Volume 296, number I, 16-20 FEDS 10566 1992 Fcdem~ion of European Biochemical Societies 00145793/92/S5.00
January 1992
0
Isola.tion and characterization of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) from human rheumatoid synovial fluid Anja Ostlzues, Vera KtGiuper, Renate Oberhoff, Uthcrsiry
oj,/‘Biclt$~ld, Faatlt_v of Ch~istry,
Dqxcrttw~t Received
Heinz Reinke and Harald Tschesche
of Biorhnistry,
12 November
P, 0. Box 8610. W-4800 Bklef~ld I, Gcrrnur~j
1991
The lissuc inhibitors of mclalloprotuinascs TIMP- I and TIMP-2 were purilied to itpparcni homogcncity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolaled by dissocialion of non-covalen1 gclaiinaseiTIMP con~plcxes. TIMP-I migrnlcd as a single polypcplidc wilh M, 23 500 on SDS-PAGE, while the M, of TIMP-2 was 21 000. The inhibitory aclivily was stable under heat and acid pH. N-lcrminal sequence data were oblained for the first IS residues of both inhibitors and showed identhy to the human libroblast inhibitors TlMP-I and TIMP-2. This is the first demonsh-ation that TIMP-I and TI1VIP-2can be directly purified from human rheumatoid synovial fluid. The complex formation between lhe melalloproleinase inhibitors and lcucocyte mcialloproteinascs was shown by immunobloliing. Tissue inhibitor of mc~alloproleinase:
1, 1NTRODUCTlON The tissue inhibitor of mctalloproteinases, TIMP-1, is a glycoprotein with an apparent molecular mass of 28 500 and was isolated from difl’erent tissues and body fluids [l--4]. TIMP-2 is a non-glycosylated protein with a molecular mass of 21 000, in most cases isolated as a complex with the proenzyme of gelatinase, i.e latent M, 72 000 gelatinase from different culture media [5-71. Both inhibitors specifically inhibit the matrix metalloproteinases, collagenase, gelatinase, stromelysin and PUMP- 1, which degrade extracellular matrix components such as collagens, proteoglycan, fibronectin and others. It has been found that TIMP- 1 forms a complex with latent Ad, 92 000 gelatinase, while TIMP-2 is able to form a complex with M, 72 000 gelatinase. All TIMP complexes can be activated e.g. by APMA and then generate gelatinolytic activity, which can be inhibited by the addition of more TIMP-1 or TIMP-2 [6,8,9]. These results indicate that regulation of the extracellular matrix degradation is more complex than assumed before and that both inhibitors TIMP-1 and TIMP-2 play important roles in controlling matrix metalloproteinase activity in vivo. A decrease in TIMP-1 and TIMP-2 levels can be expected to initiate uncontrolled degradation of the extracellular matrix as observed in pathological conditions, such as rheumatoid arthritis.
Correspondence address: H. Tschcsche, Universittit Biclefeld, FakultBt fllr Chcmic, Abteilung Biochemic, Postfach 8G40, W-4800 Biclcfcld 1, Germany. Fax: (49) (521) 1066164.
