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Cartilage chondrolysis by fibronectin fragments causes cleavage of aggrecan at the same site as found in osteoarthritic cartilage
Osteoarthritis and Cartilage (1997) 5, 450-453 ~ 1997 Osteoarthritis Research Society
1063-4584]97]060450 + 04 $12.00]0
OSTEOARTHRITIS and CARTILAGE SHORT COMMUNICATION Cartilage c h o n d r o l y s i s by f i b r o n e c t i n f r a g m e n t s c a u s e s c l e a v a g e of a g g r e c a n at t h e s a m e site as f o u n d in o s t e o a r t h r i t i c c a r t i l a g e Summary/Introduction
[11] and inflammatory joint diseases [12]. Accordingly, the enzymatic activity that may be responsible for this has been called 'aggrecanase', but the responsible proteinase has not yet been identified or isolated. Our objective here was to determine whether the Fn-fs caused cleavage at the physiologically relevant 'aggrecanase' site or at the MMP site in bovine cartilage aggrecan. The use of bovine cartilage was appropriate since proteoglycan synthesis suppression, cartilage proteoglycan degradation and release of active MMP-3 occurs to similar extents in both bovine and human knee cartilage explants [5-7]. We report here that cleavage was detected at the aggrecanase site, as well as other sites, but not at the MMP site. Thus, these data support a potential physiological role for Fn-fs in cartilage pathology in osteoarthritis or inflammatory joint disease. The data also support the relevance of the use of Fn-fs in animal models of knee joint damage as described by us earlier [2] as well as in in vitro explant culture models [1, 5-7].
FIBRONECTIN proteolytic fragments (Fn-fs) have very potent cartilage chondrolytic activities toward cultured cartilage explants in vitro [1] and when injected into rabbit knee joints [2]. Their relevance to cartilage pathology is supported by their presence at elevated levels in synovial fluids of patients with osteoarthritis and rheumatoid arthritis [3, 4]. Their relevance is also supported by the discovery that their effects are mediated through other factors, thought to have roles in cartilage pathology, catabolic cytokines. Cytokines have been detected in conditioned media of Fn-ftreated human cartilage [5] and neutralization with antibodies directed toward specific cytokines blocks cartilage damage by Fn-fs and induction of matrix metalloproteinases (MMPs) [5]. The major MMP responsible for cartilage damage appears to be MMP-3 (stromelysin-1), since antibodies to MMP-3 greatly slow cartilage damage caused by the Fn-fs [6, 7] and conditioned media of Fn-f treated cartilage contains levels of up to 800 nM of active MMP-3 [7]. Since MMPs cleave at the the Asn3(*~ 36~bond* in the interglobular domain of aggrecan [8], it would be expected that this cleavage would occur in Fn-f treated cartilage as well. However, this site is not cleaved in normal cartilage aggrecan turnover in vitro and in vivo [9] or in adult cartilage explants treated with interleukin-1 (IL-1) or retinoic acid [9-11]. In the latter cases, cleavage occurs exclusively at Glu:~92-Ala393. Further, the expected N-terminal Ala aggrecan fragments are found in human synovial fluids of patients with both osteoarthritis
Methods, Results and Discussion Cartilage was treated with a 29-kDa Fn-f, which was isolated as described [1] and shown to be free of several proteolytic activities as described [6], and detectable levels of IL-I~, IL-I~, IL-6 and tumor necrosis factor (TNF)-~ [5] and to retain activity after treatment with detoxi-Gel (Pierce Chemical Co., Rockford, IL, U.S.A.) to remove endotoxin. Culturing of bovine articular cartilage slices with 100 nM Fn-f in Dulbecco's modified Eagle's medium (DMEM) was performed as described [1]. After 2 days of treatment of cartilage with the Fn-f, media were removed for analysis of aggrecan. During this time, assays of aggrecan released into media, by means of the dimethylmethylene blue assay, showed that about 45% of the total aggrecan had been degraded and
Received 11 March 1997; accepted 1 July 1997. Address correspondence to: Gene A. Homandberg, Ph.D., Rush Medical College at Rush-Presbyterian-St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612-3864, U.S,A. Supported by the Arthritis Foundation, Greater Chicago Chapter and the National Institute of Arthritis, Musculoskeletal and Skin Diseases SCOR G r a n t AR39239-03.
