Biol. Chem. Hoppe-Seyler Vol. 371, pp. 549-555, July 1990
Monoclonal Antibodies Specific for Neutrophil Proteinase 4 Production and Use for Isolation of the Enyzme Kjell OHLSSON, Carina LINDER and Margareta ROSENGREN Lunds Universitet, Institutionen for kirurgisk patofysiologi, Malmö Allmänna Sjukhus. Malmö
(Received 2 March 1990)
Summary: Four stable hybridoma cell lines producing monoclonal antibodies specific for neutrophil proteinase 4 (NP4) were established and one monoclonal antibody was chosen to produce an immunoaffinityresin for the purification of NP4. In a precipitation assay system these antibodies bound NP4 in a dosedependent manner, but did so neither with neutrophil elastase nor with cathepsin G. NP4 was purified and electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single MT = 30000 polypeptide.The purified en-
zyme digested fibrin but not elastin and it cleaved Boc-Ala-ONp readily (Km = 0.47mM) at neutral pH, but had no effect on Suc-[Ala]3Nan and W-Suc-[Ala]2-Pro-Phe-pNA. The proteolytic activity was inhibited by DFP, and 2 with a K{ of 10~9M for the NP4«iPI complex. The NH2-terminal sequence and the amino-acid composition of NP4 were distinct from those of elastase and cathepsin G. Neutrophils contain large amounts of NP4 as judged by the comparable amounts of elastase- and 4complexes present in inflammatory exudates.
Monoklonale Antikörper spezifisch für neutrophile Proteinase 4. Ihre Herstellung und Verwendung zur Enzymisolierung Zusammenfassung: Es wurden vier stabile HybridZellinien gewonnen, die für neutrophile Proteinase 4 (NP4) spezifische monoklonale Antikörper bildeten. Mit einem dieser monoklonalen Antikörper wurde ein Immunaffinitätsharz für die Reinigung von NP4 hergestellt. In einem Präzipitations-Testsystem banden diese Antikörper NP4 dosisabhängig, nicht aber neutrophile Elastase oder Cathepsin G. NP4 wurde gereinigt. Nach Elektrophorese des affinitätsgereinigten Enzyms in Natriumdodecylsulfat-Polyacrylamidgelen wurde ein einzelnes Polypeptid mit Mr = 30000 erhalten. Das gereinigte Enzym verdaute Fi-
brin, aber nicht Elastin. Es spaltete Boc-Ala-ONp ohne weiteres (Km = 0.47mM) bei neutralen pH-Werten, hatte aber keine Wirkung auf Suc-[Ala]3Nan und N-Suc-[Ala]2-Pro-Phe-pNA. Die proteolytische Aktivität wurde gehemmt durch DFP, «iPI und a2M mit einem #rWert von 10'9M für den NP4-a1PI-Komplex. Die N-terminale Sequenz und die Aminosäurezusammensetzung von NP4 unterschieden sich von denen der Elastase und des Cathepsins G. Neutrophile Zellen enthalten große Mengen an NP4 gemessen an den vergleichbaren Mengen an Elastase- und NP4-«iPIKomplexen in Entzündungsexsudaten.
Key words: «j-proteinase inhibitor, a 2 -macroglobulin, leukocyte proteinases, peritonitis, polymorphonuclear leukocytes.
Abbreviations: NP4 = neutrophil proteinase 4 (EC 3.4.21.-); «iPI = arproteinase inhibitor; 2 = «i-macroglobulin; Suc-[Ala]3Nan = N-succinyl-Ala-Ala-Ala 4-nitroanilide; A/-Suc-[Ala]2-Pro-Phe-pNA = W-Suc-Ala-Ala-Pro-Phe-4-nitroanilide; Boc-Ala-ONp = Bocalanine 4-nitrophenyl ester; Z-Ala-Nap = /V-benzyloxycarbonyl-L-alanine ß-naphthyl ester; DFP = diisopropylfluorophosphate; SDS-PAGE = sodium dodecyl sulfate polyacrylamid gel electrophoresis; SLPI = secretory leukocyte proteinase inhibitor; DTT= dithiothreitol.
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550
K. Ohlsson, C. Linder and M. Rosengren
The azurophil granules of human neutrophil leukocytes contain large quantities of two fairly well characterized serine proteases; elastase^1"4^ and cathepsin Ql5-6] and small amounts of specific collagenase^. A third protease^9'10^, which for a period was thought to be specific collagenase^11'12^ but was later characterized as a serine protease[13], has been partly purified from leukemic polymorphonuclear leukocytes. This enzyme was also demonstrated in the azurophil granules of the neutrophils[14] and was tentatively named neutrophil proteinase 4 (NP4)[15].The enzyme has been shown to be secreted in large amounts also from normal neutrophils during phagocytosis1161 in experimental situations but has also been found to be released in large quantities during purulent inflammations such as peritonitis^. Further knowledge about NP4 is, however, very limited, perhaps because it has proved quite difficult to isolate. The purpose of the present investigation was therefore to produce monoclonal antibodies to NP4 and to use them for immunoaffinity purification of the enzyme from normal human neutrophil leukocytes. Material and Methods Chemicals Electrophoresis reagents, DextranTSOO, Protein-A-Sepharose CI4B, and CNBr-activated Sepharose CL-4B were produced by Pharmacia LKB Biotechnology, Uppsala, Sweden. Class specific goatanti mouse immuno-globulins were obtained from Nordic inimuno-logic Laboratories, P.O. Box 22, 5000 AATilburg, the Netherlands. Tissue culture medium and fetal calf serum were supplied by Gibco Laboratories LifeTechn., Inc., 3175 Stately Road, Grand Island, N.Y., 14072 U.S.A. Pansorbin was obtained from Calciobiochem Corporation, P.O. Box 12087, San Diego, California, 92112-34180 U.S.A. All other chemicals and proteins were purchased from Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178, U.S.A. Antisera against NP4112', neutrophil elastase'181, £xrproteinase inhibitor (aiPI) and a2-macroglobulin (a2M) are obtained by immunization of rabbits as described earlier'21. Peritoneal exudates from 5 patients with purulent peritonitis were collected during surgery. The samples were centrifuged within 30 min and the supernatants were stored at - 70 °C. Experimental procedures The leukocytes were purified from the buffy-coats obtained from healthy blood donors. 1200 ml of buffy coat was mixed with 400 ml 6% DextranT-500 (Pharmacia, Uppsala, Sweden) in 0.15M NaCl. The suspension was incubated for 45 min at room temperature in a glass funnel. The supernatant was then separated off and was centrifuged at 250 x g for 10 min. The cell pellet was resuspended in 0.87% NH4C1 and incubated for 5 min to lyse the erythrocytes.The leukocytes were pelleted by centrifugation at 250 χ g for 10 min and washed twice with 0.15M NaCl.The leukocytes were then lysed by incubation over night at + 4 °C in 100 m/of 0.2M sucrose containing 10000 units heparin and 0.5 mg DNAase (deoxyribonuclease 1 from bovine pancreas type IV).The granule fraction was obtained by centrifugation at 40000 x g for 50 min and stored at-20 °C. Four such portions of leukocyte granules were usually used for each
