus infiuenzae (type 1) Haemophilus influenzae (type 2) Neisseria gonorrhoeae (type 1) Neisseria gonorrhoeae (type 2) Neisseria meningitidis (type 2) Streptococcus oralis Streptococcus pneumoniae Streptococcus sanguis
T M S S S S S NR M M
62c NR d 90c, 108e 100c 112c 106-110 c,e 100c 100c NR 100c, 186e
5.0 NR NR NR NR 8.6 NR NR NR 5.45
3.4 NR 0.7 1.5 0.6 1.8 NR NR NR 23
18,33 33 9,34,35 34,36 22,34 8,22,34 36,37 38 37 10,34,38,39
M, Metalloprotease; S, serine protease; T, thiol protease. b Determined against human IgAt. c Determined by chromatography or SDS-PAGE. d NR, Not reported. e Determined by sequence analysis. Properties of Bacterial Immunoglobulin A 1 Proteases T a b l e 133-39 s u m m a r i z e s s o m e o f t h e i n f o r m a t i o n k n o w n a b o u t t h e c h e m i s t r y o f t h e b a c t e r i a l IRA l p r o t e a s e s , i n c l u d i n g t h e c l a s s o f p r o t e a s e to w h i c h t h e g i v e n IgA~ p r o t e a s e b e l o n g s a n d t h e m o l e c u l a r w e i g h t , w h e r e r e p o r t e d . D a t a a r e n o t a v a i l a b l e for all o f t h e IgA~ p r o t e a s e s t h a t h a v e b e e n d e s c r i b e d . S o m e o f t h e h i g h l i g h t s o f t h e d a t a a r e t h e f a c t s t h a t (1) three of the four classes of proteases are represented, with aspartic protea s e s b e i n g t h e e x c e p t i o n , a n d (2) t h e s e e n z y m e s s h o w a w i d e r a n g e o f m o l e c u l a r w e i g h t s a n d i s o e l e c t r i c p o i n t s . It a p p e a r s t h a t this set o f p r o t e aRes w i t h s i m i l a r l y n a r r o w r a n g e s o f s u b s t r a t e s p e c i f i c i t y is a c t u a l l y a g r o u p i n g o f v e r y d i v e r s e e n z y m e s w h i c h w e r e c l u s t e r e d into a g r o u p b a s e d solely on that one characteristic. 33 E. V. G. Frandsen, J. Reinholdt, and M. Kilian, Infect. Immun. 55, 631 (1987). 34 W. W. Bachovkin, A. G. Plaut, G. R. Flentke, M. Lynch, and C. A. Kettner, J. Biol. Chem. 265, 3738 (1990). 35j. Pohiner, C. Maercker, H. Apfel, and T. F. Meyer, in "Frontiers of Mucosal Immunology" (M. Tsuchiya, H. Nagura, T. Hibi, and I. Moro, eds.), Vol. 1, p. 567. Elsevier Science Publ., Amsterdam, 1991. 36 M. H. Mulks, unpublished data, 1982. 37M. Kilian, J. Mestecky, R. Kulhavy, M. Tomana, and W. T. Butler, J. Immunol. 124, 2596 (1980). t8 j. Reinholdt, M. Tomana, S. B. Mortensen, and M. Kilian, Infect. Immun. 58, 1186 (1990). 39 A. G. Plaut, J. V. Gilbert, and I. Heller, Adv. Exp. Med. Biol. 107, 489 (1978).
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Substrates. By definition, IgAl proteases proteolyze human IgAl and not IgA 2, IgM, IgG, or other serum proteins such as albumin. 2,3 For the proteases that have been tested against IgA from other mammals, IgAl from higher primates (i.e., chimpanzee and gorilla) can be hydrolyzed, but not IgAl from other primates or from a wide range of other mammalian species. 2'3 The type 2 IgA 1 protease from N. gonorrhoeae contains three sequences within the 169-kDa proenzyme that are cleaved by autoproteolysis during secretion of the mature protease. 8 Synthetic peptides homologous to two of the sequences are also cleaved by the gonococcal type 2 protease, although similar peptides homologous to the hinge region of human IgA1 are not.19 In addition, we have demonstrated that several proteins found in the isolated membranes of gram-negative bacteria are degraded by the type 2 IgA 1 protease of N. gonorrhoeae. 4° 4o R. J. Shoberg and M. H. Mulks, Infect. Immun. 59, 2535 (1991).