16
Gclatinase;
Rheumatoid
synovial Ruid
2. MATERlALS AND METI-IODS GO0 ml rheumatoid synovial fluid was dialyzed against 50 mM Tris-HCI pH 7.5.I mM CaC12, 300 mM NaCl and 0.02% NaN, (buffel A). The sample was supplemented with hyaluronidasc und stirred overnight a~ 20°C. This solution was cenirifuged for 20 min a~ 48 000 x g LOrcmovc insoluble material and was ihen applied to an anionexchange column (DEAE-ASO; 3.2 x IO cm) equilibrated with buffel A. The break-through fractions containing the gclatinasc/TIMPcomplexes free ofhyaluronic acids were adjusted IO0.5 M NaCl and stirred with 30 ml gelaiin Sepharosc for 1 h al 4°C. The IoJdcd gelatin sepharose wuswushcd with 50 mM Tris-HCI pl-I 7.5, I mM CaCI,. 500 mM NaCI, 0.02% NaN, (buffer 13)and filled into a column (1.6 x 30 cm), The gelatinasc/TlMP complcxus were eluled by adding 500 mM N&l and 2.5% (v/v) Me,SO to buffer B. The cluatc was concentrated by ultraiilma~ion using an Amicon PM IO membrane. The dissocialion of lhc gclatinascfllMP complexes was performed by gilliltraiion chromatography using Ultrogcl AcA 44 (2.6 x 77 cm) in 20 mM Tris-I-ICI pH 7.5. 50 mM NKI. 0. I4 NaDodSO, (buffer C). The inhibitor conlaining fractions wcrc dialyzed agains 20 m&l Tris-HCI pH 5.5, 1 mM CaCI?. IO mM NaC1,0.005% (w/v) Triton X-100,0.02% NaN, (buffer D). This solution was subjected LOa Q-Scpharosc fast-flow column (1.6 x 3 cm). TIMP-I was retarded by this column, while adsorbed TIMP-2 could be cluied with a linear gradient of0.01 to 0.2 M NaCl (80 ml). TlMP-1 or TIMP-2 fractions wcrc concentrated by ulrraliltration using an Amicon PM5 membrane, dialyzed against 20 mM Tris-HCI pH 7.5, I mM CaCI,. 0.005% Tritan X-100.0.02% NaN, and used for further characlerization. 2.2. SDS-PAGE SDS-PAGE was performed according to the method of Lnemmli [IO], Proteins were stained with silver as described by Heukeshoeven and Dernick [I I].
Amino-terminal
sequence determinations
were performed as reccm-
Volume 296, number
1
ly published [I?] using a microsequencer Germany).
2. IO. Drtecriorr of TIMP-f
(Model 810, Knauer, Berlin,
3.
measuring
the clcavugc
of the
(dinitrophcnyl-Pro-Ciln-Gly-lie-Ala-Gly-CilnD-Arg-OH) [I41 by active PMNL gclutinasc or collagenasc in the prcscnce or abscncc or mctalloproteinase inhibilor. 1.7. Purijicrtriott C$ PMNL gchrittuw UJU/ CO~~~L’IIUSL’ PMNL gclatinase and collagcnasc were purified according methods dcscribcd in [IS, I cl].
to the
2.8. Clturocrcri:ctriott of rrhhir pdyclutrol mtri-PMNL gnlutittctse clrtd C~O~I~CWWottriscrrt
The specificity of the antibodies
was recently published in [ 131.
1.9. Pwpurutiott ctttd rhoracreri:utiott
oJ rubhit poi_sclortul TIMP-I
otfriscru
The method t-or purilication scribed [I 31.
and charactcriration
I
by itrrtttrrttohIottirt~
I
I
10
20
was recently dc-
1
39
RESULTS AND DISCUSSlON
The metalloproteinase inhibitors TIMP-1 and TIMP2 were purified to homogeneity from human rheumatoid synovial fluid by a series of chromatographic steps (Table 1). Purification could be carried out, as TIMP’ complexes of latent and active gelatinase were bound to gelatin sepharose column, aIlowing easy separation of the complexes from most contaminating proteins. The isolated geIatinasc/TiMP complexes were dissociated by gelfiltration in the presence of 0.1% SDS (Fig. I). This demonstrates that gelatinase/TTlMP complexes exist in a non-covalent form. In contrast to other reports [6,7] the isolated gelatinase was active, which could bc due to using human rheumatoid synovial fluid or activation by SDS. The separation of TIMP-I from TIMP-2 was achieved by chromatography on Q-Sepharose fast-flow. While TIMP-I was not or only weakly bound to this column, TIMP-2 was eluted between 0.08 and 0.15 M NaCI. Further contaminating protein eluted with 0.5 M NaCi or I .OM NaCl (Fig. 2). This chromatographic procedure yielded pure TIMP-1 ahd TIMP-2 free of any other proteins. This is documented by SDSPAGE (Fig. 3) showing a single protein band for each inhibitor with a molecular mass of 28 SOD(TIMP-I) or 21 000 (TIMP-2). N-terminal sequence determination
XI. ASSII,V of tttclullu(~rorrittu.s~itrhihiror activity was detcrmincd
1992
This was carried out as for the dctcction or matrix mclul!opiotcina&iIMP complcxcs. but without incubation with enzyme solutions.