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lost from the cultured cartilage. C a r t i l a g e was also c u l t u r e d in the p r e s e n c e of 100 U/ml h u m a n r e c o m b i n a n t I L - l a ( C o l l a b o r a t i v e Research, Bedford, MA, U.S.A.) for 4 days in D M E M , w h i c h was s u p p l e m e n t e d as described previously [10]. D u r i n g this time, t h e r a t e of loss of a g g r e c a n f r o m the e x p l a n t s i n c r e a s e d threefold o v e r t h a t of control c a r t i l a g e w i t h o u t cytokine. For all cultures, a g g r e c a n was purified from the m e d i a as described e l s e w h e r e [10]. Briefly, the c o n d i t i o n e d m e d i a were s u b j e c t e d to CsC1 density g r a d i e n t centrifugation. T h e a g g r e c a n was t h e n d e g l y c o s y l a t e d enzymically a n d subjected to sodium dodecyl sulfate p o l y a c r y l a m i d e gel electrophoresis (SDS/PAGE) in a 2-12% g r a d i e n t gel. The u n s t a i n e d gels were t h e n blotted to P r o B l o t t m e m b r a n e s (Perkin Elmer, Applied Biosystems Division, Foster City, CA, U.S.A.), a n d the blots were s t a i n e d w i t h C o o m a s s i e blue R-250 to localize the protein. The N - t e r m i n a l sequence of the a p p r o p r i a t e b a n d was d e t e r m i n e d by using an Applied Biosystems model 477A p r o t e i n s e q u e n c e r (operated in the gas phase) with a model 120A online h i g h - p e r f o r m a n c e liquid c h r o m a t r o g r a p h y (HPLC) a n d PE Nelson (San Jose, CA, U.S.A.) Model 2600 c h r o m a t o g r a p h y software. Bovine a g g r e c a n i s o l a t e d from Fn-f t r e a t e d c u l t u r e s showed on SDS gels, two m a j o r bands, one of a b o u t 200-kDa a n d one of slightly less t h a n 200-kDa. Both b l o t t e d b a n d s were cut out a n d s u b j e c t e d to a m i n o acid sequencing. The lower m o l e c u l a r weight b a n d showed a sequence of A R G S V I L X A K (Table I) identical to the bovine a g g r e c a n sequence t h a t h a d b e e n identified p r e v i o u s l y [10] and t h a t m a t c h e d the complete bovine p r e c u r s o r sequence ( G e n B a n k U76615), b e g i n n i n g at residue 393. T h e p r i m a r y sequence showed a n initial coupling of 5.2 pmol, with no s e c o n d a r y sequence o b s e r v e d a b o v e 0.5pmol. This s e q u e n c e m a t c h e s the A R G S V I L T V K P I F E V sequence found in the h u m a n cartilage-specific a g g r e c a n core p r o t e i n b e g i n n i n g at residue 393* [13] (accession n u m b e r M55172) with the e x c e p t i o n of the r e p l a c e m e n t of A l a for Val in cycle nine. A v e r y s i m i l a r sequence of A R G N V I L T A K K P I F D M h a s b e e n r e p o r t e d for the r a t cartilage-specific a g g r e c a n core p r o t e i n p r e c u r s o r (accession number A92623) w i t h the e x c e p t i o n of the r e p l a c e m e n t of Asn for Ser in cycle 4 [14]. No residue could be identified in cycle 8 of the b o v i n e protein, w h i c h is *The numbering for the precursor protein is given rather than that for the mature protein, the latter of which is often cited. For example, the mature protein is 19 residues shorter than the precursor. Thus, the aggq'ecanase site is Glu392 Alaaga in the precursor as used here, rather than GluaTa-AlaaT~as typically given for the mature protein.
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Table I Phenylthiohydantoin amino acid yield (pmol)* from the sequencing of the <200-kDa and 200-kDa fragments from Fn-f treated cartilage Cycle Residue (< 200-kDa) Residue (200-kDa) Ala (6.02) 1 Ala (4.59) Arg (4.03) 2 Arg (2.66) Gly (4.70) 3 Gly (4.11) Ser (+)~: 4 Ser (+)t 5 Val (2.29) Val (3.04) 6 Ile (1.66) 7 Leu (2.50) 8 Xxxw 9 Ala (2.05) 10 Lys (1.19) *pmol values are background corrected, +PTH-Ser was recoveredonly as the dithiothreitol-dehydroalanine adduct at a level equivalent to 1.38 pmol Ser. +PTH-Ser was recovered primarily as the dithiothreitol-dehydroalanine adduct at a level equivalent to 1.88pmol Set. A small shoulder near elation of PTH-Ser was observed. w is an unidentified residue.
