Characterization of metalloproteinases and tissue inhibitors of metalloproteinases in human plasma.

1992, Val. 28, pp. 213-230 Reprints available directly from the publisher Photocopying permitted by license only Q 1992 Gordon and Breach Science Publishers S.A. Printed in the United States of America Connective Tissue Reseurch,

CHARACTERIZATION OF METALLOPROTEINASES AND TISSUE INHIBITORS OF METALLOPROTEINASES IN HUMAN PLASMA DEMETRIUS MOUTSIAKlS,t*s PAUL MANCUSO, HENRY KRUTZSCH,II WILLIAM STETLER-STEVENSON,II and STANLEY ZUCKERtts Connect Tissue Res Downloaded from informahealthcare.com by UB Kiel on 11/06/14 For personal use only.

+

Departments of Research? and Medicine,$ Department of Veterans Affuirs Medical Center, Northport, New York, USA; §Department of Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA; IlDepartment of Pathology, National Cancer Institute, Bethesda, Maryland, USA (Received September 18, 1991; in revised form February 19, 1992; accepted April 7 , 1992)

In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinasesitype IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin- 1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. TIMP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein. KEYWORDS: type IV collagenase, gelatinase, stromelysin, TIMF’, cu2-macroglobulin

INTRODUCTION Recent studies suggest that metalloproteinases, especially gelatinase/type IV collagenase, play an important role in diseases associated with excessive tissue breakdown. Secretion of type IV collagenases (72 kDa and 92 kDa) by tumor cells has been shown to correlate with lung colonizing potential in animal models.l-4 In contrast, Tissue Inhibitor of Metalloproteinases (TIMP-1 and TIMP-2) appear to exert an inhibitory effect on the invasive and metastatic properties of cancer cells.5.6 In addition to its role in cancer metastasis, the 72 kDa gelatinasekype IV collagenase produced by mesenchymal cells has a physiologic function in tissue remodeling.7-8 The gelatinase species of Mr of 92 kDa, 135 kDa and 225 kDa secreted by neutrophils and macrophages likewise participate in the inflammatory response .9JO Stromelysin (MMP-3)

Address for correspondence: Stanley Zucker, M.D. (#151), Department of Veterans Affairs Medical Center, Northport, NY 11768.

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D. MOUTSIAKIS et al.

and interstitial collagenase (MMP-1) are also implicated in the pathogenesis of rheumatoid arthritis” and osteoarthritis.12 The fate of gelatinases following secretion by cells in vivo is unknown. Gelatinases are thought to be secreted by cells in a latent form which requires removal of an activation peptide to convert the inactive precursor to a lower molecular weight active enzyme.’? Activated gelatinases are then subject to inactivation in tissues and plasma by TIMP-1, TIMP-2, or a2-M.14Interactions between gelatinases and TIMPs appear quite complicated as a result of data showing that latent 72 kDa and 92 kDa gelatinases are secreted by cells in non covalent complexes with TIMP-2 and TIMP-1, respectively.15-18 Although the biological effects of metalloproteinases are primarily limited to the local site of enzyme production, recent studies have shown that gelatinasedtype IV collagenases can readily be identified in plasma19.20 and quantitated by immunoassay.21-22TIMP, which was initially characterized23as a B, serum component (B, anticollagenase), can likewise be accurately measured in plasma.24 Based upon the above observations, we propose that levels of gelatinases (latenuactivated), TIMP and gelatinase-inhibitor complexes in plasma may be a reflection of those found in tissues, and hence, may serve as a means of monitoring proteinase activity in disease. The goal of this report is to characterize these metalloproteinases and inhibitor complexes in human plasma,