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preparation of enzyme, representing about 1.5 x 1010 neutrophil leukocytes. Partial purification of N P4 for use as an immunogen NP4 was purified as earlier described'11'.The enzyme preparation obtained by this method was estimated by SDS-PAGE to be approximately 90% pure. Production of monoclonal antibodies BALB/c mice (Bommice, Bomholtg rd Breeding and Research Center Ltd., Bomholtvej 10, DK-8680 Ry, Denmark) were immunized by subcutaneous injection of 10 μg of purified NP4 in complete Freund's adjuvant, repeated 5 times at 3 week intervals.Two final intravenous injections of 20 /xg each were given three days and one day before the fusion. Hybridomas were produced by fusion of SP2/0, non-secreting BALB/c myoloma cells with spleen lymphocytes from two out of five immunized mice'19'20'. Four very strongly positive "first hybridomas" were obtained. Selection of antibody-producing hybridomas Hybridoma culture fluids were tested for antibody using the solid phase ELISA technique. Microtiter plates were coated with 50 μΐ of the partly purified NP4, 10 /zg/m/. Hybridomas producing antibodies specific for the enzyme were selected for cloning. The subclass type of the monoclonal antibodies produced was determined by the double immunodiffusion test'2Il Cells harvested from cultures of hybridomas producing monoclonal antibodies against NP4 were injected intraperitoneally into Pristane-treated (Aldrich, Beerse, Belgium) BALB/c mice for the production of ascitic fluid.This procedure was approved by the ethical board. Enzyme assays Fibrinolytic activity was measured using fibrin-agarose plates preheated to destroy any plasmin(ogen) present and clastinolytic activity was measured with the elastin-agarose plate method'22'.The hydrolysis of Boc-alanine 4-nitrophenylester (Boc-Ala-ONp) in 0.05M sodiumphosphate buffer, pH 6.5 at 25 °C was monitored in a photometric assay at 347.5 nm'23'. For the preparation of the 0.02M substrate stock solution 61 mg Boc-Ala-ONp was dissolved in 10 ml spectroquality methanol. W-Suc-Ala-Ala-Pro-Phe-pNA124' and Suc-[Ala]3-Nan'25' were used to test for cathepsin G and leukocyte elastase activity, respectively. For the titration of NP4 with human c^PI (kindly supplied by C-B Laurell) or secretory leukocyte proteinase inhibitor (SLPI)'26^ constant amounts of enzyme were incubated with increasing amounts of inhibitor. After incubating the enzyme inhibitor mixture for 30 min at 25 °C in O.lMTris/HCl buffer, 0.15M NaCl, at pH 7.8 the enzyme activity was determined after the addition of 10 μΐ of the Boc-Ala-ONp stock solution.The final volume was 1.0 m/.The effect of diisopropylfluorophosphate (DFP) on the enzyme activity was also tested with Boc-Ala-ONp as substrate. 5 μΐ of 1.15M DFP solution was added to 50 μΐ enzyme solution giving a 105 molar excess of DFP versus NP4.The reaction mixture was incubated for 30 min at 25 °C before measuring the enzyme activity. A mixture with 0.15M NaCl replacing the DFP served as control. Reaction mixtures of human «iPI or plasma and NP4 were also analysed using crossed immunoelectrophoresis'27'. Enzyme activity in immuno-precipitates, after crossed immunoelectrophoresis using antiserum against o^M, was localized using TV-benzyloxycarbonyl-L-alanine /3-naphtyl ester (Z-Ala-Nap) as substrate'28'. Electrophoresis Monoclonal antibodies and NP4 preparations were checked for purity by SDS-PAGE, run under reducing conditions, (Phast Gel
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Vol. 371 (1990)
gradient 10-15%, Pharmacia LKB Biotechnology). Reaction mixtures of NP4 and ajPI were also analysed. Apparent molecular masses were estimated on the basis of a standard curve consisting of the following proteins: phosphorylase b 94000, bovine serum albumin 67000, ovalbumin 43000, carbonate dehydratase 30000, soy-beantrypsin inhibitor 20100 and a-lactalbumin 14400 Da (Electrophoresis Calibration Kit LW, Pharmacia LKB). Isoelectric focusing was performed in PhastGel IEF3-9 (Pharmacia LKB) according to the manufacturers. All gels were stained using the PhastGel Silver Kit (Pharmacia LKB). Indirect immunoprecipitation ofNP4 by monoclonal antibodies NP4 (5 /ig) was incubated in a total volume of 100 μ! with an increasing amount of monoclonal antibody and bovine serum albumin (to equalize the protein content in each tube) in 0.05MTris/HCl buffer, 0.005M EDTA, at pH 7.4, at room temperature for 30 min. The reaction mixture was further incubated at room temperature for 30 min after the addition of 10 μΐ of 20 yag/m/ rabbit anti-mouse IgG and at 4 °C for 30 min after the addition of 60 μΐ of Pansorbin suspension. The immune complexes were precipitated by centrifugation at 4 °C. Aliquots of the resulting supernatant were taken for the measurement of protease activity with Boc-Ala-ONp as substrate. The control experiments were carried out with normal mouse IgG instead of monoclonal antibody, for the initial step of the incubation. Amino-acid analysis The purified protein was reduced for l h with 25mM DTTin 0.5M Tris/HCl with 6M guanidine-HCl at pH 8.0 and then carboxymethylated for 2 h with 50mM iodoacetic acid in O.SMTris at pH 8. The carboxymethylated protein was hydrolysed in OM HCl in evacuated, sealed tubes at 110 °C for 24, 48 and 72 h.The hydrolysates were analysed in a Beckman 6300 amino-acid analyser. Half-cystine was determined as cysteic acid after performic acid oxidation129'. Amino-acid sequence analysis The NH 2 -terminal sequence of about 30 μ-g of carboxymethylated protein was analysed using an Applied Biosystems 470 A gas-liquid solid phase sequenator with a standard program'30'. Other methods Protein concentrations were determined according to Lowry using bovine serum albumin as standard'311. Immunodiffusion analyses were performed in agarose gel as described by Ouchterlony'211. Monoclonal antibodies were labelled with 125I according to a lactoperoxidase method'32' to the specific activity of about
Results
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Isolation of Neutrophil Proteinase 4
£
0.0 Monoclonal antibody [μθ\ Fig.l. Binding of NP4 by MabF3:B3. Purified NP4 (5 μ%) was incubated with increasing amounts of purified monoclonal antibodies and then with rabbit antimouse IgG (see Methods). After precipitation of the immune complexes with Pansorbin aliquots of the supernatant fluid were taken for measurement of NP4 activity.
analysis of the monoclonal antibodies revealed that they were all IgGl antibodies. Immuno-affinity purification ofNP4 Mab F3:B3 ascites fluid was purified by affinity chromatography on a Protein A Sepharose C1-4B column ( 1 x 5 cm) equilibrated with 1.5M glycine/ NaOH, 3M NaCl, at pH 8.9.The column was washed with this buffer after application of the ascites fluid and the IgG was eluted step wise with O.lM citric acid/ NaOH at pH 6.0, pH 5.0, pH 4.0 and pH 3.O. IgGl was eluted at pH 6.O. The monoclonal antibodies were then covalently attached to CNBr-activated Sepharose CI-4B in order to produce an antibodyaffinity resin for the purification of NP4.
