Proteolytic Activities of Human ADAMTS-5: COMPARATIVE STUDIES WITH ADAMTS-4

Christi Gendron,Masahide Kashiwagi,Ngee Han Lim,Jan J Enghild,Ida B Thøgersen,Clare Hughes,Bruce Caterson,Hideaki Nagase

doi:10.1074/jbc.m701523200

Abstract

Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.

Highlights

Destruction of articular cartilage is a feature of various arthritides, including rheumatoid and osteoarthritis, that results in joint impairment and disability
We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities
The proteinases that are responsible for aggrecan degradation in cartilage are matrix metalloproteinases (MMPs)4 and “aggrecanases,” members of the ADAMTS family [1, 2]

Summary

EXPERIMENTAL PROCEDURES

Materials—Human embryonic kidney cells (HEK 293-EBNA), pCEP4 vector, and the Zero Blunt TOPO PCR kit were from Invitrogen. ADAMTS5-1 was eluted from the column with 50 mM Tris-HCl (pH 7.5) containing 1 M NaCl, 10 mM CaCl2, 0.02% NaN3, and 0.02% Brij-35. Proteins in the conditioned media were precipitated using a final concentration of 3.3% trichloroacetic acid and subjected to Western blot analysis with the anti-FLAG M2 antibody to detect ADAMTS-5 and its mutants. HTB-94 cells were transiently transfected with the pCEP4 expression vector containing cDNA encoding ADAMTS-5 or its variants and cultured in serum-free DMEM with or without heparin (100 ␮g/ml) for 48 h. Aggrecanase Assays—Aggrecan (750 nM) was incubated with ADAMTS4-2 (TS4-2), full-length ADAMTS-5, or an ADAMTS-5 domain deletion mutant in 100 ␮l of aggrecanase reaction buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM CaCl2, 0.02% NaN3, and 0.02% Brij 35) at 37 °C for the indicated period of time. The 29-kDa fibromodulin fragment was sequenced in a manner similar to that described above for the Cm-Tf fragments

RESULTS

Aggrecan digestion

ADDITIONS AND CORRECTIONS