Biochemical and Immunological Characterization of the Secreted

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 260, No. 4, Issue of February 25, pp. 2493-2500,1985 Printed in U.S.A.

Biochemical and Immunological Characterization of the Secreted Forms of Human Neutrophil Gelatinase” (Received for publication, June 20, 1984)

Margaret S . HibbsS, Karen A. Hasty#, Jerome M. Seyer, Andrew H.Kang, and Carlo L. Mainardin From the Connective Tissue Research Laboratories, Departments of Medicine and Biochemistry, University of Tennessee Center for the Health Sciences and Veterans AdministrationMedical Center, Memphis, Tennessee 38104

Human neutrophils contain a neutral metallopro- tible to degradation by a number of proteinases, there has teinase which degrades denaturedcollagens and poten- also been described a group of neutral metalloproteinases tiates the action of interstitial collagenase. This gela- termed “gelatinases” which appear to have greater specificity tinase is rapidlysecreted from neutrophils stimulated for denatured collagens (5-7).While it has been postulated with phorbol myristate acetate. The secreted enzyme that these enzymes may play an important role in collagen has been purified by a combination of chromatography degradation, they have been relatively ignored due to the on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of ubiquity of proteinases which possess gelatinolytic activity. Several lines of evidence, however, suggest that these en24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl zymes are importantin the degradation of extracellular matrix sulfate-polyacrylamide gel electrophoresis of the pu- proteins. Gelatinases have been frequently found to be present rified enzyme showedthree bands with relativemolec- in situations where interstitial collagenases have been idenular weights of 225,000, 130,000, and 92,000. Elec- tified (5-7)and have been shown to act synergistically with trophoresis in the presence of a reducing agent re- interstitial collagenase in the degradation of the interstitial vealed a single band of M , = 92,000. All the proteins collagens (8).Several groups have reported gelatinases which seen on the unreduced gel were found to contain pro- are capable of degrading type V collagen (8-lo),a collagen teolytic activity against gelatin and native type V colaction of interstitial collagenases (11, lagen. Polyclonal antibodies were prepared against thewhich is resistant to the 12). Thus, gelatinase may beimportant not only in the further M, = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three degradation of interstitial collagens but also in the degradaproteins. Furthermore,the identical three proteins tion of native type V collagen. Since its description by Sopata and Dancewicz in 1974 (7), were identified by the antibodies when crude culture medium was immunoblotted. human neutrophil gelatinase has been examined by several The purified enzyme was inhibited byEDTA and investigators (8, 13-16). Reports of the estimated molecular 1,lO-phenanthroline but not byserine orthiol protein- weight of this enzyme as determined by molecular sieve chroase inhibitors, suggesting that the enzyme is a metal- matography have ranged from 105,000-250,000.Estimates of loendoproteinase. The enzyme had little or no activity molecular weight by SDS-PAGE’ have resulted in values from against common protein substrates such as bovine serum albumin or casein. Native type I collagen was 92,000-150,000.Explanations for these differences include not cleaved under conditions where native typeV col- the method utilized to isolate the crude enzyme from the neutrophil, differences in purification schemes, or partial lagen was extensively degraded. degradation of the enzyme by contaminating proteinases within the preparation. Perhaps the reason these discrepancies has notbeen resolved isthat it has been difficult to obtain The collagenous scaffold of the extracellular matrix is pre- large quantities of this enzyme. dominantly composed of the interstitial collagens-types I, Neutrophil gelatinase has been shown to be readily secreted 11, and I11 collagens (1). These structuralproteins are resistant from the neutrophil in response to soluble stimuli (17).By to degradation by most proteinases: their proteolysis is de- manipulation of culture conditions, it is possible to obtain the pendent on a specific group of enzymes classified as mam- gelatinase with minimal contamination of serine proteinases malian collagenases (EC 3.4.23.6) (2). These proteinases ini- and nonsecretory products of the neutrophil. Utilizing such tiate the degradation of interstitial collagens by cleaving the an approach, we have isolated neutrophil gelatinase by inductriple helical molecule at a single site resulting in 3/4 and 1/4 ing neutrophils to secrete this enzyme in short-term culture fragments which spontaneously denature at body temperain the presence of the secretagogue,phorbol myristate acetate. tures (3, 4). Although these denatured fragments are suscepAfter purification, the gelatinase was found to consist of three * This work was supported by National Institutes of Health Grants molecular weight species with identical substrate specificity AM01138,AM16506, and HL27614 and funds from the Veterans and immunological cross-reactivity. These data support the Administration. The costs of publication of this article were defrayed concept that neutrophil gelatinase consists of multiple molecin part by the payment of page charges. This article must therefore ular weight species which are derived from the same enzyme. be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a Clinical Investigator Award from The National Institute for Arthritis, Diabetes, and Digestive and Kidney Diseases. 5 Postdoctoral Fellow of the Arthritis Foundation. ll Clinical Investigator for the Veterans Administration.

’

The abbreviations used are: SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; PMSF, phenylmethanesulfonyl fluoride; BSA, bovine serum albumin; Tris-buffered saline, 0.05 M Tris-HCI, 0.15 M NaCl, pH 7.5.