MATERIALS AND METHODS Commercial Reagents Trypsin-TPCK, DFP, TLCK, SBTI, PMSE EDTA, APMA, p-nitrophenyl phosphate, and E-64 were obtained from Sigma Chemical Co. (St. Louis MO). Gelatin Sepharose, Superose 12 HR 10/30, and Protein A Sepharose were obtained from Pharmacia LKB Biotechnology AB (Uppsala, Sweden). 3H-human placenta type IV collagen (propionate-2,3-3H) was obtained from New England Nuclear (Boston, MA). Affigel-10 was obtained from Bio-Rad (Richmond, CA). Concanavalin A Ultrogel was obtained from Reactifs IBF SOC.Chim. (Pointet Girard 92390 Villeneuve la Garenne, France). Matrex gel green A and Centricon microconcentrators were obtained from Amicon Division, W. R. Grace & Co. (Danvers, MA.). Alkaline phosphatase conjugated polyclonal donkey antibody against sheep IgG was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Biotinylated affinity purified goat anti-rabbit IgG, alkaline phosphatase conjugated streptavidin, BCIP, and NBT were obtained from Bethesda Research Laboratories Life Technologies (Gaithersburg, MD). Affinity purified, biotin labeled, goat antibody directed against mouse IgA, IgG, and IgM were obtained from Kirkegaard & Perry Laboratories Inc. (Gaithersburg, MD). Recombinant fibroblast anticollagenase (TIMP-l), derived from an E . coli P-strain expression system, was kindly provided by Dr. David Carmichael (Synergen, Boulder CO). Preparation of Antibodies Rabbit polyclonal antibodies to recombinant TIMP- 1 were prepared as described by Welgus ef al.25 and affinity purified using Affigel 10 bound recombinant TIMP as per manufacturer’s instructions. Peptides corresponding to amino acid regions 1 to 17 (APSPIIKFPGDVA PKTD) and 67 to 89 (TMRKPRCGNPDVANYNFFPRKPK) of human 72 kDa type IV


METALLOPROTEINASES IN HUMAN PLASMA

215

procollagenase and amino acid regions 1 to 18 (APRQRQSTLVLFPGDLRT) and 396 to 414 (EALMYPMYRFTEGPPLHK) of human 92 kDa type IV procollagenase were synthesized on a Biosearch 9600 peptide synthesizer (Biosearch Inc., Nevato, CA) as previously described.26 Rabbit polyclonal antibodies to unique peptide regions in human 72 kDa type IV collagenase and 92 kDa type IV collagenase were prepared and affinity purified using native peptides as previously described.26 The unique specificity of antibodies to the initial 17 amino acids at the amino terminal of 72 Da progelatinase was confirmed by demonstrating immunostaining of the 72 kDa latent enzyme, but not the 62 kDa activated enzyme.13 Similarly, antibodies to the amino terminal peptide of 92 kDa progelatinase reacted with the 92 kDa latent enzyme, but did not react with the 85 and 75 kDa activated enzymes. Antibodies to internal peptide sequences of the 72 kDa (67-89) and 92 kDa (396-414) type TV collagenase reacted with both latent and activated enzymes, respectively. None of the specific antibodies employed in this study cross reacted with other metalloproteinases or inhibitors (72 kDa and 92 kDa type IV collagenases, interstitial collagenase, stromelysin, or TIMPs). TIMP-2 was purified from human melanoma cells as previously described15 and was used to prepare rabbit antibodies directed against TIMP-2.26 Purified human 72 kDa gelatinase was prepared from A2058 melanoma cell conditioned media.27 Purified 92 kDa gelatinase was prepared as described. l8 Polyclonal sheep antibodies to stromelysin- 1 were kindly provided by Dr. Hideaki Nagasi.28 Isolation of 72 kDa and 92 kDa Gelatinases Individual plasma specimens from healthy volunteers were prepared by centrifugation of EDTA anticoagulated blood. Heparinized human plasma was used for purification of gelatinases. Plasma was cold-filtered to remove the cold insoluble globulin and applied to a gelatin Sepharose column. The column was washed with 50 mM Tris buffer, pH 8.0, containing 5 mM CaCl,, 0.5 M NaCl, 0.05% Brij 35,0.02% NaN,, and 1 mM DFF! Protein peaks were monitored at OD 280 nm. Bound proteins were eluted in two steps using 2% and 7% DMSO, dialyzed, and concentrated (Centricon-10). The bound fractions eluted with 2% DMSO were applied to a concanavalin A Ultrogel column, and eluted with a-methyl mannoside.' Further enrichment of the 92 kDa gelatinase was achieved by application of the concentrated sample onto a Superose 12 gel filtration column using an FPLC apparatus (Pharmacia) operating at a flow rate of 0.5 mlimin. Isolation of the 72 kDa gelatinase was achieved by applying the 7% DMSO eluent from gelatin Sepharose directly onto a Superose 12 gel filtration column.29 Isolation of Stromelysin The flow-through from gelatin Sepharose was applied to a Matrex green A column. The column was eluted using a two step gradient of 0.3 M and 2 M NaCl.28 Assays for the Degradation of Matrix Proteins Assays for the degradation of soluble native type IV collagen were performed using 3Hpropionated collagen type IV at 33 "C as described previously.27 Assays for the degradation of heat denatured collagen (gelatin) at 37 "C were performed using rat skin type I collagen