[44] Elastase Assays By LYNN RUST, CALVIN R. MESSING, and BARBARAH. IGLEWSKI
Introduction The elastin protein is the primary constituent of the elastic fiber, the predominant connective tissue of the lung, blood vessels, and skin. Characteristic of elastin is the desmoisine cross-linking structure, formed by the covalent binding of four lysyl residues. Elastin includes a high concentration of the small hydrophobic amino acids including glycine, alanine, and valine. It is the most insoluble protein in the human body.1 Owing to the unusual structure and insolubility of elastin, few proteases have elastolytic activity, whereas elastases frequently have proteolytic activity against a variety of substrates. The neutral metalloproteinase of Vibro vulnificus is active against azocasein and elastin. 2 The metalloenzymes Bacillus thermoproteolyticus thermolysin and Pseudomonas aeruginosa elastase also have general proteolytic as well as elastolytic activity. In addition to elastin, P. aeruginosa elastase is capable of degrading t j. M. Davidson, in " C o n n e c t i v e T i s s u e Disease: Molecular Pathology of the Extracellular M a t r i x " (J. Uitto a n d A. J. Perejda, eds.), p. 423. Dekker, N e w York, 1987. 2 M. H. K o t h a r y and A. S. Kreger, J. Gen. Microbiol. 133, 1783 (1987).
METHODS IN ENZYMOLOGY,VOL. 235
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IgA 1 PROTEASES
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[43] Bacterial Immunoglobulin A 1 Proteases B y MARTHA H . M U L K S a n d RUSSELL J. SHOBERG
Introduction The immunoglobulin A1 (IgA0 proteases are extracellular bacterial enzymes that specifically cleave human IgA of the IgA 1 subclass to yield intact Fabt~ and Fco~ fragments. 1-4 Bacteria which produce IgA1 proteases include the causative agent of gonorrhea, Neisseria gonorrhoeae; the three most common causative agents of bacterial meningitis, Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae; a variety of oral microorganisms including Streptococcus sanguis, S. mitis, S. oralis, Preootella (Bacteroides) sp., and Capnocytophaga sp.; as well as Ureaplasma urealyticum and Clostridium ramosum. Because many of the organisms that produce IgA 1 proteases are mucosal pathogens of humans and IgA 1 proteases specifically degrade the major class of immunoglobulin found in mucosal secretions, the production of IgA 1 proteases has been postulated to be a virulence factor for these bacteria. Each IgA l protease cleaves a single peptide bond within the hinge region of human IgA 1 (Fig. 1). Several of the species which produce IgA1 proteases, including N. gonorrhoeae, 5 N. meningitidis, 6 and H. influenzae, 7 produce at least two different types of IgA 1 proteases, as defined by the specific peptide bond cleaved, although, with very few exceptions, a given isolate produces only one type of protease. The genes encoding IgA 1 proteases from N. gonorrhoeae, 8 H. influenzae, 9 and S. sanguis 1°have been cloned and sequenced. Studies on nucleotide sequence homology and on inhibition of protease activity by specific antisera have shown that the IgA 1 proteases from Neisseria and Haemo1 M. Kilian, J. Mestecky, and M. W. Russell, Microbiol. Rev. 52, 296 (1988). 2 S. J. Kornfeld and A. G. Plaut, Rev. Infect. Dis. 3, 521 (1981). 3 M. H. Mulks, in "Bacterial Enzymes and Virulence" (I. A. Holder, ed.), p. 81. CRC Press, Boca Raton, Florida, 1985. 4 A. G. Plaut, Annu. Rev. Microbiol. 37, 603 (1983). 5 M. H. Mulks and J. S. Knapp, Infect. lmmun. 55, 931 (1987). 6 M. H. Mulks, A. G. Plaut, H. A. Feldman, and B. Frangione, J. Exp. Med. 152, 1442 (1980). 7 M. H. Mulks, S. J. Kornfeld, B. Frangione, and A. G. Plaut, J. Infect. Dis. 146, 266 (1982). 8 j. Pohlner, R. Halter, K. Beyreuther, and T. F. Meyer, Nature (London) 325, 458 (1987). 9 K. Poulsen, J. Brandt, J. P. Hjorth, H. C. ThCgersen, and M. Kilian, Infect. Immun. 57, 3097 (1989). 10 j. V. Gilbert, A. G. Plaut, and A. Wright, Infect. lmmun. 59, 7 (1991).
METHODSIN ENZYMOLOGY,VOL. 235
Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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ENZYME AND TOXIN ASSAYS
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A 220
225
I
230
J
235
I
I
, sTP sPscc,
IgAl
°""rumI,
240
I
| /
N, gonorrhoele 1 N, menl~gltldl$1
H. Influenzae 2 N. gonorrhoeae 2 N.meningitid|l 2 U. urealytlcum 5 H, aegyptlui H. Influenzae 1 4
s. ,,~g,,.
