Production, purification and characterization of canine prostromelysin.
One of the best studied animal models of osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether stromelysin might play a role in this model, it was necessary to purify the enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a metalloproteinase that was demonstrated to be canine prostromelysin by Northern blot, protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express stromelysin mRNA in response to recombinant human IL-1, but expressed stromelysin mRNA only upon treatment with dog monocyte-conditioned medium (dMCM). The aminophenylmercuric acetate (APMA)-activatable metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and dye matrix affinity chromatography. The purified canine metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine metalloproteinase was inhibited by 1,10-phenanthroline or recombinant human tissue inhibitor of metalloproteinase (TIMP). The amino-terminal sequences of the canine pro- and APMA-activated enzymes were compared with those of human, rabbit and rat stromelysin. The striking homologies among the sequences demonstrated that the purified canine metalloproteinase was indeed canine prostromelysin. A rabbit anti-canine prostromelysin polyclonal antiserum was generated and used to localize the enzyme within cultured dog synoviocytes and articular cartilage stimulated with dMCM. The reagents developed in this study should be useful for examining the expression and distribution of prostromelysin in canine models of osteoarthritis.
Bayne
E K
EK
Department of Biochemical and Molecular Pathology, Merck, Sharp and Dohme Research Laboratories, Rahway, NJ 07065.
Hutchinson
N I
NI
Walakovits
L A
LA
Donatelli
S
S
MacNaul
K L
KL
Harper
C F
CF
Cameron
P
P
Moore
V L
VL
Lark
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D002358:Cartilage, Articular / Q000201:enzymology*; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics; D003470:Culture Media / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology; D004195:Disease Models, Animal; D004285:Dogs / Q000235:genetics / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010003:Osteoarthritis / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D011817:Rabbits / Q000235:genetics; D051381:Rats / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D017385:Sequence Homology; D013045:Species Specificity; D013268:Stimulation, Chemical; D013583:Synovial Membrane / Q000166:cytology / Q000201:enzymology*; D019714:Tissue Inhibitor of Metalloproteinases
D003470:Culture Media; D016207:Cytokines; D004792:Enzyme Precursors; D006023:Glycoproteins; D010618:Phenanthrolines; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; C010024:4-aminophenylmercuriacetate; D017364:Collagenases; D008666:Metalloendopeptidases; C048047:prostromelysin; D010662:Phenylmercuric Acetate; C025205:1,10-phenanthroline