2493

2494

Characterization of Secreted Human Neutrophil Gelatinase EXPERIMENTALPROCEDURES

Materials Materials were obtained from the following sources: Trizma Tris base, dextran, Ficoll-metrizoate, Brij-35, dimethyl sulfoxide, aminophenylmercuric acetate, phenylmethanesulfonyl fluoride, bovine serum albumin, phorbol myristate acetate, and gelatin from Sigma; DE52 from Whatman; Ultrogel AcA-34 from LKB; Sepharose 4B and G-200 from Pharmacia; 4-chloronaphthol, protein assay kit, and all reagents for polyacrylamide gel electrophoresis including high molecular weight markers from BioRad; phosphate-buffered saline, Hanks' balanced salt solution, and Freund's adjuvant from Grand Island Biological; peroxidase-labeled goat anti-rabbit antibody from Cappell; and all radiochemicals from New England Nuclear. Protein standards for column calibration were obtained from Boehringer Mannheim. All other chemicals were reagent grade. Methods Collagen Preparation-Type I collagen was extracted from fetal calf skin and purified by the method of Glimcher et al. (18). The purified collagen was radiolabeled with ["C]acetic anhydride according to the method of Cawston and Barrett (19). Type V collagen was prepared from a pepsin extract of human amnion by the method of Rhodes and Miller (20). Collagenolytic Assays-Gelatinolytic activity was determined by the method of Harris and Krane (6). One hundred gg of denatured I4C-labeled type I collagen was incubated in areactionmixture containing 0.08 M Tris-HC1, 0.005 M CaC12, 0.02% NaN3, pH 7.6, in a final volume of 150 p1 at 37 "C. Type I collagen was heat-denatured a t 60 "C for 15 min immediately prior to addition to the incubation mixture. At the termination of the assay, the samples were cooled to 4 "C and then the undigested gelatin and large molecular weight fragments were precipitated by the addition of trichloroacetic acid to a final concentration of 15% (w/v). After centrifugation at 10,000 X g for 5 min, an aliquot of the supernatant was counted in a Packard liquid scintillation counter. Interstitial collagenase activity was determined in a reconstituted fibril assay as described by Nagai et al. (21).Reconstituted fibrils containing 100 pg of "C-labeled type I collagen were incubated with the test fractions in a reaction mixture containing 0.08 M Tris-HC1, 0.005 M CaCI2, 0.02% NaN3, pH 7.6, in a final volume of 300 pl. The samples were incubated for 18 h at 37"C after which the intact fibrillar collagen was precipitated by centrifugation at 10,000 X g for 5 min. An aliquot of the supernatant was counted in a liquid scintillation counter. Assessment of degradation of soluble type I collagen was performed utilizing the identical reaction mixture as described for the fibril assay; however, the total volume was 150 p1 and the incubation was done at 27 "C. After termination of the assay by the addition of EDTA (10 mM final concentration), thereaction products were analyzed by SDS-PAGE. Type V collagenolytic activity was determined by incubating 100 pg of type V collagen with the enzyme preparation in a reaction mixture containing 0.08 M Tris-HC1, 0.005 M CaC12, 0.02% NaN3, pH 7.6, for 18 h at or below 32.5 "C. Previous viscometric studies have indicated that this temperature is safely below the apparent melting point of pepsin-extracted type V collagen (10). The reaction was terminated by the addition of EDTA to a final concentration of 10 mM. The reaction products were analyzed by SDS-PAGE. All collagenolytic assays were done with preparations which were pretreated with 2 mM PMSF. Further addition of PMSF during the assay did not affect the activity seen. In assays to assess the total enzyme present (activeand latent),aminophenylmercuric acetate was added to a final concentration of 1 mM. Isolation of Enzyme-Leukocyte preparations were obtained by sedimentation of whole blood through 3% dextran (w/v) in 0.15 M NaCl to remove the red blood cells (22). After the cells were washed in phosphate-buffered saline, they were resuspended in Hanks' balanced salt solution containing Ca2+ and M$+ at lo7 cells/ml and incubated with 50 ng/ml of phorbol myristate acetate for 15 min at 37 "C. After this incubation, the cells were pelleted by centrifugation and the aspirated supernatants were treated with 2 mM PMSF (final concentration). The supernatantswere stored at -20 "C. In some experiments, the mononuclear cells were separated from the polymorphonuclear cells by centrifugation on a double density cushion of Ficoll-metrizoate (1.077 and 1.119 p ) at 400 X g for 30 min (23). The cell layers were aspirated, washed in phosphate-buffered