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labelled in vitro with 3H-formaldehyde and denatured.29Stromelysin activity was identified using a 3H-carboxymethyl transferrin substrate.29 Activation of plasma metalloproteinases was routinely achieved by incubating samples with trypsin-TPCK for five minutes at 22 "C, and then inactivating the trypsin with fivefold excess SBTI.29 APMA (1.5 mM) activation of metalloproteinases was achieved following 3 hours incubation at 22°C. Activity was expressed as degradation of 2 pg substrate over 18 hours for type IV collagen, or 2 hours for gelatin, except where noted. Inhibition of enzymatic activity was performed by preincubating samples (without TLCK added initially) for 30 minutes at 22°C with either EDTA, 1,lO-phenanthroline,E-64, DFP, TLCK, TIME or a2M prior to the addition of substrate. Protein concentrations were determined by the method of Bohlen ef aZ.30 SDS Polyacrylamide Gel Electrophoresis

A discontinuous system for non-reduced and reduced (P-mercaptoethanol) SDS-PAGE was employed as described by Laemmli,31 and run for thirty minutes using the BioRad Laboratory Minivertical Slab Cell according to manufacturer's instructions. To avoid disruption of native complexes, nonreduced samples were not boiled before electrophoresis; samples reduced with P-mercaptoethanol were boiled for 3 minutes prior to electrophoresis. After SDS-PAGE, the gels were stained with silver28 or transferred to Immobilon P filters. Molecular weight standards were run concurrently and approximate molecular weights were determined by plotting the relative mobility of known proteins by the method of Weber and Osborn.32 Molecular weight standards employed were the following: myosin (200,000), E . coli P-galactosidase (116,250), rabbit muscle phosphorylase b (92,000), bovine serum albumin (66,200) and hen egg white ovalbumin (45,000) (BioRad). SDS-Gelatin Zymography Gelatin zymography in SDS gels was performed using a 7.5% polyacrylamide gel that had been cast in the presence of gelatin (from calf skin, 02. % final concentration) as previously described.29.33 Samples were not boiled. Electrophoresis was performed as usual, and Triton X-100 was exchanged with the SDS. Gels were incubated for 18 hours at 37°C in incubation buffer containing 50 mM Tiis-HC1, pH 7.6, 5 mM CaCl,, 1 pM ZnCl,, 0.02% NaN,, 1% Triton, and 1 mM APMA. Gels were stained with Coomasie Blue and destained in the usual manner. Lytic bands appeared as bands of negative staining. Immunoblotting of Proteins Immunostaining for gelatinases, TIMP-1, TIMP-2 and stromelysin-1 were performed using affinity purified rabbit or sheep polyclonal antibodies directed against purified proteins or synthesized peptides. Electrophoresis was performed in 0.1% SDS and proteins were transferred overnight to an Immobilon P filter (Millipore Co., Bedford, MA). Filters were blocked with BLOTTO (5% nonfat dry milk, 0.01% antifoam A34) at room temperature. Primary antibodies diluted in BLOTTO were added and the filters were incubated overnight at 4 "C. After thorough washing with TBS containing Tween 20, filters were incubated for one hour at room temperature with biotinylated affinity purified goat anti rabbit IgG or donkey anti sheep IgG diluted in BLOTTO. Filters were washed and incubated for an hour


217

with alkaline phosphatase conjugated streptavadin. Filters were washed in TBS and then washed with Developing Buffer (100 mM NaCI, 50 mM MgCl,). The alkaline phosphatase reaction was visualized using BCIP and NBT. Prestained molecular weight markers were run on each gel. Immunoassay (ELISA)


The concentration of antigenically reactive 72 kDa gelatinase was determined in a sandwich ELISA technique employing affinity purified rabbit polyclonal antibodies and murine monoclonal antibodies to purified 72 kDa type IV procollagenase as recently described.35