k n o w n to be Thr, an a m i n o acid w h i c h is typically r e c o v e r e d in a low yield. The r a p i d i n c r e a s e in b a c k g r o u n d , typical for h i g h m o l e c u l a r w e i g h t proteins, also c o m p l i c a t e d the analysis. T h e 200-kDa b a n d showed the same N - t e r m i n a l c l e a v age site as the < 2 0 0 - k D a f r a g m e n t (Table I). The p r i m a r y sequence c o r r e s p o n d e d to an initial c o u p l i n g of 7.4 pmol, w i t h no s e c o n d a r y s e q u e n c e o b s e r v e d a b o v e 0.5pmol. T h e s e q u e n c e was t e r m i n a t e d after five cycles b e c a u s e the sequence, ARGSV, was identical to the s e q u e n c e described a b o v e and defined a unique site in the b o v i n e a g g r e c a n sequence. The similar sequences for the two different size f r a g m e n t s suggests e i t h e r c a r b o x y l - t e r m i n a l h e t e r o g e n e i t y or v a r i a t i o n in a m o u n t of glycosylation. Since the Fn-f o p e r a t e s t h r o u g h c a t a b o l i c cytokines, including IL-1 [5], and IL-lc~ t r e a t m e n t of c a r t i l a g e has b e e n r e p o r t e d to c a u s e c l e a v a g e at t h e a g g r e c a n a s e site [10], we a t t e m p t e d to d e t e r m i n e if I L - I ~ would g e n e r a t e the s a m e c l e a v a g e as did the Fn-f. The m a j o r f r a g m e n t i s o l a t e d f r o m IL-I~ t r e a t e d c a r t i l a g e was a b o u t 200-kDa on the SDS gel and this yielded a m i x t u r e of t h r e e sequences (Table II). T h e p r i m a r y sequence, w i t h an initial c o u p l i n g of 4 pmol, h a d the s a m e a m i n o - t e r m i n a l sequence as described a b o v e for t h e a g g r e c a n a s e c l e a v a g e product. C l e a v a g e at this site was also c o n s i s t e n t w i t h the m o l e c u l a r w e i g h t of the band. As s h o w n earlier, the residue in cycle 8 could n o t be identified, nor could the residue in cycle 14. A secondary sequence, (R)AXAFPXV, was identified at a lower c o u p l i n g of only 1.9 pmol. This sequence was found to m a t c h the b o v i n e cartilage-
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Short Communication Table II Phenylthiohydantoin amino acid yield (pmol)* from the sequencing of the 200-kDa fragment from IL-la treated cartilage Cycle
Primary sequence
Secondary sequence
Tertiary sequence
1 (Ala)t (4.12) Arg (1.55) (Gly) (6.39) 2 (Arg) (0.97) Ala (1.69) (Ser) (0.52) 3 Gly (3.25) Xxxw Val (0.80) 4 (Ser) (+)$ Ala (1.37) Ile (1.03) 5 Val (1.66) Phe (1.41) Leu (1.10) 6 Ile (1.47) Pro (1.63) Xxxt 7 Leu (1.35) Xxxt (Ala) (0.67) 8 Xxxt Val (1.13) Lys (0.69) 9 Ala (1.29) Pro (0.74) 10 Lys (0.91) Xxxt 11 Pro (1.21) Phe (1.00) 12 Asp (1.03) Xxxt 13 Phe (0.57) Val (0.61) 14 Xxxt 15 Val (0.63) *pmol values are background corrected. tA residue shown in parentheses is assigned with a lowerconfidencelevel. Xxx is an unidentified residue. :~PTH-Ser was recoveredas the dithiothreitol dehydroalanineadduct at a level equivalent to 0.67 pmol Ser.