Neutrophil granules from about 1.5 x 1010 leukocytes were extracted twice with 50 ml of 0.05MTris/HCl buffer, l.OM NaCl at pH 8.0, by freeze-thawing 5 times. The two extracts were combined and all elastase and cathepsin g was recovered by affinity chromatography onTrasylol-Sepharose CI-4B as described earlier^. The non-binding proteins were loaded onto the Mab F3:B3 resin. Non-specific binding protein was eliminated by rinsing the column with the Tris/HCl buffer and specific elution of NP4 was accomplished using reversed flow elution with O.lM glycine/HCl at pH 2.5, 3M NaCl. The fraction tubes contained 150 μΐ l.OMTris/HCl, pH 8.0 per ml eluate. Brought to you by | New York University Bobst Library Technical Se Authenticated Download Date | 5/23/15 3:00 AM
Production of monoclonal antibodies Five stable hybridoma cell-lines producing antibody that bound rapidly in solution to native NP4 were established. These cell-lines were designated Mab F3:B3,F3:B7/2,F3:B7/4,F3:B7/5,F3:B8andtheywere used for the preparation of ascites fluid. The antibodies bound NP4 in the immunoprecipitation technique as described in the Methods section (Fig-1). All monoclonal antibodies bound to NP4 in an enzyme-linked immuno-sorbent assay but not to elastase and cathepsin G (data not shown). Subclass
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1.0 ·-
30kDa 0.5
Fig. 2. Absorbance curves obtained on laser densitometry of SDS-PAGE electrophoretograms of purified NP4 ( ), purified «iPI ( ) and a reaction mixture of NP4 and a molar excess of «iPI( ).
The purified enzyme appeared to be homogeneous as judged by electrophoresis on 10-15% SDS-PAGE gradient gels, followed by silver staining (Pharmacia/ LKB Biotechnology). A polypeptide of about Mr 30000 was obtained (Fig. 2). The amount of purified NP4 obtained was about 9 mg compared with 12 mg of elastase and 6 mg of cathepsin G. The approximate yield of NP4 (% of starting activity) in the immunopurification procedure was 85%. NP4 migrated completely to the cathode in a polyacrylamide isoelectric focusing gel of pH range 3-9. The purified NP4 showed the reaction of complete immunologic identity with the corresponding enzyme earlier purified from leukemic cells when tested by immunodiffusion analyses^ using polyclonal antisera and radiolabelled monoclonal antibody (Fig. 3).
C Fig. 3. Immunodiffusion analyses of (1) the old preparation of NP4 from leukemic leukocytes, (2).The NP4 preparation from normal leukocytes described in this paper, (3) the neutrophil granulae extract, (4) the non-binding proteins from the Trasylol-Sepharose CL-4B columns and (5) the non-binding proteins from the Mab F3:B3 resin. Well (A) contains a mixture of polyclonal antiserum against NP4 and 125I-labelled Mab F3:B3.The right part of the picture shows the autoradiograph of the left.
Amino-acid composition of the purified protein The amino-acid composition presented in the table is the corrected result of one 24-h and one 72-h hydrolysis of the NP4. The values for serine and threonine are the calculated results of 0-h hydrolysis. The protein was shown to contain glucosamine, but this finding was not further evaluated.
N H2-terminal sequence of the purified NP4
The results from Edman degradations of the NH2-terminal of the neutrophil proteinase are presented in Fig. 4. A single sequence was obtained. It is distinct The amino-acid composition of cathepsin G and from the one of neutrophil elastase and cathepsin G leukocyte elastase are given for comparison, showing although considerable homology is evident. The senot only a higher glutamic acid and a lower arginine quence of the first 20 NH2-terminal amino acids of the content in NP4 than in the other two enzymes, but corresponding enzyme purified earlier from leukemic cells[11] is identical (Fig. 4). also other differences. Brought to you by | New York University Bobst Library Technical S Authenticated Download Date | 5/23/15 3:00 AM
Isolation of Neutrophil Proteinase 4
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NP4
10 20 He Val Gly Gly His Glu Ala Gin Pro His Ser Arg Pro Tyr Met Ala Ser Leu Gin Met Arg Gly Asn Pro Gly
Elastase
lie Val Gly Gly Arg Arg Ala Arg Pro His Ala Trp Pro Phe Met Val Ser Leu Gin Leu Arg Gly Gly His Phe
Cathepsin G
l i e He Gly Gly Arg Glu Ser Arg Pro His Ser Arg ProThr Met Ala Thr Leu Glu Ele Glu Ser Pro Ala Gly
NP4 (Leukemic cells)
Me Val Gly Gly His Glu Ala Gin Pro His Ser Arg Pro Tyr Met Ala Ser Leu Gin Met
Fig. 4. NHi-terminal amino-acid sequence analysis of NP4 and a comparison with the amino-acid sequence of human neutrophil leukocyte elastase'·'9', cathepsin G'6' and NP4 from leukemic cells.
Table. Amino-acid composition of human neutrophil leukocyte proteases. Residues/molecu e
Amino acid
Aspartic acid Threonine Serine Glutamicacid Proline Glycine Alanine Half-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan a
This paper.
b
Ref. (6J .
c
NP4a
Cathepsin Gb
Elastase0
27 16 16 30 18 27 21 4 24 3 13 26 4 15 13 4 16 n.d.
19 12 15 24 13 20 13 6 16 5 13 19 5 7 6 4 34 3
24 7 13 18 10 28 24 6 25 2 11 20 3 9 4 1 22 2
Ref.|4).
plotted against the inhibitor concentration. The best fit between the theoretical line and the data is provided with the K, for the αιΡΙ-ΝΡ4 complex set at 10~9M. A 100-fold molar excess of SLPIgavea 10% decrease of the NP4 activity in the assay but DFP blocked the enzyme activity completely. SDS-PAGE electrophoretograms, run under reducing conditions, of reaction mixtures of NP4 and a^Pl were analysed using laser densitometry (UltroScan XL Laser Densitometer, Pharmacia/LKB, Sweden). The single, homogenous peak obtained on analyses of the NP4 preparation disappears completely on addition of «iPI in molar excess indicating that the isolated NP4 is fully active. The simultaneous appearance of a new component corresponding to a molecular mass of about 80000 Da indicates the formation of a stable complex between NP4 and a^PI (Fig. 2). On crossed immuno-electrophoresis of reaction mixtures of plasma and NP4 using antiserum against aiPl, the presence of two c^PI components is seen. One is located in the a\ region, representing free inhibitor. The second is located in the a2 zone.This component also binds 125I-labelled monoclonal antibodies, as evidenced by the blackening of the precipitate on autoradiography, and thus contains ΝΡ4-αιΡΙ complexes (Fig. 5A, B). The a2M precipitates showed the same pattern with or without the addition of NP4 to the plasma and they did not contain labelled monoclonal antibodies. After the addition of the enzyme the a2M precipitate showed, however, strong enzymatic activity on Z-Ala-Nap indicating the binding of NP4 by «2M.
Enzyme activity The purified protease hydrolysed native fibrin but showed no activity against insoluble elastin. The degradation rate for fibrin corresponded to about 40% of that for human neutrophil elastase on a molar basis. The fibrinolytic activity was blocked by «2M in a dosedependent way. The Km value for Boc-Ala-ONp found was 0.47mM. The pH optimum for this substrate was close to 7.5. NP4 showed no activity against Suc-[Ala]3-Nan and Sue Ala2-Pro-Phe-pNA. oLiPl-bound leukocyte proteases in inflammatory NP4 was titrated with human OL\-protease inhibitor using the assay with Boc-Ala-ONp as the substrate. exudates Initial measurements indicated that «iPI inhibits NP4 Fig. 5C shows the presence of three electrophoretic via the formation of a one-to-one molar complex with fractions of aiPl in inflammatory peritoneal exuthe enzyme. To determine the K\, a standard amount dates. Apart from the one with the mobility of free of the enzyme was mixed with various amounts of «iPI, a second is seen in the a2 region and a third in aiPl and allowed to come to equilibrium.The residual the /32 region. The second component is also precipifree enzyme was determined by assay with Boc-Ala- tated by polyclonal antiserum against NP4 and binds 125 ONp as substrate, and the amount of free enzyme was I-labelled monoclonal antibodies against this enBrought to you by | New York University Bobst Library Technical Se Authenticated Download Date | 5/23/15 3:00 AM
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Discussion
Λ Β
The results presented in this report demonstrate that monoclonal antibodies against NP4 are specific for this enzyme. Neither elastase nor cathepsin G is cross-reactive with these antibodies. The production of monoclonal antibodies that will bind rapidly in solution to enzymatically active NP4, and their successful use as an affinity resin, has greatly simplified the purification of the enzyme from normal neutrophils. The enzyme purified has a neutral pH optimum and its enzymatic activity is blocked by DFP. Other properties in common with the corresponding enzyme purified earlier from leukemic neutrophils^11·13^ are similar amino-acid composition and an identical Nterminal amino acid sequence. Furthermore, the two enzymes show identical immuno-precipitation patterns in solution when using monoclonal antibodies and also in gels when using a combination of monoclonal antibodies and polyclonal antibodies. Both are inhibited by c^PI and α:2Μ[Ι2].