saline, resuspended in Hanks' balanced salt solution at lo7 cells/ml and handled as indicated previously. The purity of both cell populations was monitored by differential counts of Wright's-stained material. Each cell population was found to contain >95% of the indicated cell population. Chromatographic Techniques-All chromatographic procedures were performed at 4 "C. Leukocyte culture supernatants from 10 units of blood were concentrated 5-fold by pressure dialysis using an Amicon chamber equipped with a YM-10 membrane. The concentrate was dialyzed against 0.05 M Tris-HC1, 0.005 M CaC12,0.05% Brij-35, 0.02% NaN3, pH 7.6, and then applied to a column (2.5 X 10 cm) of DE52 which had been previously equilibrated in the same buffer. The column was washed with the starting buffer until the absorbance at 280 nm reached base-line, and thebound fraction was eluted with 0.5 M NaCl in the starting buffer. Flow rate was 60 ml/h and 10-ml fractions were collected. Gelatin-Sepharose was prepared by linking heat-denatured (15 min at 60 "C) type I collagen to CNBr-activated Sepharose (24, 25). The gelatinolytic activity isolated by DEAE-cellulose chromatography was applied to a gelatin-Sepharose affinity column (1.5 X 10 cm) which had been previously equilibrated in 0.05 M Tris-HC1, 0.5 M NaCl, 0.005 M CaC12, 0.05% Brij-35, 0.02% NaN3, pH 7.6. The column was washed with starting buffer until the absorbance reached base-line, and then thebound fraction containing the gelatinolytic activity was eluted with 0.05 M Tris-HC1, 1 M NaC1, 0.005 M CaC12, 0.05% Brij35,0.02% NaN3, pH 7.6, containing 5% dimethyl sulfoxide (v/v). Flow rate was 40 ml/h and 10-ml fractions were collected. The fractions containing gelatinolytic activity from the gelatinSepharose column were pooled, dialyzed against 0.05 M Tris-HC1, 0.5 M NaCI, 0.005 M CaC12, 0.02% NaN3, pH 7.6, and concentrated 18fold by pressure dialysis as previously described. This concentrated material was applied to a column of AcA-34 (2 X 90 cm) which had been previously equilibrated in 0.05 M Tris-HC1, 0.5 M NaC1,0.005 M CaC12, 0.02% NaN3, pH 7.6. Flow rate was 16 ml/h and 10 mlfractions were collected. The column had been previously calibrated using the following protein standards: aldolase (160 kDa), BSA (65 kDa), ovalbumin (45 kDa), chymotrypsinogen (25 kDa), and cytochrome c (12.5 kDa). V, was determined using blue dextran and VTHzowas established by the inclusion of 3H20in each column run. Gel Electrophoresis-SDS-PAGE was performed using a slab gel apparatus (BioRad) according to the method of Lammeli (26). The gels were stained in 0.1% Coommassie Brilliant Blue in 50% methanol, 10% acetic acid (v/v) anddestained in 10% methanol, 10% acetic acid (v/v). Demonstration of Enzyme Activity in SDS Gels-Gels to examine gelatinase activity were prepared in the usual manner except that gelatin was included in the running gel (2 mg/ml final concentration), the samples were not boiled prior to electrophoresis, and the gel was cooled to 4 "C during electrophoresis. After electrophoresis, the gels were washed twice in 50 mM Tris-HC1,5 mM CaC12,1pM ZnC1,2.5% Triton X-100 (v/v), pH7.6, for 15 min (27). After the gels were rinsed briefly (5 min) in the above buffer without Triton X-100, they were incubated in a buffer containing 50 mM Tris-HC1, 5 mM CaC12, 1 g M ZnC12, 1 mM aminophenylmercuric acetate, 1% Triton X-100, 0.02% NaNa, pH 7.6, for 1 h at 37 "C. Following the incubation, the gels were stainedwith Coomassie Brilliant Blue and destained as described above. Zones of enzymatic activitywere indicated by negative staining. To determine the type V collagen-degrading activity of the proteins seen on SDS-PAGE, the enzyme preparation was electrophoresed on a 6% polyacrylamide gel in the presence of SDS. After completion of the electrophoresis, the gel was washed as described above to remove the SDS. The washed gel was sliced into 1-mm sections using a gel slicing device (Hoefer) and each slice was incubated with 100 pg of type v collagen in 50 mM Tris-HC1, 5 mMCaC12, 1 p M ZnC12, 1 mM aminophenylmercuric acetate, 1% Triton X-100, 0.02% NaN3, pH 7.6, in a total volume of 150 ~1 for 18 h at 32.5 "C. After terminatlon of the reaction by addition of EDTA to a final concentration of 10 mM, the reaction products were analyzed on 7.5% polyacrylamide gels. Preparation of Polyclonal Antibodies-Six hundred gg of purified gelatinase was separated on a preparative6% polyacrylamide gel. The Coomassie Brilliant Blue-stained protein bandswere cut from the gel and homogenized in the presence of 0.05 M Tris-HC1, 0.15 M NaC1, 0.08% SDS (w/v), pH 7.6, using a ground glass homogenizer (28). The eluted material was emulsified with an equal volume of complete Freund'sadjuvant and injected intradermally into New Zealand

Characterization of Secreted Human Neutrophil White rabbits (3.5-5 kg). The rabbits were boosted with similarly obtained materia1 in Freund's incomplete adjuvant 1 month later. Immunoblotting-Samples to be analyzed by immunoblotting were separated on SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose (200 mA for 2 h). After the excess protein-binding sites were saturated with 3% BSA in 0.05 M TrisHCI, 0.15 M NaCl, pH 7.5 (Tris-buffered saline), for 1 h a t 22 "C, the nitrocellulose was incubated with antisera (1:lOO dilution) in the above buffer for 18 h at 4 "C. After extensive washing with Trisbuffered saline, the samples were incubated with peroxidase-labeled goat anti-rabbit antibody (1:2000 dilution) in Tris-buffered saline containing 3%BSA for 2 h a t 22 "C. After washing, the immunoblots were visualized by the addition of 4-chloronaphthol which gives an insoluble reaction product. Measurement of Peptide B o d Hydrolysis-Quantitative assessment of the degradation of various substrates was obtained by fluorescamine labeling ofnewly formed primary amino groups (29). Protein substrates were dissolved in 0.1 M borate buffer, pH 7.8, containing 0.005 M CaC12 at a concentration of 2 mg/ml. After denaturation of the substrates in a boiling water bath for 15 min, the substrates were incubated with active gelatinase at a 1:200 enzyme:substrate ratio at 37 "C. At various intervals, aliquots were withdrawn and added to tubes containing1,lO-phenanthroline (5 mM final concentration). After the samples were diluted 1:lOO with alkaline water, an equal volume of fluorescamine (0.1 mg/ml in acetonitrile) was added during constant mixing. The samples were analyzed on a Waters fluorescence monitor using glycine as a standard. Miscellaneous Procedures-Protein was determined by the dye binding technique (BioRad) using BSA as a standard (30).

