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Human leukocyte elastase is unable to cleave synovial fluid fibronectin
O s t e o a r t h r i t i s and Cartilage Vol. 1 N o . 1
33
a c t i v a t o r o f c o n n e c t i v e t i s s u e d e g r a d i n g e n z y m e s in L1fluids (SF) f r o m o s t e o a r t h r i t i c (OA) p a t i e n t s B. MCLAUGHLIN AND J. B. WEISS
Rheumatic Diseases Centre, Hope Hospital, Salford, U.K. ~Yt~ formation in OA requires conversion of ~ ! ~ cartilage to bone which involves an angio~ i ~ : p ~ b c e s s . Previous work has indicated a relationb~tween ESAF levels in serum and SF in OA (Ann ~ . : : D i s 1988; 47: 881-885). The presence of ESAF in ~ f i : : p l a t e cartilage has been established. F has been measured by its known ability to ~ t e pro-co lagenase (Biochem Biophys Acta 1991; ~7315~:!~295-298).
We now report that ESAF is able to activate all the proforms of connective tissue degrading enzymes and to reactivate all known complexes of these enzymes with their natural inhibitors. As ESAF has no intrinsic enzyme activity (not being a protein) and is active in fentogram quantities, its diffusion from a reopened growth plate could account for degradative changes in OA cartilage.
~ l o n i n g and e x p r e s s i o n of h u m a n s t r o m e l y s i n in a b a c u l o v i r u s / i n s e c t cell system Fu-ZoN CHUNG, MARSHA A. VARTANIAN, J. HOLLIS JORDAN, DALE L. OXENDER AND VIJAYKUMAR M. BARAGI
Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, MI, U.S.A. H~an stromelysin, a proteoglycan-degrading matrix ~lloproteinase is implicated in connective tissue d ~ a d a t i o n associated with various pathological condii~i~ns, including arthritis, periodontitis, tumor progresSiOn and metastasis, was expressed in a baculovirus/ ii~sect cell system. A cDNA containing the entire coding ~ g i o n of human fibroblast stromelysin gene was ~i~alyzed by sequencing and found to be identical to the ~ l i s h e d sequences. The full-length gene was cloned itito a baculovirus transfer vector (pVL1393) and ~0transfected with wild type virus into Sf9 insect cells. Recombinant clones were isolated by the limiting dilu:~ion method and identified by PCR amplification of viral :~NA, precipitated from medium of infected cells. Sf9 cells infected with wild type or recombinant virus were
harvested 48-72 h post-infection and the media subjected to SDS-PAGE and Western blot using a polyclonal antibody against native stromelysin. The medium from insect cells infected with recombinant baclovirus revealed the presence of two proteins with Mr = 52 and 57 kDa. As a control, the medium from cells infected with wild type virus did not show cross-reaction with the stromelysin antibody. Furthermore, upon activation with 4-aminophenylmercuric acetate, the medium from cells infected with the recombinant baculovirus showed catalytic activity against proteoglycans. Taken together, these data indicate that the baculovirus/insect cell system can be used to express the fulllength stromelysin gene.
H u m a n l e u k o c y t e e l a s t a s e is u n a b l e to cleave s y n o v i a l fluid fibronectin X. CHEVALIER, N. GROULT AND W. HORNEBECK
Laboratoire de Biochemie cellulaire, Crdteil, France Fragmentation of fibronectin (FN) by proteinases may result in fragments with new properties such as chemotactic and enzymatic activity. Among these enzymes, fibronectin is highly sensitive to elastases. The purpose of this study was to determine whether in synovial fluid (SF) from patients with rheumatoid arthritis, human leukocyte elastase (HLE) was able to cleave FN and therefore generate fibronectin fragments: SF was collected and immediately centrifuged. HLE was added to SF (1 #g/ml) during 72 h of incubation (37°C) and to purified plasmatic FN in a Tris buffer (1 #g HEL/1 mg FN). Fragmentation of FN was then observed by Western blot.
Results showed that: (1) plasma FN is fragmented as soon as 1 h into fragments of 45 and 29 kDa, (2) synovial fluid FN was not fragmented by HEL even after 72 h of incubation. Using a specific substrate [Suc-(Ala) 3.pNA], elastase activity expressed as /iU/ml (compared to pig pancreatic elastase) was not significantly increased in samples of synovial fluid with HLE (0.42 pU/ml+0.05) compared to ~.samples of synovial fluid without HLE (0.4 #U/ml + 0.03). This study demonstrates t h a t in synovial fluid of patients with rheumatoid arthritis, high level of a serine-protease inhibitor protects fibronectin against enzymatic activity of elastase.