B. melaninogenicui C. ochracea 2
B
l i - -
i--i--
U H
Fc
i
i Fd
~
u
iu
i ,mm~"__
!
u
~
i L i
C
i
1
2
i
3
i
4
5
6
FZG. I. (A) Amino acid sequence of the human IgA] hinge region showing peptide bonds cleaved by bacterial IgAl proteases? -4 (B) Diagrammatic representation of IgAl protease digests of human IgA~ as seen on SDS-PAGE under reducing conditions, showing the relative mobilities of Fca and Fdc~fragments produced by IgA 1proteases of different specificities. Lane C, undigested control; lanes 1-6, cleavage patterns corresponding to digestion by proteases of the six different peptide bond specificities shown in (A). H, IgA 1 heavy chain; Fc, Fcc~ fragment; Fd, heavy chain portion of the Faba fragment; L, light chain. Horizontal dashed lines indicate where the gel would be cut for quantitative assays; section l (brace) contains intact heavy chain, whereas section 2 contains Fca and Fdct fragments.
philus species are closely related at both the gene and the protein level, whereas the IgA 1 protease from S. sanguis is distinctly different. ~°-~4 II R. Halter, J. Pohlner, and T. F. Meyer, E M B O J. 8, 2737 (1989). 12j. M. Koomey and S. Falkow, Infect. Immun. 43, 101 (1984). 13 K. Poulsen, J. P. Hjorth, and M. Kilian, Infect. Immun. 56, 987 (1988). 14 H. Lomholt, K. Poulsen, D. A. Caugant, and M. Kilian, I'roc. Natl. Acad. Sci. U.S.A. 89, 2120 (1992).
[43]
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Assay Procedures M a n y different methods of analyzing a samplc for the presence of IgAl protease have been developed, including immunoelectrophorcsis, 15 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), 7 enzyme-linked immunosorbent assay (ELISA), 16,17and high-performance liquid chromatography (HPLC).18 All have the following in common: the enzyme sample must be reacted with human IgA l as substrate and the results of that reaction analyzed for either degradation of intact IgA Ior appearance of IgA1 cleavage products. Synthetic peptide homologs of IgAl protease cleavage sites have also been tested and can serve as substrates for IgA~ proteases. ~9 However, thcsc peptide substrates are not available commercially or readily synthesized. Therefore, currently the only substrate used for the detection and quantitation of IgA~ protease activity is human IgA1. Preparation of Substrate
N u m e r o u s procedures have been described for the purification of IgA from normal human serum, from colostrum, and from plasma, serum, or other fluids of patients with IgA myeloma (see Ref. 20 for a review). The method outlined below is relatively simple and reliable, especially when used to purify myeloma IgAl from patient sera. Procedure
1. To 3 ml of IgA 1 myeloma serum, slowly add 2 ml of a saturated solution of ammonium sulfate in water, stirring constantly. Continue to stir slowly for 30 min at room temperature. Centrifuge for 20 min at 8000 g and 4 °. Discard the supernatant. Wash the precipitate once with 40% saturated ammonium sulfate and resuspend the washed precipitate in 3.0 ml o f 0.9% NaC1. Dialyze the sample extensively against 0.9% NaCI to r e m o v e all ammonium sulfate. 2. T o the dialyzed sample ( - 3 . 0 ml), add 6 ml of 60 m M sodium acetate buffer, p H 4.8. Dropwise, add 9.0 ml of caprylic acid, stirring constantly, at room temperature. Continue to stir slowly for 30 min. Centrifuge for 20 min at 8000 g and 4 °. Carefully remove the supernatant and discard the 15s. K. Mehta, A. G. Plant, N. J. Calvanico, and T. B. Tomasi, Jr., J. Irnmunol. 111, 1274 (1973). 16j. Reinholdt and M. Kilian, J. lmrnunol. Methods 63, 367 (1983). 17M. S. Blake and C. Eastby, J. Immunol. Methods 144, 215 (1991). 18S. B. Mortensen and M. Kilian, J. Chromatogr. 296, 257 (1984). 19S. G. Wood and J. Burton, Infect. Immun. 59, 1818 (1991). 2oj. Mestecky and M. Kilian, this series, Vol. 116, p. 37.