Gelatinase

io 20 fraction number

2495

io

FIG. 2. Gelatin-Sepharose chromatography. The poolfrom DEAE-cellulose containing gelatinase activity was applied to a column (1.5 X 10 cm) of gelatin-Sepharose equilibrated in 0.05 "I'risHCL, 0.5 M NaCl, 0.005 M CaC12, 0.05%Brij-35,0.02% NaN3, pH 7.6. Bound protein was eluted with 0.05 M Tris-HC1, 1 M NaC1, 0.005 M CaC12,0.05% Brij-35, 0.02% NaN3, pH 7.6, containing 5% dimethyl sulfoxide (arrow)-, absorbance 280 nm; W. . ..W, gelatinase activity.

buffer allowed the gelatinolytic activity obtained from the DEAE-cellulose column to be applied directly to the gelatinSepharose column. The workof Sopata (13) has indicated that a gelatinRESULTS Sepharose affinity column is a valuable tool in the isolation Purification of Neutrophil Gelatinuse-In previous studies, of neutrophil gelatinase. In contrast to Sopata who linked we have found that neutrophil gelatinase can be secreted in tryptic peptides of commercially obtained gelatin to CNBrresponse to specific stimuli(31). This technique has been activated Sepharose, we prepared ouraffinity column by employed in thepresent studies to generate culture superna- linking intact denatured 01 chains of type I collagen to CNBrtants which are rich in collagenolytic proteinases but rela- activated Sepharose. The gelatinolytic activity from the tively devoid of nonsecretory proteins of the neutrophil. Leu- DEAE-cellulose column was pooled and applied to a column kocyte preparations, obtained from whole blood by dextran of gelatin-Sepharose which had been equilibrated in 0.05 M sedimentation, were cultured with 50 ng/ml of phorbol myr- Tris-HC1, 0.5 M NaC1,0.005 M CaC12, 0.05% Brij-35, 0.02% istate acetate for 15 min at 37 "C as described under "Meth- NaN3, pH 7.6. After extensive washing with thestarting ods." The culture supernatants from 10 units of blood were buffer, the gelatinolytic activity was eluted with 0.05 M Trisconcentrated by pressure dialysis, dialyzed against 0.05 M HC1, 1 M NaCl, 0.05 M CaC12, 0.05% Brij-35, 5% dimethyl Tris-HC1,0.005 M CaCI2, 0.05%Brij-35,0.02% NaN3, pH 7.6, sulfoxide, 0.02% NaN3, pH 7.6. This stepwise elution gave a and applied to a column of DE52 which had previously been sharp peak of gelatinolytic activity (Fig. 2). The strong bindequilibrated in the same buffer. Under these conditions, the ing of gelatinase to this column allowed isolation of latent interstitial collagenase did not bind to DEAE-cellulose and gelatinase at a specific activity of 24,193 units/ml (Table I), was separated from the gelatinolytic activity which eluted as a specific activity which exceeds the values previously rea sharp peak of activity when 0.5 M NaCl in the starting ported by other investigators (8, 13, 15). The gelatinolytic buffer was applied (Fig. 1).No enhancement of purification activity in this purified preparation was totally inhibited by of the gelatinase could be obtained when agradient was EDTA and 1,lO-phenanthroline while no significant inhibiutilized. In addition, elution with 0.5 M NaCl in the starting tion was noted in the presence of N-ethylmaleimide (final concentration, 10 mM) or PMSF (final concentration, 2 mM), indicating that enzymatic activity was attributable to a metalloproteinase (Table 11). To assess the composition of the material obtained from the gelatin-Sepharose column, 5 gg of this material was electrophoresed on an 8% polyacrylamide gel (0.75 mm thick) in the presence and absence of 2-mercaptoethanol. As can be seen in Fig. 3, the unreduced sample contained three protein bands with molecular weights of 225,000, 130,000, and 92,000 (panel A ) . When the sample was run in the presence of a reducing agent, only one band with a molecular weight of 92,000 was seen (panel B ) . These findings suggested that all three bands seen in the unreduced gel were related. However, FIG. 1. DEAE-cellulose chromatography. Culture superna- since the preparations contained three bands, further purifitants, concentrated 5-fold by pressure dialysis, were applied to a cation was attempted utilizing molecular sieve chromatograDEAE-cellulose column (2.5 X 10 cm) equilibrated in 0.05 M TrisHCI, 0.005 M CaCIZ, 0.05% Brij-35, 0.02% NaN3, pH 7.6. Bound phy. Several different matrices were utilized in an attempt to protein was eluted with 0.5 M NaCl in the starting buffer (arrow). -, absorbance at 280 nm; A- - -A,collagenase activity; B. . . .B, separate these proteins. Initially, the material obtained from gelatinase activity. the gelatin-Sepharose column was concentrated 18-fold, di-

Characterization of Secreted Human Neutrophil Gelatinuse

2496

TARLE I Purification of ge/atinase from human neutrophils Enzyme preparations were preactivatedby incubation with 1 mM aminophenvlmercuric acetate for 1 h at 137 "C and then incubated with gelatin for 30 min at 37 "C. Units of activity are defined as p g of gelatin degraded/min/ mg of protein. No activity was detected in the absence of an activating agent. I'reparatinn Total artivity SpecificProtein activity Purificat inn Recover? C' units mp unitslrnp -f d d 58.6 1. Starting material 87,783 1,498 35,631 110 23.8 5.5 40 2. DEAE-cellulose 3. Gelatin-Sepharose 68,998 2.85 24,193 412 78 .~