NP4 is a glycoprotein with an MT value determined to about 30000 in the present study. The value initially reported as being twice this figure for the enzyme from leukemic cells may perhaps be explained by dimerization^. NP4 is distinct from elastase and cathepsin G, apart from immunologically, also concerning its amino-acid composition and its NH2-terminal amino acid sequence^~6l Furthermore, NP4 does not hydrolyse low molecular mass amide substrates typical for elastase or for cathepsin G. NP4 digests fibrin, however, and is thus a true endoproteinase, while it has no activity on elastin. αχΡΙ, besides α2Μ, appears to be an Fig. 5. (A) Precipitation patterns obtained on crossed imimportant inhibitor of NP4 as judged by the relatively muno-electrophoresis of human plasma after the addition of low Ki (~ 10~9M) which, however, is higher than the NP4 using rabbit anti human NP4 and 125I-labelled Mab F3:B3 corresponding value for c^PI-elastase, namely in the gel. ~ 10~10Mi33]. The complexes are stable on reducing (B) shows the autoradiograph of (A). (C) shows crossed imSDS-PAGE electrophoresis. On agarose gel elecmuno-electrophoresis with antiserum against «iPI of a specitrophoresis the NP4 complexes with a{Pl migrate as men of peritoneal exudate collected at operation from a patient a2-globulins, while the corresponding elastase comwith peritonitis. (D) shows the precipitate patterns obtained on crossed immuno-electrophoresis of the same exudate as in (C). plexes are found in the /32-region[18]. This suggests Part (1) contains antibodies against human «iPI and part (2) that, although the isoelectric point of NP4 is at least contains a mixture of rabbit anti-human NP4 and 125I-labelled about pH 9 as judged from the isoelectric focusing Mab F3: B3 .The center peak of «iPI in complex was due to NP4 analyses, it is probably below the isoelectric point of since it was specifically eliminated by anti NP4 antibodies and elastase. Furthermore, SLPI is a potent inhibitor of furthermore bound labelled Mab F3:B3 as shown by the autoradiograph (E). leukocyte elastase[26i, but we could not demonstrate any significant inhibition of NP4 by SLPI. During recent years a series of papers have dealt with the release of leukocyte elastase in different inflamzyme (Fig. 5D, E). It thus probably consists mostly of matory conditions and the proposed important role of NP4-C*!?! complexes. The β2 component is precipi- this enzyme in pathophysiology[34~38]. Judging from tated by polyclonal antibodies against neutrophil elas- the patterns obtained on crossed immunoelectase and it thus represents «iPI complexes with this trophoresis, similar amounts of o^PI-bound elastase enzyme (results not shown). and NP4 are present in the peritoneal exudates from Brought to you by | New York University Bobst Library Technical S Authenticated Download Date | 5/23/15 3:00 AM
Vol.371 (1990)
Isolation of Neutrophil Proteinase 4
patients with purulent peritonitis. Thus, NP4 may contribute significantly to the inflammatory process over and above its consumption of important inhibitors. In conclusion, NP4 appears to be a quantitatively prominent component of the neutrophils, comparable with elastase and cathepsin G, as judged from the amounts of enzyme obtained on purification from leukocytes and found released in inflammatory processes such as peritonitis. The availability of monoclonal antibodies will facilitate further studies of the structure and function of NP4. Note Added in Proof (23 May 1990): After submission of this manuscript a paper was published about a serine proteinase from human neutrophils, denoted p29b, with a substrate specificity similar to that of NP4140'. We found among the references a preceeding paper'41' describing p29b as an antibiotic protein with N-terminal amino acid sequence homology to elastase and cathepsin G leading the authors to suspect that p29b was a proteinase. We find that the sequences for the first 20 Nterminal amino acids, with a single exception, of p29b and NP4 are identical, strongly indicating that we are dealing with identical proteins. The amino-acid analyses were performed by Ingrid Dahlqvist at the Dept. of Clinical Chemistry, University of Lund, Malmö General Hospital, Malmö. This project was supported by the Medical Faculty, Lund University, The Swedish Medical Research Council (projects B8917X-03910-17B and B89-17K-08715-01A), The Swedish Cancer Society (project 1300-17K-08715-01A), The Swedish Association against Heart and Chest Diseases, the Greta and Johan Kock Foundations, the Alfred Österlund Foundation, and The Swedish Tobacco Company.
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Ohlsson, K. & Olsson, I. (1977) J. Lab. Clin. Med. 89, 269-277. 13 Ohlsson, K. (1980) in Collagenase in Normal and Pathological Connective Tissues (Woolley, D.E. & Evanson, J.M., eds.) pp. 209-222, John Wiley & Sons Ltd. 14 Ohlsson, K., Olsson, I. & Spitznagel, J.K. (1977) HoppeSeyler's Z. Physiol. Chem. 358, 361-366. 15 Barett, A.J. & McDonald, J.K. (1980) in Mammalian Proteases: A Glossary and Bibliography pp. 189-190, Academic Press, London. 16 Ohlsson, K. & Olsson, I. (1977) Scand. J. Haematol. 19, 145-152. 17 Ohlsson, K. (1976) Surgery 79, 652-657. 18 Ohlsson, K. & Olsson, I. (1974) Scand. J. Clin. Lab. Invest. 34, 349-355. 19 Kohler, G. & Milstein. C. (1975) Nature (London) 256, 495-497. 20 Kennett. R.H., Denis, K.A., Tung. S.A. & Klinman. N.R. (1978) Curr. Top. Microbiol. Immunol. 81, 77-91. 21 Ouchterlony, 0. (1948) Acta Pathol. Microbiol. Scand. 25, 186-191. 22 Ohlsson, K. & Olsson, A.S. (1976) Hoppe-Seyler's Z. Physiol. Chem. 357, 1153-1161. 23 Visser, L. & Blout, E.R. (1972) Biochim. Biophys. Acta 268, 257-260. 24 Nakajima, K. & Powers, J.C. (1979) J. Biol. Chem. 254, 4027-4032. 25 Bieth, J., Spiess, b. &Wermuth, C.G. (1974) Biochem. Med. 11, 350-357. 26 Thompson, R.C. & Ohlsson, K. (1986) Proc. Natl. Acad. Sei. U.S.A. 83,6692-6696. 27 Ganrot, . . (1972) Scand. J. Clin. Lab. In vest. 29,39 - 47. 28 Häkansson, H.O. & Ohlsson, K. (1988) Biol. Chem. Hoppe-SeylerW, 309-315. 29 Moore, S. (1963) /. Biol. Chem. 238,235-237. 30 Takeda, A., Kaji, H., Nakaya, K., Aouki, Y, Nakamura, Y. & Samejima,T. (1985) Biochem. Int. 11,557-564. 31 Lowry, O.K., Roseprough, N.J., Farr, A.L. & Randall, R.J. (1959)7. Biol. Chem. 193, 265-275. 32 Thorell, J. & Johansson, B.G. (1971) Biochim. Biophys. Acta 251,363 -367. 33 Bieth, J., Pichoir, M. & Metais, P. (1970) FEBS Lett. 6, 319-321. 34 Johnsson, U., Ohlsson, K. & Olsson, I. (1976) Scand. J. Immunol. 5, 421-426. 35 Senior, R.M., Tegner, H., Kühn, C., Ohlsson, K., Starcher, B.C. & Pierce, J.A. (1977) Am. Rev. Resp. Dis. 116,469 -475. 36 Aasen, A.O. & Ohlsson, K. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 683-690. 37 Egbring, R., Schmidt, W, Fuchs, G. & Havemann, K. (1977)5/ood49,219-231. 38 Fritz, H. (1980) in Ciba Foundation Symposium (vol. 75) (Evered, D. &Whelen, J., eds.) pp. 351-375, Excerpta Medica, Amsterdam. 39 Sinha, S., Watorek, W., Karr, S., Giles, J., Bode, W. & Travis, J. (1987) Proc. Natl. Acad. Sei. U.S.A. 84, 22282232. 40 Campanelli, D., Detmers, P.A., Nathan, C.F. & Gabay, J.E. (1990)7. Clin. Invest. 85, 904-915. 41 Gabay, J.E., Scott, R.W., Campanelli, D., Griffith. J., Wilde, C., Marra, M.N., Seeger, M. & Nathan, C.F. (1989) Proc. Natl. Acad. Sei. USA 86, 5610-5614.