546
ENZYME AND TOXIN ASSAYS
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precipitate. Dialyze the supernatant against 5 mM potassium phosphate buffer, pH 8.0. 3. Apply the sample to an anion-exchange column [e.g., Pharmacia (Piscataway, NJ) FPLC (fast protein liquid chromatography) Mono Q or Whatman (Clifton, NJ) DE-52] equilibrated with 15 mM Tris-phosphate buffer, pH 8.0. Elute the column with a linear buffered pH gradient, using 15 mM Tris-phosphate, pH 8.0, as the starting buffer and 0.3 M Tris-phosphate, pH 4.0, as the final buffer. 4. Screen each fraction by Ouchterlony double-diffusion analysis for IgG, IgA, IgM, and albumin, using appropriate antisera and controls. Pool fractions containing IgA, concentrate, and screen again by Ouchterlony to confirm the purity of the IgA. If necessary repeat the chromatography step to remove contaminating material. The major contaminant is usually albumin, which can also be removed by passage over an Affi-Gel blue (Bio-Rad, Richmond, CA) affinity column. An alternative procedure that is especially useful in the purification of IgA 1 from normal human serum is affinity chromatography over a jacalin-Sepharose column. Jacalin, or jack bean lectin, selectively binds human IgA 1 but not IgA2.21
Quantitative Assay Using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis The following assay technique is based on physical separation of the IgA~ cleavage products (Faba and Fco0 from the intact IgA~ molecule by electrophoresis. 7,22 Quantitative results are achieved by using trace amounts of radiolabeled IgA~ as part of the reaction mixture.
Reagents Pure human IgA1, at a concentration of 2.0 mg/ml 125I-labeled human IgA~, approximately 400/zCi/ml Assay buffer: 50 mM Tris-HC1, pH 7.5, containing 10 mM MgCI2, 10 mM CaCI2, and 0.05% bovine serum albumin (radioimmunoassay grade) SDS-PAGE sample buffer: 62.5 mM Tris-HCl, pH 6.8, containing 12.5% glycerol, 1.25% SDS, 1.25% 2-mercaptoethanol, and 0.006% bromphenol blue 21 D. L. Skea, P. Christopoulos, A. G. Plaut, and B. J. Underdown, Mol. lmmunol. 25, 1 (1988). 2z D. A. Simpson, R. P. Hausinger, and M. H. Mulks, J. Bacteriol. 170, 1866 (1988).
[43]
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Procedure
1. Mix 2.5/.d of the enzyme sample to be assayed, 2.5/.d of human IgA l (5/zg), 2.5/.d of [125I]IgA1 (--I /.~Ci), and 5/zl of assay buffer, and incubate the mixture in a 37° water bath. After incubation, add 50/zl of SDS-PAGE sample buffer and boil for 5 min to stop the reaction and reduce the IgA~ to polypeptide monomers. 2. Electrophorese the samples on a 9% polyacrylamide gel containing 0.1% SDS in a Laemmli buffer system 23 to separate the IgA 1 cleavage products. Note that we generally use 50 mM Tris, 384 mM glycine, 0.1% SDS as electrode buffer rather than the standard Laemmli recipe. 3. Stain the gel with 0.05% Coomassie Brilliant Blue in 35% methanol-10% acetic acid and destain in 35% methanol-10% acetic acid. Dry the stained gel onto Whatman 3MM paper and autoradiograph onto Kodak (Rochester, NY) XAR-5 film to localize the labeled IgA 1 bands. Using the autoradiograph as a cutting guide, excise the sections of each lane of the gel containing (1) the intact IgA1 heavy chain ( - 5 6 kDa) and (2) the F c a and Fda fragments ( - 2 5 - 3 5 kDa), as shown in Fig. lb. The Fdo~ is defined as the heavy chain portion of the Fabo~ fragment. Regions of the gel containing light chains ( - 2 2 kDa) or secretory component ( - 8 0 kDa) are discarded. The amount of radioactivity [counts per minute (cpm)] in each band is measured using a 3/counter. 4. The percent cleavage of the IgA1 in each sample is calculated as follows: cpm in Fd and Fc fragments = % fragments cpm in intact heavy chain + cpm in Fd and Fc fragments 5. The amount of IgA l cleaved is then calculated as % fragments in sample - % fragments in negative control % fragments in positive control ×/xg IgA l per digest =/~g IgA l cleaved The positive control is a sample in which complete cleavage of the available IgAl has occurred, and it provides a value for the maximum percentage of total radioactivity that is found in IgA1 fragments. This value is usually 75-80% rather than 100%. 6. Units of IgAl protease activity are expressed as micrograms IgA~ cleaved per minute. 23U. K. Laemmli, Nature (London) 227, 680 (1970).