TARIX I1 Inhihition

purifipd gelatinase 0.124 p g of purified enzyme was preactivated with 1 mM aminophenylmercuric acetate for 1 h at 37 "C and then incubated with100 UP o f gelatin for BO min at 37 "C. Gelatin deEraded Inhihitor of

enlmin

None PMSF (2 mM) N-Ethvlmaleimide (10 2.49 mM) EDTA ( I O mM) 1.10-Phenanthroline (5 mM)

-

2.55 2.49 0.04 >0.01

fraction number

4

200 *

FIG.4. AcA-34 chromatography. Material isolated from gelatin-Sepharose was applied toa column (2.0 X 90 cm) of Ultrogel AcA34 equilibrated in 0.05 M Tris-HCI, 0.5 M NaCI, 0.005 M CaC12,0.025 NaN3, pH 7.6. Molecular mass markers were: V,, blue dextran; 158 absorbance kDa, aldolase, 68 kDa, BSA; 45 kDA, ovalbumin. -, 280 mn; 0- - -0, gelatinase activity.

Additional attemptstoseparatethethreebands included chromatography on Sephadex G-200 in the presence of 1 M NaCI. In this case, the relative molecular mass was found to be 180,000-200,000, but no separationof the three bandswas accomplished. Thus, the three bands did not appear dissociated under nondenaturing conditions and the relative molecular mass of the complex was dependent on the nature of the molecular sieve and chromatographic conditions utilized. In addition, an80-90% loss of material occurred at this step with no furtherpurification being accomplished; thus, molec4 t ular sieve chromatography was omitted in subsequent purification schemes. Demonstration of Enzyme Activity in SDS Gels-While these three molecular weight species appeared tocoexist as a complex under nondenaturing conditions, it was important to determine which of the bands seen on SDS gels contained gelatin-degrading activity. Thus, the gelatinolytic activity of the three bands was examined by including the substrate in the SDS-polyacrylamide gel system and after electrophoresis incubating the gel underconditions in which the enzyme activity could be detected using the techniquesdescribed under "Methods." After incubation for 1 h at 37 "C, the gels A B were stained in 0.1% Coomassie Brilliant Blue. The zones of FIG. 3. SDS-PAGE of purified gelatinase. 5 pg of gelatinase negative staining indicating gelatinolytic activity are seen in was separated on a 8% polyacrylamide gel (0.75 mm thick) in the Fig. 5. Three bands of lysis were visualized which correlated presence (panel R ) andahsence (panel A ) of 2-mercaptoethanol. with the protein bands obtained on the stained gels. These Molecular mass markers were run ona separate tract of the same gel and are indicated hy the arrows (ovalbumin, 45 kDA; BSA, 68 kDA; results indicated that all three proteins contain gelatinolytic activity, eliminating thepossibility that one (or more) of the phosphorylase b, 116 kDA: and myosin, 200 kDa). bands represented aninactive subunit or carrierprotein. As the work of Murphy et al. ( 8 ) has indicated that neutroalyzed against 0.05 M Tris-HCI, 0.5 M NaCl, 0.005 M CaCI2, 0.02% NaN:I, pJ-l 7.6, and applied to a previously calibrated phil gelatinase degrades type V collagen, it was of interest to seen on gels under column of Ultrogel AcA-34. Gelatinolytic activity eluted with determinewhetherthethreeproteins an apparent molecular weight of 150,000-160,000 (Fig. 4). nonreducing condition were capable of degrading type V col-