Prof. Kjell Ohlsson, Carina Linder and Margareta Rosengren, Lunds Universitet, Institutionen for kirurgisk patofysiologi, Malmö Allmänna Sjukhus, S-21401 Malmö, Sweden.
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Monoclonal Antibodies Specific for Neutrophil Proteinase 4 Production and Use for Isolation of the Enyzme Kjell OHLSSON, Carina LINDER and Margareta ROSENGREN Lunds Universitet, Institutionen for kirurgisk patofysiologi, Malmö Allmänna Sjukhus. Malmö
(Received 2 March 1990)
Summary: Four stable hybridoma cell lines producing monoclonal antibodies specific for neutrophil proteinase 4 (NP4) were established and one monoclonal antibody was chosen to produce an immunoaffinityresin for the purification of NP4. In a precipitation assay system these antibodies bound NP4 in a dosedependent manner, but did so neither with neutrophil elastase nor with cathepsin G. NP4 was purified and electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single MT = 30000 polypeptide.The purified en-
zyme digested fibrin but not elastin and it cleaved Boc-Ala-ONp readily (Km = 0.47mM) at neutral pH, but had no effect on Suc-[Ala]3Nan and W-Suc-[Ala]2-Pro-Phe-pNA. The proteolytic activity was inhibited by DFP, and 2 with a K{ of 10~9M for the NP4«iPI complex. The NH2-terminal sequence and the amino-acid composition of NP4 were distinct from those of elastase and cathepsin G. Neutrophils contain large amounts of NP4 as judged by the comparable amounts of elastase- and 4complexes present in inflammatory exudates.
Monoklonale Antikörper spezifisch für neutrophile Proteinase 4. Ihre Herstellung und Verwendung zur Enzymisolierung Zusammenfassung: Es wurden vier stabile HybridZellinien gewonnen, die für neutrophile Proteinase 4 (NP4) spezifische monoklonale Antikörper bildeten. Mit einem dieser monoklonalen Antikörper wurde ein Immunaffinitätsharz für die Reinigung von NP4 hergestellt. In einem Präzipitations-Testsystem banden diese Antikörper NP4 dosisabhängig, nicht aber neutrophile Elastase oder Cathepsin G. NP4 wurde gereinigt. Nach Elektrophorese des affinitätsgereinigten Enzyms in Natriumdodecylsulfat-Polyacrylamidgelen wurde ein einzelnes Polypeptid mit Mr = 30000 erhalten. Das gereinigte Enzym verdaute Fi-
brin, aber nicht Elastin. Es spaltete Boc-Ala-ONp ohne weiteres (Km = 0.47mM) bei neutralen pH-Werten, hatte aber keine Wirkung auf Suc-[Ala]3Nan und N-Suc-[Ala]2-Pro-Phe-pNA. Die proteolytische Aktivität wurde gehemmt durch DFP, «iPI und a2M mit einem #rWert von 10'9M für den NP4-a1PI-Komplex. Die N-terminale Sequenz und die Aminosäurezusammensetzung von NP4 unterschieden sich von denen der Elastase und des Cathepsins G. Neutrophile Zellen enthalten große Mengen an NP4 gemessen an den vergleichbaren Mengen an Elastase- und NP4-«iPIKomplexen in Entzündungsexsudaten.
Key words: «j-proteinase inhibitor, a 2 -macroglobulin, leukocyte proteinases, peritonitis, polymorphonuclear leukocytes.
Abbreviations: NP4 = neutrophil proteinase 4 (EC 3.4.21.-); «iPI = arproteinase inhibitor; 2 = «i-macroglobulin; Suc-[Ala]3Nan = N-succinyl-Ala-Ala-Ala 4-nitroanilide; A/-Suc-[Ala]2-Pro-Phe-pNA = W-Suc-Ala-Ala-Pro-Phe-4-nitroanilide; Boc-Ala-ONp = Bocalanine 4-nitrophenyl ester; Z-Ala-Nap = /V-benzyloxycarbonyl-L-alanine ß-naphthyl ester; DFP = diisopropylfluorophosphate; SDS-PAGE = sodium dodecyl sulfate polyacrylamid gel electrophoresis; SLPI = secretory leukocyte proteinase inhibitor; DTT= dithiothreitol.