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ENZYME AND TOXIN ASSAYS
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Example. An undiluted sample of supernatant from a late log phase broth culture of N. gonorrhoeae yields 25% fragments in a 20-min incubation. The percentage of fragments in the negative control is 5% and that in the positive control is 80%. The assay reaction mix included 2.5 /xl enzyme sample and 5/xg IgA~. For the calculation, (25% - 5%) x 5 ~g IgAl = 25/xg IgA1 cleaved/min/ml (80%) × 20 m i n x 0.0025 ml = 25 units/ml of sample
Comments. The SDS-PAGE procedure is the only method for the assay of IgAl protease activity which permits both quantitation of units of IgAl protease and determination of the type of cleavage, based on the size of the Fda and Fct~ fragments produced. The method can also be used for qualitative assays on culture supernatants or on suspensions of bacterial colonies. For qualitative assays, we recommend using only the radiolabeled substrate to yield increased sensitivity. The electrophoresis-based assays are the method of choice in use today by most laboratories studying IgA~ proteases. The technique allows for multiple samples to be processed simultaneously, offers consistency, is amenable to qualitative or quantitative studies, and is relatively rapid. The use of radiolabeled IgA~ limits the amount of IgA~ necessary for assays, but it can present a problem for laboratories that do not wish to employ radioisotopes. Detection of IgA1 bands by immunoblotting with antiserum against the a chain is an alternative, 24 although quantitation by densitometric scanning of developed blots is generally less sensitive and less reproducible than assay with radiolabeled substrate. Qualitative Plate Assay for Immunoglobulin A1 Protease Solid-phase plate assays can be used for screening large numbers of isolates simultaneously and are most often used to select an IgAl proteaseproducing isolate from a population of nonproducers. The first described method utilizes radiolabeled IgA1 immobilized on polyacrylamide beads via an anti-Fcot antibody. 25 The technique involves overlaying bacterial colonies on an agar plate with a layer of immobilized IgA~ in top agar or agarose. Secreted IgA~ protease from producing colonies will cleave the immobilized IgAl, liberating Fabot, which is diffusible. Diffused Faba fragments are blotted onto a nitrocellulose membrane, which is then autoradiographed. IgA~ protease-producing isolates are identified by a locationcorresponding radioactive signal. 24 T. Ahl and J. Reinholdt, Infect. lmmun. 59, 563 (1991). 25 j. V. Gilbert and A. G. Plaut, J. lmmunol. Methods 57, 247 (1983).
[43]
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549
Reagents 125I-Labeled human IgA1, approximately 400/zCi/ml Immunobead (Bio-Rad) reagent specific for human IgA Phosphate-buffered saline, pH 7.5 (PBS) 2% Agarose in PBS Nitrocellulose filters
Procedure 1. Reconstitute the lyophilized polyacrylamide beads at a concentration of 1 mg/ml in PBS. Let stand at least 30 rain at room temperature. 2. For each 100-mm petri plate to be assayed, mix 2 ml bead suspension plus 4-10 ~Ci [125I]IgA1 (10-20/zl). Incubate for 90 min at 37° with gentle shaking. Wash the beads three times with PBS to remove unbound IgA. Resuspend the beads to a concentration of I mg/ml in PBS and warm to 50° for 5 min. 3. For each plate to be assayed, melt 2 ml of sterile 2% agarose in PBS and cool to 50 °. 4. Rapidly mix 2 ml of the 125I-labeled bead suspension plus 2 ml agarose and pour onto the surface of the plate to be assayed. Swirl gently to distribute an even layer of agarose, being careful not to disturb the bacterial colonies. It is helpful to have the culture plates at 37° to prevent the agarose from solidifying too rapidly. Incubate at 37° in a moist chamber, such as a sealed box lined with damp paper towels. The incubation time will vary with the bacteria to be assayed: N. gonorrhoeae colonies need approximately 30 min, whereas Escherichia coil clones expressing iga genes may need 12-16 hr. 5. Lay a dry nitrocellulose filter over the agarose, being careful to avoid trapping any air bubbles, and mark the orientation of the filter. Incubate at 37° for 20 min in a moist chamber. Lift off the filter and wash twice in PBS to remove any adherent agarose or other debris. Air-dry the filter and autoradiograph onto Kodak XAR-5 film. 6. I g A 1 protease-producing colonies release radiolabeled IgAl fragments, which are picked up by the nitrocellulose. Therefore, exposed areas on the autoradiograph correspond to IgA~ protease-producing colonies.