45

Characterization of Secreted Human Neutrophil

Gelatinase

2497

these molecular weight species could only be resolved on SDSPAGE, the polyclonal antibodies were obtained by immunization with material isolated from a preparative gel as described under “Methods.” The ability of these antibodies to react with the different protein species was tested by separating the proteins ona 8% polyacrylamide gel, transferring the proteins tonitrocellulose by Western blotting, and developing the blots as described under “Methods.” The results areseen in Fig. 7. Antiserum 69 was preparedagainst the 130-kDa protein and antiserum 70 against the 92-kDa protein. While the preimmune serafailed to show any reaction products (not shown), both antisera recognized all three proteins in the unreduced sample (panel A ) . The reduced sample (panel B ) showed only the 92-kDa species. These data indicate that the three species of gelatinase contained immunologically similar epitopes andagain suggest that they representmultiple forms of the same proteinase. Studies Using Purified Cell Populations-Since the starting material was obtained from unfractionated leukocyte preparations, itremained possible that themononuclear cells within the preparation could contribute a portion of the enzyme activity and possibly account for the heterogeneity of the proteins isolated. To exclude this possibility, neutrophils and mononuclear cells were separated usinga double density cushion of Ficoll-metrizoate (23). Each preparationobtained in this mannerwas >95% the indicated cell type. The isolated cells were incubated a t 10’ cells/ml in the presence of 50 ng/ ml of phorbol myristate acetate as described above and the cell culture supernatantswere tested for gelatinolytic activity. While the neutrophil cell culture supernatants were capable of degrading 27.9 pg of gelatin/min/lO’ cells, no gelatinolytic activity was detected in the mononuclear cell fraction. Furthermore, when both culture supernatants were analyzed for immunoreactivity by immunoblotting, the mononuclear cell supernatants showed no reactivity with the antisera (datanot shown). However, the neutrophil culture supernatants demonstrated a pattern of reactivity which was identical to the reactivity seenwith the purifiedgelatinase (Fig. 8). These data indicate that crude culture supernatants obtained from purified neutrophils contained theidentical molecular weight forms of gelatinase seen in the purified preparation. They also indicate that themononuclear cells contained within the FIG. 5. Gelatinase activity of the proteins seen on SDSPAGE. Purified enzyme was electrophoresed on a 6%polyacrylamide leukocyte fractioncontributeneither biochemically or imgel containing gelatin (final concentration, 2 mg/ml) in the running munologically detectable gelatinase activity. Thus, the gelagelat 4 “C and then washed to remove the SDS as described under tinase activity detected in leukocyte preparations in short“Methods.” Gel was incubated for 1 h at 37 “C. Zones of enzyme term culture appears tobe exclusively derived from the polyactivity are indicated by negative staining. morphonuclear leukocytes. Substrate Specificity-As a variety of neutral proteinases lagen. T o examine this, the enzymewas electrophoresed on a possess gelatinolytic activity, the substrate specificity of this 6% polyacrylamide gel, washed as described for the gelatin enzyme was further characterized. Initially the ability of the substrate containing gels, and then sliced in 1-mm sections. proteinase todegrade BSA and casein was examined utilizing Each slice was incubated with 100 pg of type V collagen for the sensitive detection afforded by substrate-containing gels. 18 h a t 32.5 “C as described under “Methods.” After termi- In these studies the enzyme was electrophoresed on gels which nation of the assayby addition of EDTA toa final concentra- contained either BSA or casein in the running gel (2 mg/ml) tion of 10 mM, the reaction products were analyzed on a 7.5% and handled as described previously for the gelatin gels except polyacrylamide gel. The fractions representing the upper two- that they were incubated for 6 h a t 37 “C. Under these thirds of the original gel are seen in Fig. 6. Three discrete conditions minimal degradation of casein was detected while zones of lysis of type V collagen were presentandthey no activity against BSA was noted (not shown). In order to correspond to the protein bands seen on stained gels. While quantitate this specificity, peptide bond cleavage was meathese studiesreinforced the hypothesis that the three proteins sured by fluorescamine labeling of newly formed primary represented three molecular weight species of the same pro- amino groups as described under “Methods.”Gelatin was teinase, immunological support of this hypothesiswas sought. rapidly cleaved while casein was degraded a t less than oneImmunological Studies-Immunological cross-reactivity of fifth the rate of gelatin (Fig. 9). No degradation of BSA or the three species of gelatinase was examined by preparing ovalbumin was noted. Theseexperiments illustrated that polyclonal antibodies to the 130- and 92-kDa proteins. As among the common substrates degraded by nonspecific “ge-

Characterization of Secreted Human Neutrophil Gelatinme

2498

40

30

20

10

rnrn

FIG. 6. Degradation of native type V collagen by the proteins seen on SDS-PAGE. Purified enzyme was electrophoresed on a 6'6 polyacrylamide gel at 4 "C,then washed to remove the SDS. The gel was sliced in 1mm sections which were incuhated with 100 pg of type V collagen a t 32.5 "C for 18 h. Reaction products were of the original gel are shown. No degradation analyzed on 7.sri gel. The fractions representing the upper two-thirds was noted in the fractions from thelower third of the original gel.

W

69

70

69 70

A

B

FIG. 7 . Immuno-cross-reactivity ofthe polyclonal antisera.

FIG. 8. Immunoidentification ofgelatinase in polymorphoCross-reactivity of antisera prepared against the 1:QOOO protein (69) nuclear culture supernatants. Unpurified culture supernatants and 92.000 protein (70) was analyzed hy immunohlotting. Panel A (panel H ) and purified gelatinase (panel A ) were analyzed hy immushows rross-reactivity with the unreduced proteins; pnnel H repre- nohlotting using antiserum against the 92,000 protein. The proteins sents the reduced enzyme. The purified enzyme was separated on an 8 5 gel prior to immunohlotting.

were separated on a 6rI polyacrylamide gel.

EDTA (final concentration) the reaction products were separated on 7.5% polyacrylamide gels. While no degradation of native type I was noted (Fig. native t-ve collagen was extensively degraded. the Thus, proteinase showed specificity for gelatin andnativetype v collagen.

latinases," neutrophil gelatinase exhibited significant a specificity for gelatin. TO further illustrate the specificity of this proteinase against various collagenous substrates, the purified enzyme I collagen or native type V DISCUSSION was incubated with native type collagen at, a 1:200 enzyme:substrate ratio for 18 h a t 28 "c. The human neutrophil contains several proteinases which After termination of the reaction by the addition of 10 mM participate in the degradation of collagen, including elastase

