Protease analysis by neoepitope approach reveals the activation of MMP-9 is achieved proteolytically in a test tissue cartilage model involved in bone formation.
A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Lee
Eunice R
ER
Electron Microscopy Unit, Joint Diseases Laboratory, Shriners Hospital for Children, and Division of Surgical Research, Department of Surgery, McGill University, Montreal, Quebec, H3G 1A6, Canada. elee@shriners.mcgill.ca
Lamplugh
Lisa
L
Kluczyk
Beata
B
Mort
John S
JS
Leblond
Charles Philippe
CP
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001846:Bone Development; D002356:Cartilage / Q000201:enzymology* / Q000254:growth & development; D045744:Cell Line, Tumor; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation; D004838:Epiphyses / Q000201:enzymology / Q000254:growth & development; D000939:Epitopes; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism
D000939:Epitopes; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D020780:Matrix Metalloproteinase 9; D003545:Cysteine