Comments. It is necessary to adjust both incubation times and exposure times to achieve optimal sensitivity with minimal background. Strong IgA~ protease producers such as N. gonorrhoeae will need only 15-30 min of incubation for protease activity to be detectable, whereas E. coli clones, which are often very weak protease producers, may need 12-16 hr of incubation as well as increased exposure time for the autoradiograph. In any case, incubation with nitrocellulose filters should be for only 20-30
550
ENZYME AND TOXIN ASSAYS
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min, or background levels may become too high for interpretation of results. It is possible to recover viable bacteria from under the overlay agarose if exposure to radiation is kept to a minimum. 26 Alternatively, bacteria or phage to be tested can be spotted in a grid pattern onto two plates, with one plate assayed by this method and the other held in reserve for the isolation of positive colonies. Two variations have arisen from this method. One involves prior immobilization of the radiolabeled IgA l on the nitrocellulose membrane and incubation on a bacteria-covered surface. 27 After allowing for diffusion of the liberated Fabot, IgA l protease producers can be detected by a decrease in the amount of radioactive signal. This method is not as sensitive as the original assay procedure, since it is much more difficult to detect a negative area than a positive one. A second variation incorporates t~-light chain antibodies bound to the nitrocellulose membrane to improve the efficiency of Fabo~ recovery. 28 Detection of bound Fca fragments with biotinylated antibodies to Fcct eliminates the need for using radioactive materials.
Immunoassay Procedure for Quantitation of lmmunoglobulin A 1 Protease Activity Several ELISAs for the quantitation of IgA l protease activity have been developed. 16,17These methods are not optimal for screening bacterial samples for IgA1 protease activity, since they do not permit identification of protease type nor do they distinguish between IgAi protease activity and nonspecific degradation ofimmunoglobulin Al. However, these assays generally are very sensitive and reproducible, and they have been especially useful in detection of monoclonal antibodies capable of inhibition of IgA~ protease activity. 17 Reinholdt and Kilian 16 developed an ELISA that utilizes antibody to human light chain to coat the wells and to bind IgAl by its Fab end. After digestion with IgA1 protease, horseradish peroxidaseconjugated antibody against Fco~ is used to quantitate the remaining uncleaved IgA~. In this assay, decreased absorbance correlates with increased protease activity. Blake and Eastby ~7developed a similar procedure that utilizes the IgAbinding protein from group B streptococci 29 to coat the wells and to bind 26 R. 27 R. 28 T. 29 G.
J. Shoberg, Ph.D. Thesis, Michigan State Univ., East Lansing (1991). Halter, J. Pohlner, and T. F. Meyer, EMBO J. 3, 1595 (1984). A. Brown and I. G. Leak, J. lmmunol. Methods 123, 241 (1989). J. Russell-Jones, E. C. Gotschlich, and M. S. Blake, J. Exp. Med. 160, 1467 (1984).
[43]
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551
IgAl substrate by its Fc end. After digestion with IgA~ protease, remaining Fabt~ is detected and quantitated using alkaline phosphatase-conjugated antibodies to human light chain. Decreased absorbance correlates with increased cleavage of IgAl and increased protease activity. Both of these ELISA techniques offer the ability to perform multiple assays rapidly and simultaneously, using very small amounts of human IgAl and no radioactivity, with very high sensitivity. However, the methods may not be useful for assay of IgA 1proteases from oral streptococci, which may cleave only one heavy chain rather than both and thus fail to release cleaved fragments.~6,z°
Enzyme Production and Isolation Several methods have been described for the production and partial purification of neisserial IgA l proteases.17,22'27'3° All rely on a combination of chromatographic procedures, including ion-exchange, chromatofocusing, molecular sieve, and hydrophobic interaction chromatography. The procedure we use to purify the gonococcal type 2 IgA~ protease is a modification of a previously published method 22 that gives a higher yield of active IgA 1 protease. Procedure
1. Inoculate brain-heart infusion (BHI) agar containing 10 ml/liter KeUogg's supplement 31 from a freezer stock of N . g o n o r r h o e a e stored at - 7 0 ° in 2% tryptone-20% glycerol. Grow overnight at 35 ° under 5% CO z. 2. Prepare brain-heart infusion broth. Filter through a 10,000 molecular weight cutoff Amicon (Danvers, MA) membrane. Collect and autoclave the filtrate; discard the retentate. Immediately before use, add 10 ml/liter Kellogg's supplement plus 10 ml/liter of 4.2% sodium bicarbonate (BHIK). 3. Inoculate 30 ml of this medium (BHIK) from the overnight plate culture. The starting OD520 should be approximately 0.05. Incubate the culture at 35° in a water bath shaker at 150 rpm for 3-4 hr, until the ODsz0 reaches 0.8-1.0. Use the broth to inoculate 500 ml of prewarmed BHIK. Incubate at 35 ° for 3-4 hr, to an OD520of 0.8-1.2 (late log phase). Centrifuge for 30 min at 6000 g and 4 ° to pellet the bacteria. 4. Carefully decant the supernatant into a clean, ice-cold flask. Discard the cell pellet. Add 0.5 M EDTA, pH 8.0, to a final concentration of 50 mM. Plaut, this series, Vol. 165, p. 117 (1988). 31D. S. Kellogg,Jr., W. L. Peacock, W. E. Deacon, L. Brown, and C. I. Pirkle, J. Bacteriol. 85, 1274(1963). 3o A . G .