specific aggrecan core p r o t e i n p r e c u r s o r beginning at residue 425. The c o r r e s p o n d i n g h u m a n sequence is G A T A F A E V E N E T G E A and the r a t sequence is G T T V F P E A G E R T E K T . Confirmation of this cleavage site was found in a n o t h e r experiment in which this sequence was extended as described later. A tertiary sequence, (G)(S)VILX(A)KPXFXV, was observed with an initial coupling of 1.0 pmol. This is likely cleavage at residue 395, two residues C-terminal to the cleavage site for the p r i m a r y sequence. The o b s e r v a t i o n t h a t IL-I~ t r e a t m e n t of bovine cartilage g e n e r a t e d the a g g r e c a n a s e site is consistent with the role of this cytokine in Fn-f-mediated cartilage chondrolysis [5]. The observation t h a t the a g g r e c a n a s e cleavage, which occurs in c a r t i l a g e in patients with both o s t e o a r t h r i t i s and inflammatory joint diseases, as well as in IL-lc~ t r e a t e d cartilage, also occurs in Fn-f t r e a t e d c a r t i l a g e lends credibility to our d e m o n s t r a t i o n t h a t the Fn-fs operate t h r o u g h catabolic cytokines and to our proposed physiologic relevance of Fn-fs in cartilage p a t h o l o g y in vivo. The p a r a d o x t h a t antibodies to MMP-3 block Fn-f-mediated cartilage chondrolysis [6, 7], but t h a t the classical M M P site of cleavage is not found in Fn-f t r e a t e d cartilage, may be because the first cleavage of a g g r e c a n is by MMP-3 at bond 360-361 and subsequent cleavage occurs by the unidentified ' a g g r e c a n a s e ' or even by MMP-3, at bond 392-393. This would r e s u l t in loss of the residue 361-392 peptide and the a p p e a r a n c e in larger aggrecan fragments of only the a g g r e c a n a s e
site sequence beginning with Ala ~93. Such peptides with N-terminal Phe 361 have been found by F o s a n g et al. [15] in low b u o y a n t density fractions of cesium chloride density gradients of synovial fluids of patients with both inflammatory and n o n i n f l a m m a t o r y arthritis. The peptides are a few residues s h o r t e r t h a n expected, likely due to s e c o n d a r y proteolysis from the C-terminal end. Nonetheless, t h e i r work provides u n e q u i v o c a l evidence t h a t MMPs, as well as 'aggrecanase', act directly in a g g r e c a n d e g r a d a t i o n in arthritis. Since we only analyzed high b u o y a n t density fractions, these peptides would not have been detected. F u r t h e r w o r k will be needed to explain the role of MMP-3 in Fn-f-mediated cartilage damage, nonetheless these data are consistent with the possibility t h a t Fn-fs may c o n t r i b u t e to a g g r e c a n d e g r a d a t i o n in o s t e o a r t h r i t i s and inflammatory joint diseases. These data also support the n o t i o n t h a t the Fn-fs mediate t h e i r effects t h r o u g h catabolic cytokines.
Acknowledgments Ms Carla Pellegrino is acknowledged for her expert assistance in protein sequencing.
References 1. Homandberg GA, Meyers, R, Xie DL. Fibronectin fragments cause chondrolysis of bovine articular cartilage slices in culture. J Biol Chem 1992;267:3597-604. 2. Homandberg GA, Meyers R, Williams J. Intraarticular injection of fibronectin fragments
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4. 5.
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induces cartilage proteoglycan release. J Rheumatol 1993;20:1378-82. Carsons S, Lavietes BB, Diamond HS. Role of fibronectin in rheumatic diseases in fibronectin. In: Mosher DF, Ed. Biology of extracellular matrix: a Series. San Diego, CA: Academic Press, 1989; 327-361. Xie DL, Meyers R, Homandberg GA. Fibronectin fragments in osteoarthritic synovial fluid; pathologic implications. J Rheumatol 1992;19:1448-52. Homandberg GA, Hui F, Wen C, Purple C, Bewsey K, Koepp H, Huch K, Harris A. Fibronectin fragment induced cartilage chondrolysis is associated with release of catabolic cytokines. Biochem J 1997;321:751-7. Bewsey K, Wen C, Purple C, Homandberg GA. Fibronectin fragments induce the expression of stromelysin-1 mRNA and protein in bovine chondrocytes in monolayer culture. Biochim Biophys Acta 1996;1317:55-64. Homandberg GA, Hui F, Wen C. Association of proteoglycan degradation with catabolic cytokine and stromelysin release from cartilage cultured with fibronectin fragments. Arch Biochem Biophys 1996;334:325-31. Flannery, CR, Lark MW, Sandy JD. Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage. J Biol Chem 1992;267:1008-14. Ilic MZ, Handley CJ, Robinson HC, Mok MT. Mechanism of catabolism of aggrecan by arti-
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cular cartilage. Arch Biochem Biophys 1992;294: 115-22. Loulakis P, Shrikhande A, Davis G, Maniglia CA. N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-l-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage. Biochem J 1992;284:589-93. Sandy JD, Flannery CR, Neame PJ, Lohmander LS. The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain. J Clin Invest 1992;89:1512-6. Lohmander LS, Neame PJ, Sandy JD. The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum 1993;36:1214-22. Doege KJ, Sasaki M, Kimura T, Yamada Y. Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. J Biol Chem 1991;266: 894-902. Doege K, Sasaki M, Horigan T, Hasell JR, Yamada K. Complete primary structure of the rat cartilage proteoglycan core protein deduced from eDNA clones. J Biol Chem 1987;262:17757 67. Fosang AJ, Last K, Maciewicz RA. Aggrecan is degraded by matrix metalloproteinases in human arthritis. Evidence that matrix metalloproteinase and aggrecanase activities can be independent. J Clin Invest 1996;98:2292-99.