Purified TIMP-I or TIMP-1 was blotted l’rom unstained SDSPAGE on10 PVDF membranes by using the l3iometru fast blot system. The membrane wos’incubuted for 30 min with 3% DSA (w/v) in TBST-buTTcr (50 mM Tris-HCI pH 7.9, 150 mM NaCI, 0.05% Twecn 20). for IO min in TBST-buffer and 90 min with 50 ,ug latent OI activated matrix mctalloprotcinasc (PMNL collngcnasc or gclntinttsc) in TBST-buffer. The control rcmaincd in TDST-buffer. Mcmbruncs were washed with TBST-bufrcr for 5 min. inhibitor/matrix mctalloprotcinasc complexes were incubated with rabbit polyclonal antibodies specilic for PMNL-gelatinnsc or collagcnascc. After 90 min reaction time the blots were washed with TBST-bulTcr for 10 min. The antigen! antibody complexes were dctccted using anti-rabbit alkaline phosphnlasc conjugdtcd second antibodies as rcccnlly published [ 131.
Inhibitory wivity synthetic octupcptide
January
FEB.5 LETTERS
I
40
60
Fraction
fS-9
1
1
70
90
1
90
100
1
Number
Fig. I. GclatinascITIMP complex dissociation by SDS-gclfiltration on Ultrogcl AcA 44 column. Conccntratcd fractions rrom gclatinsepharose (1 ml) were applied to an Ultrogcl AcA 44 column (2.6 x 77 cm) equilibrated with huller C. Fractions (I .S ml) wcr$ collcctcd al a Ilow rate of 10 ml/h and ilntilyzcd for protein by silver stained SDS-PAGE. Gclatinasc (- - - - -) or inhibitor (--) containing fractions were combincdand dialyzed against buffer D,
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1 0 I -
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I
1
30.0 -
7,I 1”
20.1 14.4 -
4
I’
. ..- -.-
10
AJ
w
FracIien
L”
.I
Number
Fig. 2. Separation ol’TIM P- I from TI MP-2 by Q-Scpharosc. Dialyzed inhibitor conwining lkictions WCKtipplicd lo P Q-Scphi~rose column (I .6 x 3 cm) cquilibrakd with bulk D. Fractions (2 ml) were collected at a Row rate of 30 ml/h. Bound protein was cluted using a linetir grndient (0.01-0.2 M NuCI) ;Ind analyzed for TIMP-1 and TIMP-2 by silver stained SDS-PAGE and inhibitor assay. TIMP-I (- - - -) OI TIMP.2 (-_) containing frxtions were combined and used for further charactcrieation,
revealed total identity of the isolated rheumatoid synovial fluid inhibitor TlMP-1 with human fibroblast inhibitor TIMP-1 in the first 15 residues (Table 2). Furthermore, the first 15 amino acid residues of the &!I, 21 000 inhibitor shared total homology with TIMP-2 isolated from human melanoma cells [7] or recombinant TIMP-2 [I 71. Both inhibitors were stable up to pH2 and temperatures up to 100°C for 30 min, and were iaacti-
Fig. 3. Isolated human rheumatoid synovial tluid inhibitors and immunological dctcction by TIMP-I antibodies. Lnnc 1. molecular ~nilss standard: lane 2. HRSF-TIMP-I (10% SDS-PAGE. silver skncd): lane 3, HRSF-TIMP-2 (10% SDS-PAGE, siivcr stained); lane 4, HRSF-TlMP-I. in~n~unologicully dctccted by TIMP-I antibody; lane 5. HRSF-TIMP-2. no cross reaction with TIMP-I antibody.
vared by reduction and carboxamidomethylation, A specific antibody raised against the isolated TIMP-I only detected this inhibitor but not TIMP-2 (Fig. 3). Rheumatoid synovial fluid TIMP-1 and TIMP-2 are both able to inhibit active leucocyte gelatinasc and colIagenase. Autocatalytic degradation of these enzymes did not take place in the presence of either of these inhibitors. Comp!e:r formation between the active leucocyte ma-
Table 1
SlImmaryot pwllkntion
18
1992
of TIMP-I
end TIMP-2 from humn rlwumetoid synovlal fluid
FEBS LETTERS
Volume 296, number I A 1
B 2
12
c
M, x 1o-3
D
12123
: g-8
- 43:o - 30.0 - 20.1
” 14.4
., ‘.
