Research article summary (published 30 Aug 2009):

ADAM-8 isolated from human osteoarthritic chondrocytes cleaves fibronectin at Ala(271).

Full Abstract

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. CONCLUSION: 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.

Author information

Author/s: Zack, Marc D (MD); 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);

Affiliation: Pfizer Global Research and Development, 700 Chesterfield Parkway, Chesterfield, MO 63017, USA. Marc.d.zack(-atsign-)pfizer.com

Journal and publication information

Publication Type: Journal Article

Journal: Arthritis and rheumatism (Arthritis Rheum), published in United States. (Language: eng)

Reference: 2009-Sep; vol 60 (issue 9) : pp 2704-13

Dates: Created 2009/09/21; Completed 2009/10/19;

PMID: 19714641, status: MEDLINE (last retrieval date: 10/19/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

ADAM Proteins - metabolism; pharmacology Aged, 80 and over Alanine - metabolism Cells, Cultured Chondrocytes - drug effects; metabolism; pathology Culture Media, Conditioned - pharmacology Epitopes Female

Female Fibronectins - metabolism Humans Male Membrane Proteins - metabolism; pharmacology Middle Aged Osteoarthritis, Knee - metabolism; pathology Serine Endopeptidases - metabolism

Associated Chemicals: Culture Media, Conditioned (0) ; Epitopes (0) ; Fibronectins (0) ; Membrane Proteins (0) ; Alanine (56-41-7) ; Serine Endopeptidases (EC 3.4.21.-) ; fibronectinase (EC 3.4.21.-) ; ADAM Proteins (EC 3.4.24.-) ; ADAM8 protein, human (EC 3.4.24.-)

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