RESULTS Activation of Plasma Gelatinases On bioassay of 25 individual human plasma samples, preincubation with trypsin (followed by inactivation of the trypsin with excess soybean trypsin inhibitor) significantly enhanced the degradation of soluble radiolabeled gelatin substrate; (3H)gelatin degradation increased from a baseline of 0.4k0.3 Fgiml plasma (meankSD) to 4.2k3.2 pg/ml plasma following activation with trypsin. Although APMA was a better activator of purified gelatinases, trypsin activation yielded higher enzymatic activity from crude plasma samples. On zymography of plasma, gelatinolytic bands were visualized at 72 kDa, 92 kDa, 135 kDa, and 270 kDa. Metalloproteinase Isolation and Characterization Purification of the 72 kDa gelatinase from plasma was achieved by gelatin Sepharose chromatography followed by gel filtration chromatography. Application of 2000 ml of cold filtered plasma to a gelatin Sepharose column resulted in virtually complete binding of 72 and 92 kDa gelatinase species to the column (Fig. 1). Subsequent elution of bound proteins with a two step DMSO gradient resulted in almost complete separation of the 92 kDa gelatinase species (Fig. 2), eluting in the 2% DMSO fraction, from the 72 kDa gelatinase species, eluting in the 7% DMSO fraction. A 270 kDa gelatinase band was present in both the 2% and 7% DMSO fractions. Application of the 7% DMSO eluent to a Superose 12 gel filtration column (Fig. 3 ) resulted in the separation of the 72 kDa gelatinase from the 270 kDa gelatinase as indicated by gelatin zymography (Fig. 4A). Immunoassay (ELISA) of the gel filtration fractions from the 7% DMSO eluent identified a protein peak in the chromatogram which corresponded with the second of two gelatinolytic peaks identified by bioassay (Fig. 3). An earlier peak of gelatinolytic activity, corresponding to the 270 kDa gelatinase did not react with antibody directed against the 72 kDa gelatinase, but did react with antibody directed against the 92 kDa gelatinase [data not shown]. On silver stain of the purified 72 kDa gelatinase under non reducing SDS-PAGE conditions, two bands of Mr of 72 kDa and 62 kDa were noted (Fig. 4A). The prominence of the 62 kDa silver stained band in lane 3 as compared to the dominant 72 kDa gelatinolytic band in lane 2 is explained by a 2 month delay before SDS-PAGE which resulted in autoactivation of the enzyme.36 In a second plasma purification of 72 kDa gelatinase, another major protein band was noted

2.0 A

200

I

t .5

150

1.o

100

50


0.5

4 100

200

300

400

500

Elution Volume (mu FIGURE 1 Fractionation of plasma by gelatin Sepharose chromatography. Arrows 1 and 2 indicate elution of proteins with 2% and 7% DMSO respectively. Fractions were analyzed for absorbance at 280 nm (-) and gelatinolytic activity. Samples were activated by preincubation with trypsin followed by inactivation of trypsin with excess SBTI prior to bioassay. Gelatinolytic activity was determined by incubating activated samples with 2 pg 3Hgelatin for 2 hours at 37°C. Activity was expressed as pg gelatin degraded per ml sample.

at Mr 20 kDa on reduced SDS-PAGE which corresponds with the TIMP-2 antigen (Fig. 4B). Activation of latent 72 kDa plasma gelatinase with APMA resulted in conversion to a 62 kDa protein band. Total enzyme recovery and enrichment of 72-62 kDa gelatinase are shown in Table I. Relative enrichment values represent the combined influence of true enrichment (purification) as well as removal of plasma metalloproteinase inhibitors. Immunoblotting of protein fractions bound to gelatin Sepharose and eluted with 7% DMSO was performed using rabbit antibodies directed against human 72 kDa gelatinase (Fig. 5A). Antibodies directed against the peptide region 67-89 of the 72 kDa gelatinase identified a major band of Mr of 72 kDa (which corresponded with the intense band on gelatin zymography) and a minor band at 62 kDa. Antibodies directed against the peptide region 1 to 17 of 72 kDa gelatinase, which reacts with latent but not activated enzyme, also identified the major band of 72 kDa, but did not stain the minor band at 62 kDa (lane 2). Application of the 2%DMSO eluted proteins from gelatin Sepharose to a concanavalin A Ultrogel column resulted in binding of the 92 kDa and 270 kDa gelatinases to the column with subsequent elution of gelatinolytic proteins with a-methyl mannoside as shown in the zymogram in Figure 2. Affinity purified rabbit polyclonal antibodies directed against the peptide region 396-414 of 92 kDa gelatinase recognized three major bands (Fig. 5B) of Mr of 92 kDa, 135 kDa, and 270 kDa (which corresponds with gelatinolytic bands recognized on zymography-Fig. 2), and a minor band of Mr of 85 kDa in the native sample bound to gelatin Sepharose and eluted