BY GENE A. HOMANDBERG*,GARY DAVISt, CHARLESMANIGLIAS~AND ALKA SHRIKHANDE:~ * D e p a r t m e n t of Biochemistry, Rush M e d i c a l College at R u s h - P r e s b y t e r i a n - S t . L u k e ' s M e d i c a l Center, Chicago, Illinois and t I n s t i t u t e F o r R e s e a r c h T e c h n o l o g i e s a n d $ I n s t i t u t e F o r Bone a n d J o i n t D i s o r d e r s and Cancer, B a y e r C o r p o r a t i o n , West H a v e n , C o n n e c t i c u t , U.S.A.
1063-4584]97]060450 + 04 $12.00]0
OSTEOARTHRITIS and CARTILAGE SHORT COMMUNICATION Cartilage c h o n d r o l y s i s by f i b r o n e c t i n f r a g m e n t s c a u s e s c l e a v a g e of a g g r e c a n at t h e s a m e site as f o u n d in o s t e o a r t h r i t i c c a r t i l a g e Summary/Introduction
[11] and inflammatory joint diseases [12]. Accordingly, the enzymatic activity that may be responsible for this has been called 'aggrecanase', but the responsible proteinase has not yet been identified or isolated. Our objective here was to determine whether the Fn-fs caused cleavage at the physiologically relevant 'aggrecanase' site or at the MMP site in bovine cartilage aggrecan. The use of bovine cartilage was appropriate since proteoglycan synthesis suppression, cartilage proteoglycan degradation and release of active MMP-3 occurs to similar extents in both bovine and human knee cartilage explants [5-7]. We report here that cleavage was detected at the aggrecanase site, as well as other sites, but not at the MMP site. Thus, these data support a potential physiological role for Fn-fs in cartilage pathology in osteoarthritis or inflammatory joint disease. The data also support the relevance of the use of Fn-fs in animal models of knee joint damage as described by us earlier [2] as well as in in vitro explant culture models [1, 5-7].
FIBRONECTIN proteolytic fragments (Fn-fs) have very potent cartilage chondrolytic activities toward cultured cartilage explants in vitro [1] and when injected into rabbit knee joints [2]. Their relevance to cartilage pathology is supported by their presence at elevated levels in synovial fluids of patients with osteoarthritis and rheumatoid arthritis [3, 4]. Their relevance is also supported by the discovery that their effects are mediated through other factors, thought to have roles in cartilage pathology, catabolic cytokines. Cytokines have been detected in conditioned media of Fn-ftreated human cartilage [5] and neutralization with antibodies directed toward specific cytokines blocks cartilage damage by Fn-fs and induction of matrix metalloproteinases (MMPs) [5]. The major MMP responsible for cartilage damage appears to be MMP-3 (stromelysin-1), since antibodies to MMP-3 greatly slow cartilage damage caused by the Fn-fs [6, 7] and conditioned media of Fn-f treated cartilage contains levels of up to 800 nM of active MMP-3 [7]. Since MMPs cleave at the the Asn3(*~ 36~bond* in the interglobular domain of aggrecan [8], it would be expected that this cleavage would occur in Fn-f treated cartilage as well. However, this site is not cleaved in normal cartilage aggrecan turnover in vitro and in vivo [9] or in adult cartilage explants treated with interleukin-1 (IL-1) or retinoic acid [9-11]. In the latter cases, cleavage occurs exclusively at Glu:~92-Ala393. Further, the expected N-terminal Ala aggrecan fragments are found in human synovial fluids of patients with both osteoarthritis
Methods, Results and Discussion Cartilage was treated with a 29-kDa Fn-f, which was isolated as described [1] and shown to be free of several proteolytic activities as described [6], and detectable levels of IL-I~, IL-I~, IL-6 and tumor necrosis factor (TNF)-~ [5] and to retain activity after treatment with detoxi-Gel (Pierce Chemical Co., Rockford, IL, U.S.A.) to remove endotoxin. Culturing of bovine articular cartilage slices with 100 nM Fn-f in Dulbecco's modified Eagle's medium (DMEM) was performed as described [1]. After 2 days of treatment of cartilage with the Fn-f, media were removed for analysis of aggrecan. During this time, assays of aggrecan released into media, by means of the dimethylmethylene blue assay, showed that about 45% of the total aggrecan had been degraded and
Received 11 March 1997; accepted 1 July 1997. Address correspondence to: Gene A. Homandberg, Ph.D., Rush Medical College at Rush-Presbyterian-St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612-3864, U.S,A. Supported by the Arthritis Foundation, Greater Chicago Chapter and the National Institute of Arthritis, Musculoskeletal and Skin Diseases SCOR G r a n t AR39239-03.