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The azurophil granules of human neutrophil leukocytes contain large quantities of two fairly well characterized serine proteases; elastase^1"4^ and cathepsin Ql5-6] and small amounts of specific collagenase^. A third protease^9'10^, which for a period was thought to be specific collagenase^11'12^ but was later characterized as a serine protease[13], has been partly purified from leukemic polymorphonuclear leukocytes. This enzyme was also demonstrated in the azurophil granules of the neutrophils[14] and was tentatively named neutrophil proteinase 4 (NP4)[15].The enzyme has been shown to be secreted in large amounts also from normal neutrophils during phagocytosis1161 in experimental situations but has also been found to be released in large quantities during purulent inflammations such as peritonitis^. Further knowledge about NP4 is, however, very limited, perhaps because it has proved quite difficult to isolate. The purpose of the present investigation was therefore to produce monoclonal antibodies to NP4 and to use them for immunoaffinity purification of the enzyme from normal human neutrophil leukocytes. Material and Methods Chemicals Electrophoresis reagents, DextranTSOO, Protein-A-Sepharose CI4B, and CNBr-activated Sepharose CL-4B were produced by Pharmacia LKB Biotechnology, Uppsala, Sweden. Class specific goatanti mouse immuno-globulins were obtained from Nordic inimuno-logic Laboratories, P.O. Box 22, 5000 AATilburg, the Netherlands. Tissue culture medium and fetal calf serum were supplied by Gibco Laboratories LifeTechn., Inc., 3175 Stately Road, Grand Island, N.Y., 14072 U.S.A. Pansorbin was obtained from Calciobiochem Corporation, P.O. Box 12087, San Diego, California, 92112-34180 U.S.A. All other chemicals and proteins were purchased from Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178, U.S.A. Antisera against NP4112', neutrophil elastase'181, £xrproteinase inhibitor (aiPI) and a2-macroglobulin (a2M) are obtained by immunization of rabbits as described earlier'21. Peritoneal exudates from 5 patients with purulent peritonitis were collected during surgery. The samples were centrifuged within 30 min and the supernatants were stored at - 70 °C. Experimental procedures The leukocytes were purified from the buffy-coats obtained from healthy blood donors. 1200 ml of buffy coat was mixed with 400 ml 6% DextranT-500 (Pharmacia, Uppsala, Sweden) in 0.15M NaCl. The suspension was incubated for 45 min at room temperature in a glass funnel. The supernatant was then separated off and was centrifuged at 250 x g for 10 min. The cell pellet was resuspended in 0.87% NH4C1 and incubated for 5 min to lyse the erythrocytes.The leukocytes were pelleted by centrifugation at 250 χ g for 10 min and washed twice with 0.15M NaCl.The leukocytes were then lysed by incubation over night at + 4 °C in 100 m/of 0.2M sucrose containing 10000 units heparin and 0.5 mg DNAase (deoxyribonuclease 1 from bovine pancreas type IV).The granule fraction was obtained by centrifugation at 40000 x g for 50 min and stored at-20 °C. Four such portions of leukocyte granules were usually used for each
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preparation of enzyme, representing about 1.5 x 1010 neutrophil leukocytes. Partial purification of N P4 for use as an immunogen NP4 was purified as earlier described'11'.The enzyme preparation obtained by this method was estimated by SDS-PAGE to be approximately 90% pure. Production of monoclonal antibodies BALB/c mice (Bommice, Bomholtg rd Breeding and Research Center Ltd., Bomholtvej 10, DK-8680 Ry, Denmark) were immunized by subcutaneous injection of 10 μg of purified NP4 in complete Freund's adjuvant, repeated 5 times at 3 week intervals.Two final intravenous injections of 20 /xg each were given three days and one day before the fusion. Hybridomas were produced by fusion of SP2/0, non-secreting BALB/c myoloma cells with spleen lymphocytes from two out of five immunized mice'19'20'. Four very strongly positive "first hybridomas" were obtained. Selection of antibody-producing hybridomas Hybridoma culture fluids were tested for antibody using the solid phase ELISA technique. Microtiter plates were coated with 50 μΐ of the partly purified NP4, 10 /zg/m/. Hybridomas producing antibodies specific for the enzyme were selected for cloning. The subclass type of the monoclonal antibodies produced was determined by the double immunodiffusion test'2Il Cells harvested from cultures of hybridomas producing monoclonal antibodies against NP4 were injected intraperitoneally into Pristane-treated (Aldrich, Beerse, Belgium) BALB/c mice for the production of ascitic fluid.This procedure was approved by the ethical board. Enzyme assays Fibrinolytic activity was measured using fibrin-agarose plates preheated to destroy any plasmin(ogen) present and clastinolytic activity was measured with the elastin-agarose plate method'22'.The hydrolysis of Boc-alanine 4-nitrophenylester (Boc-Ala-ONp) in 0.05M sodiumphosphate buffer, pH 6.5 at 25 °C was monitored in a photometric assay at 347.5 nm'23'. For the preparation of the 0.02M substrate stock solution 61 mg Boc-Ala-ONp was dissolved in 10 ml spectroquality methanol. W-Suc-Ala-Ala-Pro-Phe-pNA124' and Suc-[Ala]3-Nan'25' were used to test for cathepsin G and leukocyte elastase activity, respectively. For the titration of NP4 with human c^PI (kindly supplied by C-B Laurell) or secretory leukocyte proteinase inhibitor (SLPI)'26^ constant amounts of enzyme were incubated with increasing amounts of inhibitor. After incubating the enzyme inhibitor mixture for 30 min at 25 °C in O.lMTris/HCl buffer, 0.15M NaCl, at pH 7.8 the enzyme activity was determined after the addition of 10 μΐ of the Boc-Ala-ONp stock solution.The final volume was 1.0 m/.The effect of diisopropylfluorophosphate (DFP) on the enzyme activity was also tested with Boc-Ala-ONp as substrate. 5 μΐ of 1.15M DFP solution was added to 50 μΐ enzyme solution giving a 105 molar excess of DFP versus NP4.The reaction mixture was incubated for 30 min at 25 °C before measuring the enzyme activity. A mixture with 0.15M NaCl replacing the DFP served as control. Reaction mixtures of human «iPI or plasma and NP4 were also analysed using crossed immunoelectrophoresis'27'. Enzyme activity in immuno-precipitates, after crossed immunoelectrophoresis using antiserum against o^M, was localized using TV-benzyloxycarbonyl-L-alanine /3-naphtyl ester (Z-Ala-Nap) as substrate'28'. Electrophoresis Monoclonal antibodies and NP4 preparations were checked for purity by SDS-PAGE, run under reducing conditions, (Phast Gel
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Vol. 371 (1990)
gradient 10-15%, Pharmacia LKB Biotechnology). Reaction mixtures of NP4 and ajPI were also analysed. Apparent molecular masses were estimated on the basis of a standard curve consisting of the following proteins: phosphorylase b 94000, bovine serum albumin 67000, ovalbumin 43000, carbonate dehydratase 30000, soy-beantrypsin inhibitor 20100 and a-lactalbumin 14400 Da (Electrophoresis Calibration Kit LW, Pharmacia LKB). Isoelectric focusing was performed in PhastGel IEF3-9 (Pharmacia LKB) according to the manufacturers. All gels were stained using the PhastGel Silver Kit (Pharmacia LKB). Indirect immunoprecipitation ofNP4 by monoclonal antibodies NP4 (5 /ig) was incubated in a total volume of 100 μ! with an increasing amount of monoclonal antibody and bovine serum albumin (to equalize the protein content in each tube) in 0.05MTris/HCl buffer, 0.005M EDTA, at pH 7.4, at room temperature for 30 min. The reaction mixture was further incubated at room temperature for 30 min after the addition of 10 μΐ of 20 yag/m/ rabbit anti-mouse IgG and at 4 °C for 30 min after the addition of 60 μΐ of Pansorbin suspension. The immune complexes were precipitated by centrifugation at 4 °C. Aliquots of the resulting supernatant were taken for the measurement of protease activity with Boc-Ala-ONp as substrate. The control experiments were carried out with normal mouse IgG instead of monoclonal antibody, for the initial step of the incubation. Amino-acid analysis The purified protein was reduced for l h with 25mM DTTin 0.5M Tris/HCl with 6M guanidine-HCl at pH 8.0 and then carboxymethylated for 2 h with 50mM iodoacetic acid in O.SMTris at pH 8. The carboxymethylated protein was hydrolysed in OM HCl in evacuated, sealed tubes at 110 °C for 24, 48 and 72 h.The hydrolysates were analysed in a Beckman 6300 amino-acid analyser. Half-cystine was determined as cysteic acid after performic acid oxidation129'. Amino-acid sequence analysis The NH 2 -terminal sequence of about 30 μ-g of carboxymethylated protein was analysed using an Applied Biosystems 470 A gas-liquid solid phase sequenator with a standard program'30'. Other methods Protein concentrations were determined according to Lowry using bovine serum albumin as standard'311. Immunodiffusion analyses were performed in agarose gel as described by Ouchterlony'211. Monoclonal antibodies were labelled with 125I according to a lactoperoxidase method'32' to the specific activity of about
Results
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Isolation of Neutrophil Proteinase 4
£
0.0 Monoclonal antibody [μθ\ Fig.l. Binding of NP4 by MabF3:B3. Purified NP4 (5 μ%) was incubated with increasing amounts of purified monoclonal antibodies and then with rabbit antimouse IgG (see Methods). After precipitation of the immune complexes with Pansorbin aliquots of the supernatant fluid were taken for measurement of NP4 activity.