219

FIGURE 2 SDS-gelatin zymography of plasma after elution from gelatin Sepharose and concanavalin A Ultrogel. Samples were not boiled prior to SDS-electrophoresis, After electrophoresis, Triton X-100 was substituted for SDS, and the gels were incubated overnight at 37 "C in the presence of 0.1 mM APMA. Gels were stained with Coomasie Brilliant Blue. Zones of enzymatic activity were characterized by negative staining. Sample contents are as follows: Lane 1, human plasma (diluted 1 5 ) ; Lane 2, plasma not bound to gelatin Sepharose; Lane 3, plasma bound to gelatin Sepharose and eluted with 2% DMSO; Lane 4, plasma bound to gelatin Sepharose and eluted with 7% DMSO; Lane 5,296 DMSO eluent not bound to concanavalin A Ultrogel; Lane 6 , 2 % DMSO eluent bound to concanavalin A Ultrogel. Marker proteins were run simultaneously to identify molecular weights, and are indicated on the left side of the figure.

with 2% DMSO. Antibodies directed against the peptide region 1 to 18 of 92 kDa gelatinase, which recognize only the latent proenzyme, reacted with the same three major bands, but did not react with the minor band of Mr of 85 kDa (lane 1). Upon reduction with P-mercaptoethanol, one major band was identified of Mr of 92 kDa using either antibody directed against peptide sequences from the human 92 kDa gelatinase (lanes 3 and 4). Application of the unbound pool from gelatin Sepharose onto a Matrex green A column, followed by elution with 0.3 M and 2 M NaCl, resulted in the isolation of stromelysin-1 in the 2 M NaCl fraction. Immunostaining of this fraction with antibodies directed against human stromelysin-1 identified a major band of 57 kDa and a minor band of 54 kDa (lane 1) corresponding to stromelysin- 1 (lane 2) previously purified from rheumatoid synovial cell conditioned medium (Fig. 5C). Activated stromelysin- 1 (45 kDa) and lower molecular weight degradation products (resulting from enzyme autoactivation) were detected in the purified synovial material, but not in human plasma.

220

D. MOUTSIAKIS et a1

200

kOa

0.1

8

N

0


0

0.021

5

15

(0

20

ELUTION VOLUME (m0

FIGURE 3 Fractionation of plasma bound to gelatin Sepharose and eluted with 7% DMSO by Superose 12 gel filtration chromatography. Fractions were analyzed for absorbance at 280 nm (-), gelatinolytic activity (0-0)and type IV collagenolytic activity (A-A). Activity was expressed as pg substrate degraded per ml sample. 72 kDa gelatinase antigen levels (0-o), determined by ELISA, were expressed as ng/ml. Molecular weights, as determined with marker proteins, are identified by arrows.

Inhibition of Plasma Gelatinolytic Activity Metal chelation with EDTA was shown by gelatin zymography to inhibit the 72 kDa and 92 kDa gelatinolytic bands, whereas DFP had no inhibitory activity (Fig. 6). In a (3H)gelatin degradation assay, the metal chelators, EDTA and 1, 10-phenanthroline, human 1x2-macroglobulin and TIMP- 1 effectively inhibited gelatinolytic activity in native human plasma and gelatinase enriched pools (Table 11). E-64, an inhibitor of cysteine proteinases, TLCK, an inhibitor of cysteine and serine proteinases, and PMSE an inhibitor of serine proteinases were ineffective inhibitors of gelatinoiytic activity. Identification of TIMPs in Plasma Immunostaining of the protein fraction bound to gelatin Sepharose and eluted with a combined pool of the 2% and 7% DMSO eluents was performed using rabbit antibodies



22 1

FIGURE 4 A , Gelatin zymogram and silver stain of SDS-PAGE showing 72 kDa gelatinase purified from human plasma. Samples were not boiled prior to SDS- electrophoresis. After electrophoresis, Triton X-100 was substituted for SDS, and the gels were incubated overnight at 37 "C. Gels were stained with Coomasie Brilliant Blue. Zones of enzymatic activity are characterized by bands of negative staining. Sample contents are as follows: Lanes 1-2 are from a gelatin zymogram; Lane 3 is a non-reduced sample run on SDS-PAGE and stained with silver. Molecular weights in kDa are shown on left. Samples represent plasma fractions bound to gelatin Sepharose, eluted with 7% DMSO, and applied to a Superose 12 gel filtration column. Lane 1, Superose fraction eluting at 9.5 ml; Lane 2, Superose fraction eluting at 13 ml; Lane 3 , silver stain of Superose fraction eluting at 13 ml. (specimen was stored for 2 months prior to SDS-PAGE). Marker proteins were run simultaneously to identify molecular weights, and are indicated on the left side of the figure.


