Characterization of metalloprotease cleavage products of human articular cartilage.
OBJECTIVE
To identify, characterize, and compare proteolysis peptide products generated by metalloprotease digests of human articular cartilage.


METHODS
Human articular cartilage was digested by the addition of exogenous metalloproteases, including matrix metalloproteinases 2, 3, 8, 9, 12, and 13 and aggrecanases ADAMTS-4 and ADAMTS-5. Proteolyzed peptide products were identified by proteomics methods using mass spectrometry.


RESULTS
Complete sequences of the peptides proteolyzed from human articular cartilage, including N- and C-termini and hydroxylated posttranslational modifications, were determined. A wide variety of peptides, originating from types I, II, and III collagen, biglycan, prolargin, fibromodulin, fibronectin, decorin, cartilage oligomeric matrix protein, cartilage intermediate-layer protein, megakaryocyte-stimulating factor, mimecan, aggrecan, and lumican, was analyzed following metalloprotease digestion. Release of peptides varied as a function of time, enzyme specificity, and abundance. Specific type II collagen peptide biomarkers, including those containing the three-quarter-length fragment cleavage site and those containing the domains for helical peptide of type II collagen and C-telopeptide of type II collagen, were observed after release by selected proteases.


CONCLUSIONS
The use of intact cartilage instead of purified protein substrates in the assay allowed for the identification of novel potential substrates and cleavage sites for individual enzymes under more physiologically relevant conditions. Characterization of these cartilage matrix peptides may help in the development of pharmacodynamic biomarkers of cartilage degradation, and also may contribute to an understanding of the bioactive peptides important in chondrocyte signaling.
Zhen
Eugene Y
EY
Eli Lilly & Company, Greenfield, IN 46140, USA.
Brittain
Isabelle J
IJ
Laska
Dennis A
DA
Mitchell
Peter G
PG
Sumer
Eren U
EU
Karsdal
Morten A
MA
Duffin
Kevin L
KL
D051722:ADAM Proteins / Q000502:physiology; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D000328:Adult; D000595:Amino Acid Sequence; D001168:Arthritis / Q000378:metabolism*; D058578:Biglycan; D015415:Biomarkers / Q000378:metabolism; D002358:Cartilage, Articular / Q000201:enzymology*; D024043:Collagen Type II / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005260:Female; D006801:Humans; D045726:Metalloproteases / Q000378:metabolism*; D008875:Middle Aged; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011348:Procollagen N-Endopeptidase / Q000502:physiology; D011509:Proteoglycans / Q000378:metabolism*
C546884:BGN protein, human; D058578:Biglycan; D015415:Biomarkers; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D010455:Peptides; D011509:Proteoglycans; D045726:Metalloproteases; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human