ADAM-8 isolated from human osteoarthritic chondrocytes cleaves fibronectin at Ala(271).
OBJECTIVE
Fibronectin fragments are thought to play a critical role in the initiation and progression of cartilage degradation in arthritis. In a recent study, fibronectin neoepitopes resulting from cleavage of intact fibronectin at the Ala(271)/Val(272) scissile bond, generating an approximately 30-kd fragment with the new C-terminus VRAA(271) and an approximately 50-85-kd fragment with the new N-terminus (272)VYQP, were identified in osteoarthritis (OA) cartilage. The present study was undertaken to isolate the enzymes responsible for this cleavage from human OA chondrocytes.


METHODS
Fibronectin-degrading activity in human OA chondrocyte-conditioned medium (OACCM) was purified using conventional chromatography. A fluorescent peptide was developed based on the fibronectin scissile bond (269)RAA downward arrowVal(272), and this peptide was used to track fibronectinase activity during purification. Western blotting with antibodies that detect the fibronectin neoepitopes VRAA(271) and (272)VYQP was used to confirm cleavage of intact fibronectin by the enzymatically active fractions. Mass spectrometry was used to identify the proteins found in the fibronectinase-enriched fractions, with further confirmation by Western blotting. In addition, a recombinant enzyme identified by mass spectrometry was tested by Western blotting and dimethylmethylene blue assay for its ability to produce fibronectin neoepitopes in OA cartilage.


RESULTS
Purification of OACCM by chromatography resulted in isolation of a fibronectin-degrading enzyme, and mass spectrometry identified ADAM-8 as the fibronectinase present in these preparations. Furthermore, treatment of OA cartilage with recombinant human ADAM-8 promoted cartilage catabolism.


CONCLUSIONS
The results of this study identify ADAM-8 as a fibronectinase in human OA chondrocytes. Because ADAM-8 is capable of producing the fibronectin neoepitopes VRAA(271) and (272)VYQP in human OA cartilage, this enzyme may be an important mediator of cartilage catabolism.
Zack
Marc D
MD
Pfizer Global Research and Development, 700 Chesterfield Parkway, Chesterfield, MO 63017, USA. Marc.d.zack@pfizer.com
Malfait
Anne-Marie
AM
Skepner
Adam P
AP
Yates
Matthew P
MP
Griggs
David W
DW
Hall
Troii
T
Hills
Robert L
RL
Alston
James T
JT
Nemirovskiy
Olga V
OV
Radabaugh
Melissa R
MR
Leone
Joseph W
JW
Arner
Elizabeth C
EC
Tortorella
Micky D
MD
D051722:ADAM Proteins / Q000378:metabolism* / Q000494:pharmacology*; D000369:Aged, 80 and over; D000409:Alanine / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D000939:Epitopes; D005260:Female; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism* / Q000494:pharmacology*; D008875:Middle Aged; D020370:Osteoarthritis, Knee / Q000378:metabolism* / Q000473:pathology; D012697:Serine Endopeptidases / Q000378:metabolism
D017077:Culture Media, Conditioned; D000939:Epitopes; D005353:Fibronectins; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C071222:fibronectinase; D051722:ADAM Proteins; C068490:ADAM8 protein, human; D000409:Alanine