222

D. MOUTSIAKIS er 01.

FIGURE 4 B, SDS PAGE (10% polyacrylamide-reducing conditions) and silver staining of 72 kDa gelatinase from human plasma obtained during a repeat purification as in Figure 4A. Lane 1 contains the molecular weight markers; lanes 2 and 3 contain the Superose fraction eluting at 13 ml run in the native state and after activation with APMA, respectively. The arrowhead identifies plasma TIMP-2.

directed against human TIMP-1 (Fig. 7). A weak band at approximately 205 kDa was identified in the nonreduced sample (lane 3), which upon reduction appeared as a band of 28 kDa (lane 4). Immunostaining using antibodies directed against human TIMP-2 identified several weak diffuse bands of high and low molecular weights in the nonreduced sample (lane 8). Upon reduction and boiling of the sample, a major band of 21 kDa was visible (lane 9). Most of the protein bands identified using antibodies directed against either TIMP-1 or

223

METALLOPROTEINASES IN HUMAN PLASMA TABLE I Purification of 72 kDa gelatinase from human plasma. Samples were activated prior to assay.+ ~~

Total protein Sample


Plasma Unbound to gelatin Sepharose Bound to gelatin Sepharose and eluted with 2% DMSO with 7% DMSO Superose 26-28

(m&) 1 4000.0 13900.0

25.2 4.0 0.045

Total activity ( y g substrate degraded)

( k g substrate degraded

I96 0

0.014 0.000

171 48 39

Specific activity per mg protein)

6.8 12.0 860.0

Enrichment

Recovery (7%)

1 0

100

486 857 6 1400

87 24 20

Gelatinolytic activity was measured using heat-denatured [Wmethyl] collagen type I incubated with activated samples for 2 hours. In this purification scheme, the proteins bound to gelatin Sepharose and eluted with 7% DMSO were separated by gel filtration, and collected in 0.5 ml fractions.

TIMP-2 did not correlate with the equivalent molecular weight gelatinolytic bands observed on zymography. Antibodies to TIMP- 1 did not cross react with purified TIMP-2; likewise antibodies to TIMP-2 did not cross react with recombinant TIMP-1 (lanes 5 and 10, respectively).

DISCUSSION In this study, gelatin zymography was employed to identify gelatinolytic species of 72 kDa, 92 kDa, 135 kDa and 270 kDa in human plasma. Bioassay of native plasma samples showed minimal gelatinolytic activity in the absence of enzyme activation. Trypsin was a more effective activator of gelatinolytic activity in native plasma, which contrasts with results obtained with purified progelatinase in which APMA was the better activator. One explanation for the enhanced activity of trypsin on native plasma is that trypsin is able to bind to a 2 M in plasma and may titrate out the inhibitory effect of a 2 M on gelatinases, thereby permitting unencumbered gelatinase activation. The 72 kDa gelatinase was readily purified from plasma using gelatin Sepharose chromatography followed by gel filtration. In one of two purifications, TIMP-2 copurified with MMP-2 in plasma which is similar to observations of MMP-2 being secreted from cells in a noncovalent complex with TIMP-2.'5.16 Purification of the 92 kDa gelatinase from plasma was not achieved (data not shown) using protocols that were previously successful in purifying the enzyme from tumor conditioned media. 10,18 The relative difficulty of purification of the 92 kDa versus the 72 kDa gelatinase from plasma is related in part to the approximate 50 fold lower concentration of the former enzyme in plasma.35 Rabbit polyclonal antibodies to unique peptide fragments of the 72 kDa and 92 kDa gelatinase were utilized in immunoblots to identify their respective proteins. Antibodies specific for the amino terminal of the 72 kDa and 92 kDa gelatinase were used to identify the latent forms of each enzyme. Antibodies to internal peptide sequences were used to identify both latent and activated enzymes. Using this combination of antibodies, we have been able to demonstrate that the vast majority of 72 kDa and 92 kDa gelatinases circulate in plasma in