552
ENZYME AND TOXIN ASSAYS
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5. Add approximately 200 ml of DEAE-cellulose (Whatman DE-52) resin, equilibrated in 50 mM Tris-HC1, pH 7.5, to the supernatant. Stir slowly at 4° for at least 1 hr. Centrifuge for 30 min at 6000 rpm and 4 ° to pellet the resin. Decant and save the supernatant. Wash the pelleted resin with 50 mM Tris-HCl, pH 7.5, plus 5 mM EDTA; decant and save the supernatant. Pool the supernatants and discard the used resin. 6. Concentrate the pooled supernatant by positive pressure dialysis in an Amicon stirred cell using a 50,000 molecular weight cutoff membrane. If the material is to be stored, add 20% sterile, ultrapure glycerol (BRL, Gaithersburg, MD) to the concentrated sample. 7. Using an Amicon or similar membrane filtration device (not dialysis tubing), dialyze the sample into at least 2 volumes of distilled water, to dilute the salt concentration. Bring the sample to 47.5 ml with sterile distilled water. Add 2.0 ml of ampholytes (Pharmacia), pH 8.0-10.5 range, plus 0.5 ml of ampholytes, pH 5.0-8.0 range. Separate the sample in a Rotofor (Bio-Rad) isoelectric focusing cell, according to the manufacturer's instructions. 8. Pool the fractions with IgA 1 protease activity and exchange into 50 mM Tris-HC1, pH 7.5, plus 5 mM EDTA, 20% glycerol, and 0.2% Nonidet P-40 detergent. Chromatograph over a Pharmacia FPLC Superose 12 HR 10/30 column equilibrated with the same buffer. Screen fractions for IgAl protease activity by SDS-PAGE assay and for purity by Coomassie blue-silver staining. 32 Pool fractions containing IgA1 protease and exchange into 50 mM Tris-HCl, pH 7.5, plus 5 mM EDTA and 20% glycerol, and store at - 7 0 ° . Comments. We have successfully purified type 2 gonococcal IgA 1 protease to homogeneity by the above method. Type 1 gonococcal protease can be purified by similar methods, although it is much less stable. 22 Use of 50 mM Tris-acetate, pH 7.5, plus 5 mM EDTA, 1 mM dithiothreitol (DTT), and 20% glycerol as a stabilization buffer is necessary for purification of the type 1 gonococcal protease. The IgA 1proteases from N. meningitidis 6 and H. influenzae 7 have been partially purified by a combination of anion-exchange and molecular sieve chromatography. The method described here, with modifications such as optimization of appropriate stabilization buffers for each enzyme, may also be applicable to purification of IgA 1 proteases from these bacteria.
32 A. Gorg, W. Postel, J. Weser, H. W. Schivaragin, and W. M. Boesken, Sci. Tools (Sweden) 32, 5 (1985).
[43]
BACTERIAL IGAI PROTEASES
553
TABLE I PROPERTIES OF BACTERIALIMMUNOGLOBULINA1 PROTEASES Species
Enzyme classa
Mr (× 10-3)
pl
Kmb (/,~mol)
Refs.