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lost from the cultured cartilage. C a r t i l a g e was also c u l t u r e d in the p r e s e n c e of 100 U/ml h u m a n r e c o m b i n a n t I L - l a ( C o l l a b o r a t i v e Research, Bedford, MA, U.S.A.) for 4 days in D M E M , w h i c h was s u p p l e m e n t e d as described previously [10]. D u r i n g this time, t h e r a t e of loss of a g g r e c a n f r o m the e x p l a n t s i n c r e a s e d threefold o v e r t h a t of control c a r t i l a g e w i t h o u t cytokine. For all cultures, a g g r e c a n was purified from the m e d i a as described e l s e w h e r e [10]. Briefly, the c o n d i t i o n e d m e d i a were s u b j e c t e d to CsC1 density g r a d i e n t centrifugation. T h e a g g r e c a n was t h e n d e g l y c o s y l a t e d enzymically a n d subjected to sodium dodecyl sulfate p o l y a c r y l a m i d e gel electrophoresis (SDS/PAGE) in a 2-12% g r a d i e n t gel. The u n s t a i n e d gels were t h e n blotted to P r o B l o t t m e m b r a n e s (Perkin Elmer, Applied Biosystems Division, Foster City, CA, U.S.A.), a n d the blots were s t a i n e d w i t h C o o m a s s i e blue R-250 to localize the protein. The N - t e r m i n a l sequence of the a p p r o p r i a t e b a n d was d e t e r m i n e d by using an Applied Biosystems model 477A p r o t e i n s e q u e n c e r (operated in the gas phase) with a model 120A online h i g h - p e r f o r m a n c e liquid c h r o m a t r o g r a p h y (HPLC) a n d PE Nelson (San Jose, CA, U.S.A.) Model 2600 c h r o m a t o g r a p h y software. Bovine a g g r e c a n i s o l a t e d from Fn-f t r e a t e d c u l t u r e s showed on SDS gels, two m a j o r bands, one of a b o u t 200-kDa a n d one of slightly less t h a n 200-kDa. Both b l o t t e d b a n d s were cut out a n d s u b j e c t e d to a m i n o acid sequencing. The lower m o l e c u l a r weight b a n d showed a sequence of A R G S V I L X A K (Table I) identical to the bovine a g g r e c a n sequence t h a t h a d b e e n identified p r e v i o u s l y [10] and t h a t m a t c h e d the complete bovine p r e c u r s o r sequence ( G e n B a n k U76615), b e g i n n i n g at residue 393. T h e p r i m a r y sequence showed a n initial coupling of 5.2 pmol, with no s e c o n d a r y sequence o b s e r v e d a b o v e 0.5pmol. This s e q u e n c e m a t c h e s the A R G S V I L T V K P I F E V sequence found in the h u m a n cartilage-specific a g g r e c a n core p r o t e i n b e g i n n i n g at residue 393* [13] (accession n u m b e r M55172) with the e x c e p t i o n of the r e p l a c e m e n t of A l a for Val in cycle nine. A v e r y s i m i l a r sequence of A R G N V I L T A K K P I F D M h a s b e e n r e p o r t e d for the r a t cartilage-specific a g g r e c a n core p r o t e i n p r e c u r s o r (accession number A92623) w i t h the e x c e p t i o n of the r e p l a c e m e n t of Asn for Ser in cycle 4 [14]. No residue could be identified in cycle 8 of the b o v i n e protein, w h i c h is *The numbering for the precursor protein is given rather than that for the mature protein, the latter of which is often cited. For example, the mature protein is 19 residues shorter than the precursor. Thus, the aggq'ecanase site is Glu392 Alaaga in the precursor as used here, rather than GluaTa-AlaaT~as typically given for the mature protein.