224

D. MOUTSIAKIS et a1


116-

77-

FIGURE 5 A, Identification of 72 kDa gelatinase in human plasma by Western blot technique. Immunoblotting of plasma fractions bound to gelatin Sepharose (7% DMSO eluted) was performed using rabbit polyclonal antibodies directed against unique peptide regions of human 72 kDa gelatinase. Electrophoresis was performed using an 8.75%polyacrylamide gel. Transfer and immunostaining were performed as per methods section. Lane 1, non-reduced sample blotted with antibody directed against peptide region 67 to 89; Lane 2, non-reduced sample blotted with antibody directed against peptide region 1 to 17;Lane 3, reduced sample blotted with antibody directed against peptide region 67 to 89.



225

FIGURE 5 B , Identification of 92 kDa gelatinase in human plasma by Western blot technique. Immunoblotting of plasma fractions bound to gelatin Sepharose (2% DMSO eluted) was performed using rabbit polyclonal antibodies directed against unique peptide regions of human 92 kDa gelatinase. Electrophoresis was performed using an 8.75% polyacrylamide gel. Sample contents are as follows: Lane 1, non-reduced sample blotted with antibody directed against peptide region 1 to 18; Lane 2, non-reduced sample blotted with antibody directed against peptide region 396 to 414; Lane 3, reduced sample blotted with antibody directed against peptide region 1 to 18; Lane 4, reduced sample blotted with antibody directed against peptide region 396 to 414. Prestained marker proteins were used to identify molecular weights, and are indicated on the left side of the figure.

the latent state, which is consistent with the requirement for enzyme activation in order to detect gelatinolytic activity in native plasma. The cellular sources of gelatinases in plasma have not yet been elucidated. Granulocytes have been shown in vitro to readily secrete latent gelatinases of Mr of 92 kDa, 130 kDa, and 225 kDa in response to stimuli.9 Thus, one potential candidate for the origin of these gelatinases observed in plasma is the granulocyte.19 This possibility is supported by the

226



205 116 77 46

FIGURE!5C Identification of stromelysin in plasma fraction bound to Matrex green A by Western blot technique. Immunostaining was performed using sheep polyclonal antibodies directed against human stromelysin-I. Samples were not reduced. Electrophoresis was performed using an 11% polyacrylamide gel. Sample contents are as follows: Lane I , fraction bound to Matrex green A and eluted with 2M NaCI; Lane 2, prostromelysin previously purified from rheumatoid synovial cell conditioned medium.

detection on gelatin zymography of higher amounts of the 92 kDa gelatinases in human serum as compared to plasma, suggesting that granulocytes may release gelatinase during the in vitro clotting process. The source of the 72 kDa gelatinase in plasma, however, is less clear, since normal cellular components of blood do not appear to produce a 72 kDa gelatinase.19 Thus the 72 kDa plasma gelatinase probably originates from multiple cellular sources in extravascular sites of tissue remodeling9J9 and/or the endothelial cells lining blood vessels.? This study is the first to demonstrate the presence of stromelysin-1 in human plasma. THurewitz, A. and Zucker, S. (1992). Characterization of gelatinaseltype IV collagenase in human pleural effusions. (manuscript submitted).

227



FIGURE 6 Inhibition of Gelatinolytic Activity by Metal Chelation. Both native plasma and 7% DMSO eluent from the gelatin Sepharose column were electrophoresed and gelatin zymography was performed on replicate samples. Gels were sliced and incubated with Triton buffer for 18 hours in the presence of enzyme inhibitors. Lanes 2 , 4 , and 6 contain native plasma and lanes I , 3 , and 5 contain 7% DMSO eluent. Lanes 1 and 2 are controls and contained no inhibitors; lanes 3 and 4 contained ImM DFP; and lanes 5 and 6 contained 25 mM EDTA. As shown, EDTA completely inhibited the gelatinolytic bands.