Characterization of Secreted Human Neutrophil Gelatinase

8l

2499

optimal for enzyme recovery. Chromatographyon DEAEcellulose provided both further concentration and initial purification of thegelatinase from thecrudematerial.The material obtained from this step could be directly applied to a n affinity column of heat-denatured a chains of type I collagen as the affinity ligand. The enzyme displayed a high affinity for this column which may be partially attributable to hydrophobic interactions, as washing with both high (2 M NaCl) and low (0.001 M Tris-HC1, pH 7.5) ionic strength buffers failed to elute the enzyme. Ethylene glycol (20% v/v) which Sopata (13) utilized to elute theenzyme from a similar column composed of tryptic peptides of gelatin also failed to elute theenzyme from our column. The combinationof these two chromatographic steps provided a 400-fold purification and isolation of gelatinase in a latent form with high specific activity. When the DEAE-cellulose material was radioiodinated (35) and purified on the gelatin-Sepharose column, no additional bandscould be detected by autoradiography. Thus, the purification procedure appears to yield a homogeneous preparation of gelatinolytic activity. 30 Murphy et al. (8) postulated that neutrophilgelatinase is a minutes FIG. 9. Degradation of common protein substrates. Equal complex protein consisting of subunits. The data presented concentrations of the substrates were incubated with gelatinase (en- in this report, using a more highly purified enzyme, support zymembstrate ratio 1:200) at 37 "C. Aliquots were removed at spec- that hypothesis. While multiple protein specieshavebeen ified times and formation of primary amines was quantitated by isolated,all threeproteinsare capable of degrading both reaction with fluorescamine. A-A, gelatin; M, casein; gelatin and type V collagen and all the components have U , BSA; M, ovalbumin. common antigenic determinants. These datasuggest that the multiple molecular weight forms are derived from a single proteinase. While a subunit structure is one possible explaA B nation of this phenomenon, it is also conceivable that the enzyme has undergone limited proteolysis during secretion or purification. However, the immunological identificationof the identical proteins in crude supernatants indicates that the enzyme is not being degraded during the purification procedure. Furthermore, in preliminary experiments,we have been able to detect the threemolecular weight species in both the secreted and intracellularenzyme. Further studies, however, will be required to exclude the possibility that cellular processing occurs during secretion. Although there have been several previous reports about the purificationof neutrophil gelatinase, the resultshave been somewhat conflicting. Sopata (13) has purifiedgelatinase from extracts of intact neutrophils in the absence of serine proteinase inhibitors and reporteda single species with a M, of 90,000-94,000. Utilizing similar extracts but a different purification protocol, Ranatala-Rynanen et al. (15) reported a closely spaced doublet of slightly greater molecular weight 1 2 1 2 FIG. 10. Degradation of native types I and V collagens. 100 (100,000). Murphy andco-workers (8)purified gelatinase from extracts of isolated neutrophil granules in the presence of eg of substrate were incubated with 0.5 pg of enzyme for 18 h at 28 "C. Reaction products were separated on a 7.5% polyacrylamide serine proteinase inhibitors. Theirpurified enzyme consisted pel. Lone 1. control; lane 2, collagen + enzyme; A, type I; R, type V. of three protein bands when examined on SDS gels in the absence of a reducing agent. The smallest of these was de(32-34), interstitial collagenase, and gelatinase. Each of these scribed as 110,000, but a more prominent band with a M,= collagenolytic proteinases are stored within a distinct com- 150,000 was observed. Upon reduction, the large species was partment of the neutrophil and their release to the extracel- not seen, but the 110,000 protein remained. In addition, a lular environment in response to various stimuli exhibits a prominent small component(25,000) was seen on reduction. In comparison, we havepurified the gelatinase which is differential sensitivity (31).The participation of one or more of the collagenolytic proteinases a t sites of inflammation may secreted fromisolated neutrophils inresponse to phorbol analyzed by SDS-PAGE, the purified lead to differences in the qualitative and quantitative altera- myristate acetate. When enzyme consisted of three protein bands at 225,000, 130,000, tions in collagen content. While the serine proteinase elastase the 92,000 species was has been purified and well-characterized, the purification of and 92,000. Uponreduction,only apparent. It is likely that the 92,000 species is identical to the metalloproteinases remainscontroversial. T o facilitate the purification of neutrophil gelatinase, the proteins of similar molecular mass reported by other authors secretory abilityof the neutrophil hasbeen utilized to obtain and may represent the basic subunit of the enzyme. The enriched preparationsof this enzyme. Gelatinase was purified differences between the work we present and the previous from these culture supernatants using conditions which were reports could be related to the fact thatwe utilized secreted "