‘.. ..
I
. .._
,’ :
‘, ._ ::-.,
Fig. 4. Detection or human leucocytc metallopro~einase~:rrlMP complexes by immunoblotting. Lane 1. HRSF-TIMP-I: lane 2. HRSFTIMP-I, A. bloated inllibirors incubavd for 2 h with ltitent leucocylc collagonnse and analyzed with col&cnase antibody: B, bloltrd inhibitors incubated
Ibr 7 II with latent
lcucocyte
gelatintisc
and analyzed
wilh gclatinase IgG; C. as A, bul incubatioti with activated leucocytc collapenssc; D. BS B. tx~ incubation with activakd lcucocytc gcluliIlMC.
trix mctalloproteinases and these inhibitors was also demonstrated by immunoblotting (Fig. 4). Blotted inhi-
January 1992
bitors were incubated
with activated gelatinase or collagenasc. Only a single stained band was detected for each inhibitor by the specific antibodies, which recognized the complex with the enzyme. Similar results were obtained for TIMP-I using PMNL progelatinase, while such a complex was not detected for TIMP-2. This is in agreement with the results of Goldberg [ 1S]. who found that M,. 92 000 progelatinase isolated from SV 40 transformated fibroblasts is able to form a complex with TIMP-1. In contrast PMNL procollagenasc remained unbound to TlMP-I or TIMP-2 and no complex formation was observed. In summary TIMP-1 and TIMP-2 are able to form stable complexes with activated PMNL collagennse and gelatinasc, but only TIMP-I can form a complex with PMNL progelatinase. This demonstrates that there must be a specilic binding site for TlMP-I in latent M, 92 000 gelatinase. Howard et al. have shown that M, 72 000 progelatinase forms complexes with TIMP2 and perhaps TIMP-2 is 3 stabilizer of this enzyme [9]. It would seem that TIMP-1 has the same function for Mr 92 000 progelatinase and that the matrix metalloproteinase inhibitors have more assignments than just blocking the activated enzymes. Furthermore, it was shown that TIMP-I and TIMP-2 suppressed collagen degradation by activated leucocyte collagenase (results not shown). This underlines that these inhibitors play an important role in the control of matrix metalloproteinases in tissue remodelling. These enzymes are able to degrade many extracellular matrix components. Therefore. decreased inhibitor levels could
Tabk 2 The fux
I5 N-rmdn8l
amino rcids of hum
and TIMP-2) and mparirim
I
10
5
X-S-X-S-P-V-H-P-Q-Q-A-F’X-N-A-
H-TIMP.2
C-S-C-S-P-V-H-P-Q-Q-A-F-C-N-A-
I-tRSF-TIMP- 1. human rhcumaloid synuvial fluid TIMPhuman IIMP-
HRSF-‘IIMP-2. It-TIMP-2,
repned
by Docbcfly cl nl.
I
1IS1
human rheumatoid synovial fluid TIMP-2
humnn TIMP.2 rcpncd by S~c~cr-~~erc~~wt~ci
1171 x
15
C-T-C-V-P-P-H-P-Q-T-A-F-C-N-S-
HRSF.TlMP.2
I%TIMP-I,
(IIMP-
*_~~~~~_~~~~~~~_Q._~-A-F-X-N-‘-
HRSF-TIMP- 1
P
6ynoviaJfluid inhibit
with scqucnocr uf inhibiton Fran aihcr -a.
1
H-TIMP-
tiumuoip
= unidcmiricd vmlno acid
d.
i?i
d
hz
c': d.