analysis of the monoclonal antibodies revealed that they were all IgGl antibodies. Immuno-affinity purification ofNP4 Mab F3:B3 ascites fluid was purified by affinity chromatography on a Protein A Sepharose C1-4B column ( 1 x 5 cm) equilibrated with 1.5M glycine/ NaOH, 3M NaCl, at pH 8.9.The column was washed with this buffer after application of the ascites fluid and the IgG was eluted step wise with O.lM citric acid/ NaOH at pH 6.0, pH 5.0, pH 4.0 and pH 3.O. IgGl was eluted at pH 6.O. The monoclonal antibodies were then covalently attached to CNBr-activated Sepharose CI-4B in order to produce an antibodyaffinity resin for the purification of NP4.
Neutrophil granules from about 1.5 x 1010 leukocytes were extracted twice with 50 ml of 0.05MTris/HCl buffer, l.OM NaCl at pH 8.0, by freeze-thawing 5 times. The two extracts were combined and all elastase and cathepsin g was recovered by affinity chromatography onTrasylol-Sepharose CI-4B as described earlier^. The non-binding proteins were loaded onto the Mab F3:B3 resin. Non-specific binding protein was eliminated by rinsing the column with the Tris/HCl buffer and specific elution of NP4 was accomplished using reversed flow elution with O.lM glycine/HCl at pH 2.5, 3M NaCl. The fraction tubes contained 150 μΐ l.OMTris/HCl, pH 8.0 per ml eluate. Brought to you by | New York University Bobst Library Technical Se Authenticated Download Date | 5/23/15 3:00 AM
Production of monoclonal antibodies Five stable hybridoma cell-lines producing antibody that bound rapidly in solution to native NP4 were established. These cell-lines were designated Mab F3:B3,F3:B7/2,F3:B7/4,F3:B7/5,F3:B8andtheywere used for the preparation of ascites fluid. The antibodies bound NP4 in the immunoprecipitation technique as described in the Methods section (Fig-1). All monoclonal antibodies bound to NP4 in an enzyme-linked immuno-sorbent assay but not to elastase and cathepsin G (data not shown). Subclass
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1.0 ·-
30kDa 0.5
Fig. 2. Absorbance curves obtained on laser densitometry of SDS-PAGE electrophoretograms of purified NP4 ( ), purified «iPI ( ) and a reaction mixture of NP4 and a molar excess of «iPI( ).
The purified enzyme appeared to be homogeneous as judged by electrophoresis on 10-15% SDS-PAGE gradient gels, followed by silver staining (Pharmacia/ LKB Biotechnology). A polypeptide of about Mr 30000 was obtained (Fig. 2). The amount of purified NP4 obtained was about 9 mg compared with 12 mg of elastase and 6 mg of cathepsin G. The approximate yield of NP4 (% of starting activity) in the immunopurification procedure was 85%. NP4 migrated completely to the cathode in a polyacrylamide isoelectric focusing gel of pH range 3-9. The purified NP4 showed the reaction of complete immunologic identity with the corresponding enzyme earlier purified from leukemic cells when tested by immunodiffusion analyses^ using polyclonal antisera and radiolabelled monoclonal antibody (Fig. 3).
C Fig. 3. Immunodiffusion analyses of (1) the old preparation of NP4 from leukemic leukocytes, (2).The NP4 preparation from normal leukocytes described in this paper, (3) the neutrophil granulae extract, (4) the non-binding proteins from the Trasylol-Sepharose CL-4B columns and (5) the non-binding proteins from the Mab F3:B3 resin. Well (A) contains a mixture of polyclonal antiserum against NP4 and 125I-labelled Mab F3:B3.The right part of the picture shows the autoradiograph of the left.
Amino-acid composition of the purified protein The amino-acid composition presented in the table is the corrected result of one 24-h and one 72-h hydrolysis of the NP4. The values for serine and threonine are the calculated results of 0-h hydrolysis. The protein was shown to contain glucosamine, but this finding was not further evaluated.
N H2-terminal sequence of the purified NP4
The results from Edman degradations of the NH2-terminal of the neutrophil proteinase are presented in Fig. 4. A single sequence was obtained. It is distinct The amino-acid composition of cathepsin G and from the one of neutrophil elastase and cathepsin G leukocyte elastase are given for comparison, showing although considerable homology is evident. The senot only a higher glutamic acid and a lower arginine quence of the first 20 NH2-terminal amino acids of the content in NP4 than in the other two enzymes, but corresponding enzyme purified earlier from leukemic cells[11] is identical (Fig. 4). also other differences. Brought to you by | New York University Bobst Library Technical S Authenticated Download Date | 5/23/15 3:00 AM
Isolation of Neutrophil Proteinase 4
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553
NP4
10 20 He Val Gly Gly His Glu Ala Gin Pro His Ser Arg Pro Tyr Met Ala Ser Leu Gin Met Arg Gly Asn Pro Gly
Elastase
lie Val Gly Gly Arg Arg Ala Arg Pro His Ala Trp Pro Phe Met Val Ser Leu Gin Leu Arg Gly Gly His Phe
Cathepsin G
l i e He Gly Gly Arg Glu Ser Arg Pro His Ser Arg ProThr Met Ala Thr Leu Glu Ele Glu Ser Pro Ala Gly
NP4 (Leukemic cells)
Me Val Gly Gly His Glu Ala Gin Pro His Ser Arg Pro Tyr Met Ala Ser Leu Gin Met
Fig. 4. NHi-terminal amino-acid sequence analysis of NP4 and a comparison with the amino-acid sequence of human neutrophil leukocyte elastase'·'9', cathepsin G'6' and NP4 from leukemic cells.
Table. Amino-acid composition of human neutrophil leukocyte proteases. Residues/molecu e
Amino acid
Aspartic acid Threonine Serine Glutamicacid Proline Glycine Alanine Half-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan a
This paper.
b
Ref. (6J .
c
NP4a
Cathepsin Gb
Elastase0
27 16 16 30 18 27 21 4 24 3 13 26 4 15 13 4 16 n.d.
19 12 15 24 13 20 13 6 16 5 13 19 5 7 6 4 34 3
24 7 13 18 10 28 24 6 25 2 11 20 3 9 4 1 22 2
Ref.|4).
plotted against the inhibitor concentration. The best fit between the theoretical line and the data is provided with the K, for the αιΡΙ-ΝΡ4 complex set at 10~9M. A 100-fold molar excess of SLPIgavea 10% decrease of the NP4 activity in the assay but DFP blocked the enzyme activity completely. SDS-PAGE electrophoretograms, run under reducing conditions, of reaction mixtures of NP4 and a^Pl were analysed using laser densitometry (UltroScan XL Laser Densitometer, Pharmacia/LKB, Sweden). The single, homogenous peak obtained on analyses of the NP4 preparation disappears completely on addition of «iPI in molar excess indicating that the isolated NP4 is fully active. The simultaneous appearance of a new component corresponding to a molecular mass of about 80000 Da indicates the formation of a stable complex between NP4 and a^PI (Fig. 2). On crossed immuno-electrophoresis of reaction mixtures of plasma and NP4 using antiserum against aiPl, the presence of two c^PI components is seen. One is located in the a\ region, representing free inhibitor. The second is located in the a2 zone.This component also binds 125I-labelled monoclonal antibodies, as evidenced by the blackening of the precipitate on autoradiography, and thus contains ΝΡ4-αιΡΙ complexes (Fig. 5A, B). The a2M precipitates showed the same pattern with or without the addition of NP4 to the plasma and they did not contain labelled monoclonal antibodies. After the addition of the enzyme the a2M precipitate showed, however, strong enzymatic activity on Z-Ala-Nap indicating the binding of NP4 by «2M.