Bacteroides melaninogenicus Capnocytophaga spp. Haemophilus infiuenzae (type 1) Haemophilus influenzae (type 2) Neisseria gonorrhoeae (type 1) Neisseria gonorrhoeae (type 2) Neisseria meningitidis (type 2) Streptococcus oralis Streptococcus pneumoniae Streptococcus sanguis
T M S S S S S NR M M
62c NR d 90c, 108e 100c 112c 106-110 c,e 100c 100c NR 100c, 186e
5.0 NR NR NR NR 8.6 NR NR NR 5.45
3.4 NR 0.7 1.5 0.6 1.8 NR NR NR 23
18,33 33 9,34,35 34,36 22,34 8,22,34 36,37 38 37 10,34,38,39
M, Metalloprotease; S, serine protease; T, thiol protease. b Determined against human IgAt. c Determined by chromatography or SDS-PAGE. d NR, Not reported. e Determined by sequence analysis. Properties of Bacterial Immunoglobulin A 1 Proteases T a b l e 133-39 s u m m a r i z e s s o m e o f t h e i n f o r m a t i o n k n o w n a b o u t t h e c h e m i s t r y o f t h e b a c t e r i a l IRA l p r o t e a s e s , i n c l u d i n g t h e c l a s s o f p r o t e a s e to w h i c h t h e g i v e n IgA~ p r o t e a s e b e l o n g s a n d t h e m o l e c u l a r w e i g h t , w h e r e r e p o r t e d . D a t a a r e n o t a v a i l a b l e for all o f t h e IgA~ p r o t e a s e s t h a t h a v e b e e n d e s c r i b e d . S o m e o f t h e h i g h l i g h t s o f t h e d a t a a r e t h e f a c t s t h a t (1) three of the four classes of proteases are represented, with aspartic protea s e s b e i n g t h e e x c e p t i o n , a n d (2) t h e s e e n z y m e s s h o w a w i d e r a n g e o f m o l e c u l a r w e i g h t s a n d i s o e l e c t r i c p o i n t s . It a p p e a r s t h a t this set o f p r o t e aRes w i t h s i m i l a r l y n a r r o w r a n g e s o f s u b s t r a t e s p e c i f i c i t y is a c t u a l l y a g r o u p i n g o f v e r y d i v e r s e e n z y m e s w h i c h w e r e c l u s t e r e d into a g r o u p b a s e d solely on that one characteristic. 33 E. V. G. Frandsen, J. Reinholdt, and M. Kilian, Infect. Immun. 55, 631 (1987). 34 W. W. Bachovkin, A. G. Plaut, G. R. Flentke, M. Lynch, and C. A. Kettner, J. Biol. Chem. 265, 3738 (1990). 35j. Pohiner, C. Maercker, H. Apfel, and T. F. Meyer, in "Frontiers of Mucosal Immunology" (M. Tsuchiya, H. Nagura, T. Hibi, and I. Moro, eds.), Vol. 1, p. 567. Elsevier Science Publ., Amsterdam, 1991. 36 M. H. Mulks, unpublished data, 1982. 37M. Kilian, J. Mestecky, R. Kulhavy, M. Tomana, and W. T. Butler, J. Immunol. 124, 2596 (1980). t8 j. Reinholdt, M. Tomana, S. B. Mortensen, and M. Kilian, Infect. Immun. 58, 1186 (1990). 39 A. G. Plaut, J. V. Gilbert, and I. Heller, Adv. Exp. Med. Biol. 107, 489 (1978).
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ENZYME AND TOXIN ASSAYS
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Substrates. By definition, IgAl proteases proteolyze human IgAl and not IgA 2, IgM, IgG, or other serum proteins such as albumin. 2,3 For the proteases that have been tested against IgA from other mammals, IgAl from higher primates (i.e., chimpanzee and gorilla) can be hydrolyzed, but not IgAl from other primates or from a wide range of other mammalian species. 2'3 The type 2 IgA 1 protease from N. gonorrhoeae contains three sequences within the 169-kDa proenzyme that are cleaved by autoproteolysis during secretion of the mature protease. 8 Synthetic peptides homologous to two of the sequences are also cleaved by the gonococcal type 2 protease, although similar peptides homologous to the hinge region of human IgA1 are not.19 In addition, we have demonstrated that several proteins found in the isolated membranes of gram-negative bacteria are degraded by the type 2 IgA 1 protease of N. gonorrhoeae. 4° 4o R. J. Shoberg and M. H. Mulks, Infect. Immun. 59, 2535 (1991).
[44] Elastase Assays By LYNN RUST, CALVIN R. MESSING, and BARBARAH. IGLEWSKI
Introduction The elastin protein is the primary constituent of the elastic fiber, the predominant connective tissue of the lung, blood vessels, and skin. Characteristic of elastin is the desmoisine cross-linking structure, formed by the covalent binding of four lysyl residues. Elastin includes a high concentration of the small hydrophobic amino acids including glycine, alanine, and valine. It is the most insoluble protein in the human body.1 Owing to the unusual structure and insolubility of elastin, few proteases have elastolytic activity, whereas elastases frequently have proteolytic activity against a variety of substrates. The neutral metalloproteinase of Vibro vulnificus is active against azocasein and elastin. 2 The metalloenzymes Bacillus thermoproteolyticus thermolysin and Pseudomonas aeruginosa elastase also have general proteolytic as well as elastolytic activity. In addition to elastin, P. aeruginosa elastase is capable of degrading t j. M. Davidson, in " C o n n e c t i v e T i s s u e Disease: Molecular Pathology of the Extracellular M a t r i x " (J. Uitto a n d A. J. Perejda, eds.), p. 423. Dekker, N e w York, 1987. 2 M. H. K o t h a r y and A. S. Kreger, J. Gen. Microbiol. 133, 1783 (1987).
METHODS IN ENZYMOLOGY,VOL. 235
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