451
Table I Phenylthiohydantoin amino acid yield (pmol)* from the sequencing of the <200-kDa and 200-kDa fragments from Fn-f treated cartilage Cycle Residue (< 200-kDa) Residue (200-kDa) Ala (6.02) 1 Ala (4.59) Arg (4.03) 2 Arg (2.66) Gly (4.70) 3 Gly (4.11) Ser (+)~: 4 Ser (+)t 5 Val (2.29) Val (3.04) 6 Ile (1.66) 7 Leu (2.50) 8 Xxxw 9 Ala (2.05) 10 Lys (1.19) *pmol values are background corrected, +PTH-Ser was recoveredonly as the dithiothreitol-dehydroalanine adduct at a level equivalent to 1.38 pmol Ser. +PTH-Ser was recovered primarily as the dithiothreitol-dehydroalanine adduct at a level equivalent to 1.88pmol Set. A small shoulder near elation of PTH-Ser was observed. w is an unidentified residue.
k n o w n to be Thr, an a m i n o acid w h i c h is typically r e c o v e r e d in a low yield. The r a p i d i n c r e a s e in b a c k g r o u n d , typical for h i g h m o l e c u l a r w e i g h t proteins, also c o m p l i c a t e d the analysis. T h e 200-kDa b a n d showed the same N - t e r m i n a l c l e a v age site as the < 2 0 0 - k D a f r a g m e n t (Table I). The p r i m a r y sequence c o r r e s p o n d e d to an initial c o u p l i n g of 7.4 pmol, w i t h no s e c o n d a r y s e q u e n c e o b s e r v e d a b o v e 0.5pmol. T h e s e q u e n c e was t e r m i n a t e d after five cycles b e c a u s e the sequence, ARGSV, was identical to the s e q u e n c e described a b o v e and defined a unique site in the b o v i n e a g g r e c a n sequence. The similar sequences for the two different size f r a g m e n t s suggests e i t h e r c a r b o x y l - t e r m i n a l h e t e r o g e n e i t y or v a r i a t i o n in a m o u n t of glycosylation. Since the Fn-f o p e r a t e s t h r o u g h c a t a b o l i c cytokines, including IL-1 [5], and IL-lc~ t r e a t m e n t of c a r t i l a g e has b e e n r e p o r t e d to c a u s e c l e a v a g e at t h e a g g r e c a n a s e site [10], we a t t e m p t e d to d e t e r m i n e if I L - I ~ would g e n e r a t e the s a m e c l e a v a g e as did the Fn-f. The m a j o r f r a g m e n t i s o l a t e d f r o m IL-I~ t r e a t e d c a r t i l a g e was a b o u t 200-kDa on the SDS gel and this yielded a m i x t u r e of t h r e e sequences (Table II). T h e p r i m a r y sequence, w i t h an initial c o u p l i n g of 4 pmol, h a d the s a m e a m i n o - t e r m i n a l sequence as described a b o v e for t h e a g g r e c a n a s e c l e a v a g e product. C l e a v a g e at this site was also c o n s i s t e n t w i t h the m o l e c u l a r w e i g h t of the band. As s h o w n earlier, the residue in cycle 8 could n o t be identified, nor could the residue in cycle 14. A secondary sequence, (R)AXAFPXV, was identified at a lower c o u p l i n g of only 1.9 pmol. This sequence was found to m a t c h the b o v i n e cartilage-
452
Short Communication Table II Phenylthiohydantoin amino acid yield (pmol)* from the sequencing of the 200-kDa fragment from IL-la treated cartilage Cycle
Primary sequence
Secondary sequence
Tertiary sequence
1 (Ala)t (4.12) Arg (1.55) (Gly) (6.39) 2 (Arg) (0.97) Ala (1.69) (Ser) (0.52) 3 Gly (3.25) Xxxw Val (0.80) 4 (Ser) (+)$ Ala (1.37) Ile (1.03) 5 Val (1.66) Phe (1.41) Leu (1.10) 6 Ile (1.47) Pro (1.63) Xxxt 7 Leu (1.35) Xxxt (Ala) (0.67) 8 Xxxt Val (1.13) Lys (0.69) 9 Ala (1.29) Pro (0.74) 10 Lys (0.91) Xxxt 11 Pro (1.21) Phe (1.00) 12 Asp (1.03) Xxxt 13 Phe (0.57) Val (0.61) 14 Xxxt 15 Val (0.63) *pmol values are background corrected. tA residue shown in parentheses is assigned with a lowerconfidencelevel. Xxx is an unidentified residue. :~PTH-Ser was recoveredas the dithiothreitol dehydroalanineadduct at a level equivalent to 0.67 pmol Ser.