Enzyme activity The purified protease hydrolysed native fibrin but showed no activity against insoluble elastin. The degradation rate for fibrin corresponded to about 40% of that for human neutrophil elastase on a molar basis. The fibrinolytic activity was blocked by «2M in a dosedependent way. The Km value for Boc-Ala-ONp found was 0.47mM. The pH optimum for this substrate was close to 7.5. NP4 showed no activity against Suc-[Ala]3-Nan and Sue Ala2-Pro-Phe-pNA. oLiPl-bound leukocyte proteases in inflammatory NP4 was titrated with human OL\-protease inhibitor using the assay with Boc-Ala-ONp as the substrate. exudates Initial measurements indicated that «iPI inhibits NP4 Fig. 5C shows the presence of three electrophoretic via the formation of a one-to-one molar complex with fractions of aiPl in inflammatory peritoneal exuthe enzyme. To determine the K\, a standard amount dates. Apart from the one with the mobility of free of the enzyme was mixed with various amounts of «iPI, a second is seen in the a2 region and a third in aiPl and allowed to come to equilibrium.The residual the /32 region. The second component is also precipifree enzyme was determined by assay with Boc-Ala- tated by polyclonal antiserum against NP4 and binds 125 ONp as substrate, and the amount of free enzyme was I-labelled monoclonal antibodies against this enBrought to you by | New York University Bobst Library Technical Se Authenticated Download Date | 5/23/15 3:00 AM
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Discussion
Λ Β
The results presented in this report demonstrate that monoclonal antibodies against NP4 are specific for this enzyme. Neither elastase nor cathepsin G is cross-reactive with these antibodies. The production of monoclonal antibodies that will bind rapidly in solution to enzymatically active NP4, and their successful use as an affinity resin, has greatly simplified the purification of the enzyme from normal neutrophils. The enzyme purified has a neutral pH optimum and its enzymatic activity is blocked by DFP. Other properties in common with the corresponding enzyme purified earlier from leukemic neutrophils^11·13^ are similar amino-acid composition and an identical Nterminal amino acid sequence. Furthermore, the two enzymes show identical immuno-precipitation patterns in solution when using monoclonal antibodies and also in gels when using a combination of monoclonal antibodies and polyclonal antibodies. Both are inhibited by c^PI and α:2Μ[Ι2].
NP4 is a glycoprotein with an MT value determined to about 30000 in the present study. The value initially reported as being twice this figure for the enzyme from leukemic cells may perhaps be explained by dimerization^. NP4 is distinct from elastase and cathepsin G, apart from immunologically, also concerning its amino-acid composition and its NH2-terminal amino acid sequence^~6l Furthermore, NP4 does not hydrolyse low molecular mass amide substrates typical for elastase or for cathepsin G. NP4 digests fibrin, however, and is thus a true endoproteinase, while it has no activity on elastin. αχΡΙ, besides α2Μ, appears to be an Fig. 5. (A) Precipitation patterns obtained on crossed imimportant inhibitor of NP4 as judged by the relatively muno-electrophoresis of human plasma after the addition of low Ki (~ 10~9M) which, however, is higher than the NP4 using rabbit anti human NP4 and 125I-labelled Mab F3:B3 corresponding value for c^PI-elastase, namely in the gel. ~ 10~10Mi33]. The complexes are stable on reducing (B) shows the autoradiograph of (A). (C) shows crossed imSDS-PAGE electrophoresis. On agarose gel elecmuno-electrophoresis with antiserum against «iPI of a specitrophoresis the NP4 complexes with a{Pl migrate as men of peritoneal exudate collected at operation from a patient a2-globulins, while the corresponding elastase comwith peritonitis. (D) shows the precipitate patterns obtained on crossed immuno-electrophoresis of the same exudate as in (C). plexes are found in the /32-region[18]. This suggests Part (1) contains antibodies against human «iPI and part (2) that, although the isoelectric point of NP4 is at least contains a mixture of rabbit anti-human NP4 and 125I-labelled about pH 9 as judged from the isoelectric focusing Mab F3: B3 .The center peak of «iPI in complex was due to NP4 analyses, it is probably below the isoelectric point of since it was specifically eliminated by anti NP4 antibodies and elastase. Furthermore, SLPI is a potent inhibitor of furthermore bound labelled Mab F3:B3 as shown by the autoradiograph (E). leukocyte elastase[26i, but we could not demonstrate any significant inhibition of NP4 by SLPI. During recent years a series of papers have dealt with the release of leukocyte elastase in different inflamzyme (Fig. 5D, E). It thus probably consists mostly of matory conditions and the proposed important role of NP4-C*!?! complexes. The β2 component is precipi- this enzyme in pathophysiology[34~38]. Judging from tated by polyclonal antibodies against neutrophil elas- the patterns obtained on crossed immunoelectase and it thus represents «iPI complexes with this trophoresis, similar amounts of o^PI-bound elastase enzyme (results not shown). and NP4 are present in the peritoneal exudates from Brought to you by | New York University Bobst Library Technical S Authenticated Download Date | 5/23/15 3:00 AM
Vol.371 (1990)
Isolation of Neutrophil Proteinase 4
patients with purulent peritonitis. Thus, NP4 may contribute significantly to the inflammatory process over and above its consumption of important inhibitors. In conclusion, NP4 appears to be a quantitatively prominent component of the neutrophils, comparable with elastase and cathepsin G, as judged from the amounts of enzyme obtained on purification from leukocytes and found released in inflammatory processes such as peritonitis. The availability of monoclonal antibodies will facilitate further studies of the structure and function of NP4. Note Added in Proof (23 May 1990): After submission of this manuscript a paper was published about a serine proteinase from human neutrophils, denoted p29b, with a substrate specificity similar to that of NP4140'. We found among the references a preceeding paper'41' describing p29b as an antibiotic protein with N-terminal amino acid sequence homology to elastase and cathepsin G leading the authors to suspect that p29b was a proteinase. We find that the sequences for the first 20 Nterminal amino acids, with a single exception, of p29b and NP4 are identical, strongly indicating that we are dealing with identical proteins. The amino-acid analyses were performed by Ingrid Dahlqvist at the Dept. of Clinical Chemistry, University of Lund, Malmö General Hospital, Malmö. This project was supported by the Medical Faculty, Lund University, The Swedish Medical Research Council (projects B8917X-03910-17B and B89-17K-08715-01A), The Swedish Cancer Society (project 1300-17K-08715-01A), The Swedish Association against Heart and Chest Diseases, the Greta and Johan Kock Foundations, the Alfred Österlund Foundation, and The Swedish Tobacco Company.
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Prof. Kjell Ohlsson, Carina Linder and Margareta Rosengren, Lunds Universitet, Institutionen for kirurgisk patofysiologi, Malmö Allmänna Sjukhus, S-21401 Malmö, Sweden.
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