33
a c t i v a t o r o f c o n n e c t i v e t i s s u e d e g r a d i n g e n z y m e s in L1fluids (SF) f r o m o s t e o a r t h r i t i c (OA) p a t i e n t s B. MCLAUGHLIN AND J. B. WEISS
Rheumatic Diseases Centre, Hope Hospital, Salford, U.K. ~Yt~ formation in OA requires conversion of ~ ! ~ cartilage to bone which involves an angio~ i ~ : p ~ b c e s s . Previous work has indicated a relationb~tween ESAF levels in serum and SF in OA (Ann ~ . : : D i s 1988; 47: 881-885). The presence of ESAF in ~ f i : : p l a t e cartilage has been established. F has been measured by its known ability to ~ t e pro-co lagenase (Biochem Biophys Acta 1991; ~7315~:!~295-298).
We now report that ESAF is able to activate all the proforms of connective tissue degrading enzymes and to reactivate all known complexes of these enzymes with their natural inhibitors. As ESAF has no intrinsic enzyme activity (not being a protein) and is active in fentogram quantities, its diffusion from a reopened growth plate could account for degradative changes in OA cartilage.
~ l o n i n g and e x p r e s s i o n of h u m a n s t r o m e l y s i n in a b a c u l o v i r u s / i n s e c t cell system Fu-ZoN CHUNG, MARSHA A. VARTANIAN, J. HOLLIS JORDAN, DALE L. OXENDER AND VIJAYKUMAR M. BARAGI
Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, MI, U.S.A. H~an stromelysin, a proteoglycan-degrading matrix ~lloproteinase is implicated in connective tissue d ~ a d a t i o n associated with various pathological condii~i~ns, including arthritis, periodontitis, tumor progresSiOn and metastasis, was expressed in a baculovirus/ ii~sect cell system. A cDNA containing the entire coding ~ g i o n of human fibroblast stromelysin gene was ~i~alyzed by sequencing and found to be identical to the ~ l i s h e d sequences. The full-length gene was cloned itito a baculovirus transfer vector (pVL1393) and ~0transfected with wild type virus into Sf9 insect cells. Recombinant clones were isolated by the limiting dilu:~ion method and identified by PCR amplification of viral :~NA, precipitated from medium of infected cells. Sf9 cells infected with wild type or recombinant virus were
harvested 48-72 h post-infection and the media subjected to SDS-PAGE and Western blot using a polyclonal antibody against native stromelysin. The medium from insect cells infected with recombinant baclovirus revealed the presence of two proteins with Mr = 52 and 57 kDa. As a control, the medium from cells infected with wild type virus did not show cross-reaction with the stromelysin antibody. Furthermore, upon activation with 4-aminophenylmercuric acetate, the medium from cells infected with the recombinant baculovirus showed catalytic activity against proteoglycans. Taken together, these data indicate that the baculovirus/insect cell system can be used to express the fulllength stromelysin gene.
H u m a n l e u k o c y t e e l a s t a s e is u n a b l e to cleave s y n o v i a l fluid fibronectin X. CHEVALIER, N. GROULT AND W. HORNEBECK
Laboratoire de Biochemie cellulaire, Crdteil, France Fragmentation of fibronectin (FN) by proteinases may result in fragments with new properties such as chemotactic and enzymatic activity. Among these enzymes, fibronectin is highly sensitive to elastases. The purpose of this study was to determine whether in synovial fluid (SF) from patients with rheumatoid arthritis, human leukocyte elastase (HLE) was able to cleave FN and therefore generate fibronectin fragments: SF was collected and immediately centrifuged. HLE was added to SF (1 #g/ml) during 72 h of incubation (37°C) and to purified plasmatic FN in a Tris buffer (1 #g HEL/1 mg FN). Fragmentation of FN was then observed by Western blot.
Results showed that: (1) plasma FN is fragmented as soon as 1 h into fragments of 45 and 29 kDa, (2) synovial fluid FN was not fragmented by HEL even after 72 h of incubation. Using a specific substrate [Suc-(Ala) 3.pNA], elastase activity expressed as /iU/ml (compared to pig pancreatic elastase) was not significantly increased in samples of synovial fluid with HLE (0.42 pU/ml+0.05) compared to ~.samples of synovial fluid without HLE (0.4 #U/ml + 0.03). This study demonstrates t h a t in synovial fluid of patients with rheumatoid arthritis, high level of a serine-protease inhibitor protects fibronectin against enzymatic activity of elastase.