2500

Characterization of Secreted Human

Neutrophil Gelatinase

3. Eisen, A. Z., Jeffrey, J. J., and Gross, J. (1968) Biochim. Biophys. rather thanextracted enzyme. It should be stressed that, like Acta 151,637-645 Murphy et al. (8),we incorporated serine proteinase inhibitors 4. Sakai, T., and Gross, J. (1967) Biochemistry 6, 518-528 in our preparations. This is of interest in that previous studies, 5. Seltzer, J. L., Adams, S. A., Grant, G. A., and Eisen, A. Z. (1981) where serine proteinase inhibitors were not utilized, did not J. Biol. Chem. 2 5 6 , 4662-4668 proteins, suggesting a modificaobserve the large (>110,000) 6. Harris, E. D., Jr., and Krane, S. M. (1972) Biochim. Biophys. Acta 258,566-576 tion of gelatinase by serine proteinases. 7. Sopata, I., and Dancewicz, A. M. (1974) Biochim. Biophys. Acta While a number of neutral proteinases may possess gelatin370,510-523 olytic activity, neutrophil gelatinase shows specificity for gel8. Murphy, G., Reynolds, J. J., Bretz, U., and Baggiolini, M. (1982) atin and type V collagen. Little or no proteolysis of noncolBwchem. J. 203,209-221 lagenous proteins, which are common substrates for “gelati9. Murphy, G., Cawston, T. E., Galloway, W. A,, Barnes, M. J., Bunning, R. A. D., Mercer, E., Reynolds, J. J., and Burgeson, nases,” was noted when peptide bond hydrolysis was quantiR. E. (1981) Biochem. J. 199,807-811 tated.Furthermore,Murphy et al. (8), using a less highly C. L., Hibbs, M. S., and Seyer, J. M. (1983) Clin. Res. purified enzyme, reported that the enzyme did not degrade 10. Mainardi, 31,652A other extracellular matrix proteins such as laminin, fibronec- 11. Welgus, H.G., Jeffrey, J. J., and Eisen, A. Z. (1981) J. Biol. tin, and proteoglycan. Thus, this metalloproteinase appears Chem. 256,9511-9515 to have specificity for denatured collagens and native type V 12. Woolley, D. E., Glanville, R. W., Roberts, D. R., and Evanson, J. M. (1978) Biochem. J. 169, 265-276 collagen, although further studies with noninterstitial colla13. Sopata, I. (1982) Biochim. Biophys. Acta 717, 26-31 gens will be needed to fully characterize its substratespecific- 14. Murphy, G., Bretz, U., Baggiolini, M., and Reynolds, J. J. (1980) ity. Biochem. J. 192,517-525 The physiological significance of the ability of proteinase 15. Ranatala-Rynanen, L., Nowak, F. V., and Uitto, J. (1983) Eur. J. Biochem. 134, 129-137 to degrade type V collagen remains to be determined. While several investigators (8-10) have described gelatinases which 16. Kobayashi, S., and Nagai, Y. (1978) J. Biochem. (Tokyo) 84, 559-567 are capable of degrading native type V collagen, these studies 17. Dewald, B., Bretz, U., and Baggiolini, M. (1982) J. Clin. Znuest. as well as our ownwere done with pepsin-treated type V 70,518-525 collagen. Since it has been shown that in tissue this collagen 18. Glimcher, M. J., Francois, C. J., Richards, L., and Krane, S. M. (1964) Biochim. Biophys. Acta 93,585-602 contains a globular domain which is pepsin-sensitive (36), it is possible that the loss of this domain leads to loosening of 19. Cawston, T. E., and Barrett, A. J. (1979) Anal. Biochem. 99, 340-345 triple helix in certain regions which are then susceptible to 20. Rhodes, R. K., and Miller, E. J. (1978) Biochemistry 17, 3442gelatinases. Thus, furtherunderstanding of the native confor3448 mation of type V collagen, as it exists in the tissue, will be 21. Nagai, Y., Lapiere, C. M., and Gross, J. (1966) Biochemistry 5, 3123-3130 required before the physiological significance of the degrada22. Boyum, A. (1968) Scand. J. Clin. Lab. Med. Suppl. 97, 77-89 tion of this molecule by gelatinases can be determined. 23. English, D., and Andersen, B. R. (1974) J. Zmmunol. Methods 5, Although the ability of gelatinase to influence matrix com249-252 position by the degradation of type V collagen is speculative, 24. March. S. C.. Parikh. I.. and Cuatrecasas. P. (1974) Anal. the ability of this enzyme to potentiate the interstitialcollaBiockem. 60,149-152 genase has been well documented. Murphy et al. (8) have 25. Gillet, C., Eeckhout, Y., and Vaes, G . (1977) FEBS Lett. 74, 126128 shown that the gelatinase markedly potentiates the action of 26. Lammeli, U. K. (1970) Nature (Lond.)2 2 7 , 680-685 interstitial collagenase, particularlyagainst insoluble sub- 27. Birkedal-Hansen, H., and Taylor, R. E. (1982) Biochrn. Biophys. strates. As the gelatinase would be released from the neutroRes. Commun. 107, 1173-1178 phil concomitantly with the interstitialcollagenase a t inflam- 28. Cheng, T. P., Byrd, F. I., Whitaker, J. N., and Wood, J. G. (1980) J. CeR Bioi. 86, 624-633 matory sites (17,31), its presence would accelerate the action 29. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, of the interstitial collagenase. Since the neutrophil contains W., and Weigele, M. (1972) Science (Wash. D.C.) 178, 871relatively small amounts of interstitial collagenase (8), it is 872 possible that the major physiological role of the gelatinase is 30. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 31. Hibbs, M. S., Hasty, K. A., Kang, A. H., and Mainardi, C. L. its potentiation of interstitial collagenase. ,

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Acknowledgments-We thank Laura A. Lee for her excellent technical assistance, Dr. G. P. Stricklin for his helpful comments, and Connie Carrier and Jeannie Bailey for manuscript preparation. REFERENCES 1. Bornstein, P., and Sage, H. (1980) Annu. Reu. Biochem. 49,957-

1003 2. Gross, J., and Nagai, Y. (1965) Proc. Natl. Acad. Sci. U. S. A. 54, 1197-1204

(1983) Arthritis Rheum. 26, S42 32. Mainardi, C. L., Hasty, D.L., Seyer, J. M., and Kang, A. H. (1980) J. BWl. Chem. 255, 12006-12010 33. Mainardi, C. L., Dixit, S. N., and Kang, A. H. (1980) J. Biol. Chem. 255,5435-5441 34. Gadek, J. E., Fells, G. A., Wright, D. G., and Crystal, R. G. (1980) Biochern. Biophys. Res. Commun. 9 5 , 1815-1822 35. Hunter, W. M., and Greenwood, F. C. (1962) Nature (Lond.) 194,495-496 36. Fessler, L. I., Kumamoto, C. A., Meis, M. E., and Fessler, J. H. (1981) J. Biol. Chem. 256,9640-9645