Volume
296, number
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FEBS LETTERS
lead to unbalanced matrix degradation as observed in pathological conditions such as rheumatoid arthritis. This is the first report that TIMP-I as well as TIMP-2 are present in human rheumatoid synovial fluid and that both inhibitors can be isolated from this body fluid by a simple chromatographic procedure. Future investigations need to clarify the roles of TIMP-1 and TIMP-2 in diseases such as rheumatoid arthritis. This could perhaps help to develop therapeutic approaches to inhibit matrix degradation. Ackno~~,k~dga?lcrrr.s: This wjork was
supported by the Dcutsclx Forschungs~cmcinschaft, special research proaramme SFB 223, project B2. The authors wish to thank Prof. Dr. R. Fricke for the kind gift of human rheumatoid synovial fluid, Mrs. V, Stlwcr for antibody preparation and Mrs. G. Delany for linguistic advice.
REFERENCES [I] Stricklin. G.P. and Welgus. H.G. (1983) .I. Dial. Chem. 258, 1?352-13258. [2] Cawston, T.E., Galloway, W.P., Merser, E., Murphy, G. and Reynolds, J.J. (1981) Biochem. J. 195, 159-165. [3] Murphy, G., Cawston, T.E. and Reynolds, J.J. (1981) Biochcm. 5. 195, 167-180. [4] Bunning, R.A.D., Murphy, G., Kumar. S,. Phillips, P. and Reynolds. J.J. I19541 Eur. J. Biochem. 139. 75-50. [5] De Clerck, Y,A,, Yean, T.-D., Ratzkin, B.J.. Lu, H.S, and Langley. K.E. (1989) J. Biol. Chem. 264, 17445-17453.
20
January
161Goldberg, G.I., Marmer, B.L., Grant. G.A., Eisen. AZ.
1992
Wilhelm, S. und Hc, C. (19S9) Proc. Natl. Acad. Sci. USA 86, X2978211. W.G.. Krutsch. WC. and Liotta, L.A. (1989) 171Stetlcr-Strvcnson. J. Biol. Chem. 364. l737C17373. .-. Fl Kolkcnbroch. H.. Orgcl, D.. Hccker-Kiu. A., Noack, W. and Ulbrich. N. (1991) Eur. J. Biochem. 198, 775-781, t91 Howard. E.W., Bullen. E.E. and Bandu. W.J. (1991) J. &I. Chcm. X6, 13064-I 3069. Lacmmli, U.K. (1970) Nature 227.650-685. :I:; Hcukeshoven, J. and Dernick. R. (1985) Eiectrophorcsis 6, 103112. [I’1 Rcinke, H., Fischer, St., Rr.imann. F, and Tscheschc, H. (1991) in: Methods in Protein Sequence Analysis (H. Jomvall, J.-O. H4og and A.-M. Gusiavsson eds,) Birkhauscr Vcrlag, Basal, pp. 55-66. 1131 Bergmann. U., Michaelis, J., Obcrhoff, R.. KnQuper, V.. Beckmann. R. and Tschcsche, I-I. (1989) J. Clin. Chem. Clin. Biochem. 27.35 l-359. 1141 M&i, Y., Takemoto, T., Sakakibara, S.. Hori, H. and Nagai, Y. (1977) Biochem. Med. 17. 215-221. 1151 Knaupcr. V.. Kramer, S.. Reinke. I-1. and Tschesche. H. (1990) Eur. J. Biochcm. 1989, 295-300. [I61 Wilhelm, S.M., Collier. I.E., Murmer. B.L., Eiscn. AZ.. Grant, G.A.and Goldberg, G.1. (1959) J. Biol. Chcm. 264, 17213-17221. [l7] Boone, T.C., Johnson, M.J., De Clcrck, Y.A. and Langley, K.E. (1990) Proc. Nat]. Actd. Sci. USA 87. 2800-2804. [IS] Dochcrty, A.J.P., Lyons, A., Smith, B.J., Wright, E.M., Stephens, P.E., Harris, T.J.R.. Murphy, G. and Reynolds, J.J. (I 985) Nature 3 18, 66-69.