specific aggrecan core p r o t e i n p r e c u r s o r beginning at residue 425. The c o r r e s p o n d i n g h u m a n sequence is G A T A F A E V E N E T G E A and the r a t sequence is G T T V F P E A G E R T E K T . Confirmation of this cleavage site was found in a n o t h e r experiment in which this sequence was extended as described later. A tertiary sequence, (G)(S)VILX(A)KPXFXV, was observed with an initial coupling of 1.0 pmol. This is likely cleavage at residue 395, two residues C-terminal to the cleavage site for the p r i m a r y sequence. The o b s e r v a t i o n t h a t IL-I~ t r e a t m e n t of bovine cartilage g e n e r a t e d the a g g r e c a n a s e site is consistent with the role of this cytokine in Fn-f-mediated cartilage chondrolysis [5]. The observation t h a t the a g g r e c a n a s e cleavage, which occurs in c a r t i l a g e in patients with both o s t e o a r t h r i t i s and inflammatory joint diseases, as well as in IL-lc~ t r e a t e d cartilage, also occurs in Fn-f t r e a t e d c a r t i l a g e lends credibility to our d e m o n s t r a t i o n t h a t the Fn-fs operate t h r o u g h catabolic cytokines and to our proposed physiologic relevance of Fn-fs in cartilage p a t h o l o g y in vivo. The p a r a d o x t h a t antibodies to MMP-3 block Fn-f-mediated cartilage chondrolysis [6, 7], but t h a t the classical M M P site of cleavage is not found in Fn-f t r e a t e d cartilage, may be because the first cleavage of a g g r e c a n is by MMP-3 at bond 360-361 and subsequent cleavage occurs by the unidentified ' a g g r e c a n a s e ' or even by MMP-3, at bond 392-393. This would r e s u l t in loss of the residue 361-392 peptide and the a p p e a r a n c e in larger aggrecan fragments of only the a g g r e c a n a s e
site sequence beginning with Ala ~93. Such peptides with N-terminal Phe 361 have been found by F o s a n g et al. [15] in low b u o y a n t density fractions of cesium chloride density gradients of synovial fluids of patients with both inflammatory and n o n i n f l a m m a t o r y arthritis. The peptides are a few residues s h o r t e r t h a n expected, likely due to s e c o n d a r y proteolysis from the C-terminal end. Nonetheless, t h e i r work provides u n e q u i v o c a l evidence t h a t MMPs, as well as 'aggrecanase', act directly in a g g r e c a n d e g r a d a t i o n in arthritis. Since we only analyzed high b u o y a n t density fractions, these peptides would not have been detected. F u r t h e r w o r k will be needed to explain the role of MMP-3 in Fn-f-mediated cartilage damage, nonetheless these data are consistent with the possibility t h a t Fn-fs may c o n t r i b u t e to a g g r e c a n d e g r a d a t i o n in o s t e o a r t h r i t i s and inflammatory joint diseases. These data also support the n o t i o n t h a t the Fn-fs mediate t h e i r effects t h r o u g h catabolic cytokines.
Acknowledgments Ms Carla Pellegrino is acknowledged for her expert assistance in protein sequencing.
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BY GENE A. HOMANDBERG*,GARY DAVISt, CHARLESMANIGLIAS~AND ALKA SHRIKHANDE:~ * D e p a r t m e n t of Biochemistry, Rush M e d i c a l College at R u s h - P r e s b y t e r i a n - S t . L u k e ' s M e d i c a l Center, Chicago, Illinois and t I n s t i t u t e F o r R e s e a r c h T e c h n o l o g i e s a n d $ I n s t i t u t e F o r Bone a n d J o i n t D i s o r d e r s and Cancer, B a y e r C o r p o r a t i o n , West H a v e n , C o n n e c t i c u t , U.S.A.