TABLE II Effect of proteinase inhibitors on gelatinolytic activity of plasma proteinases.+ Inhibitor added EDTA I , 10 phenanthroline a2M TIMP TLCK E-64 PMSF

Concentration

Native plasma

25 mM 1 mM 5.4 p M 1.7 p M 125 p M 0.5 mM 1 mM

100 100

100 100 0 0 0

92 kDa gelatinase

72 kDa gelatinase

80

86

96 I00 67 0 22 6

100 100 76 0 20 14

%elatinases were isolated by gelatin Sepharose chromatography. The 2% and 7% DMSO eluents are referred to as 92 kDa gelantinase and 72 kDa gelantinase, respectively. DMSO eluted fractions were activated with APMA and the native plasma sample was activated by trypsin and then inactivated by an excess of SBTI. Following enzyme activation, proteinase inhibitors were added for 30 minutes prior to the measurement of (3H)gelatin degradation. The degradation of (3H) gelatin substrate (2pglsample) was 0.66 p g for native plasma, 1.04 p g for 92kDa gelatinase, and 1.12 pg for the 72 kDa gelatinase. The data is listed as % inhibition of substrate degradation with each inhibitor.


228

D. MOUTSIAKIS er a1

RGURE 7 Identification of TIMP-1 and TIMP-2 by Western blotting technique. Immunoblotting of plasma fractions bound to gelatin Sepharose was performed using rabbit polyclonal antibodies directed against human TIMP-1 (lanes 1-5) and TIMP-2 (lanes 6-10). Electrophoresis was performed in SDS using 8.75% polyacrylamide gels. Transfer and immunostaining were performed as per methods section. Sample contents are as follows: Lanes 1,2 and 10 contain recombinant TIMP-1 (Mr=21 m a ) ; lanes 3 , 4 , 8 , and 9 contain human plasma following elution from a gelatin Sepharose column with DMSO (2% and 7% combined). Lanes 5, 6 , and 7 contain TIMP-2 (copurified with MMP-2 in complex from melanoma conditioned media.15 Lanes 1 , 3, 5 , 6 , 8 , and 10 were run under non-reduced conditions; lanes 2 , 4,7, and 9 were run under reduced conditions. Note that the TIMP-2:MMP-2 complex, purified from conditioned media, dissociated under non reducing conditions in SDS-PAGE and TIMP-2 migrated at a molecular weight of 21 kDa (lane 6). In contrast, TIMP 2 (lane 8) and TIMP-1 (lane 3), isolated from plasma migrated as multiple weak staining higher molecular weight bands under non-reducing conditions, but migrated at 21 kDa (lane 9) and 28 kDa (lane 4), respectively, under reducing conditions.

Stromelysins are more broad spectrum metalloproteinases that have been implicated in the pathophysiology of diseases associated with tissue destruction, including rheumatoid arthritis28 and cancer.36 The cellular sources of stromelysin in plasma remain to be determined. Immunoblots of plasma TIMPs bound to gelatin Sepharose demonstrated that TIMP-1 and TIMP-2 in plasma circulate in high molecular weight, SDS stable forms which revert to the native 28 kDa and 21 kDa species, respectively, upon boiling and reduction with P-mercaptoethanol. I'The high molecular weight species may be the result of an artefact caused by disulfide bridge formation between TIMP molecules produced during sample preparation. Alternatively, since free TIMP-1 and TIMP-216 do not bind to gelatin Sepharose, it is reasonable to speculate that high molecular weight forms of TIMPs in plasma are the result of inhibitor complex formation with metalloproteinases. Complexes between purified 72 kDa gelatinase and TIMP-2 and between 92 kDa gelatinase and TIMP- 1 prepared ~

+Ourtechnique for preparation of non-reduced gels for immunoblotting differs from other reports in that we did not boil samples prior to electrophoresis so as to avoid potential dissociation of enzyme: inhibitor complexes, thus permitting a comparison of protein bands of similar molecular weights on immunoblots and zymograms


229

from tumor conditioned media, however, have been reported to be unstable in SDS PAGE10.15,18 even under non reducing SDS-PAGE conditions (see Fig. 7, lane 7).

ACKNOWLEDGMENTS


This work was presented in part at the 100th National Meeting of the American Federation of Clinical Research in Washington, D.C., on 4-7 May, 1990. This work was supported by Merit Review Funds from the Department of Veterans Affairs and by a grant from the Center for Biotechnology, State University of New York at Stony Brook which is sponsored by the New York State Science and Technology Foundation. The authors would like to thank Betty DiMassimo, Rita M. Lysik, and Michael Gurfinkel for technical advice and assistance. We also thank Dr. David Carmichael for his generous provision of recombinant fibroblast anticollagenase (TIMP), and Dr. Hideaki Nagase for kind provision of purified human stromelysin- 1 and sheep antibodies to human prostromelysin.

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