Conserved sequence in the aggrecan interglobular domain modulates cleavage by ADAMTS-4 and ADAMTS-5

Abstract

Background: Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.

Methods: To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.

Results: Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4' Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.

Conclusion: We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme-substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.

General significance: Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.

Fig. 1

Quantification of aggrecan by dot blot western analysis. Dilutions of a standard solution of steer A1A1D1 aggrecan (shown) and similar dilutions of recombinant aggrecan preparations were dot-blotted on a PVDF membrane, reacted with anti-G1 and visualized by ECL. The concentration of recombinant aggrecan preparations was calculated from the A1A1D1 standard curve.

Fig. 2

Summary of recombinant aggrecan mutants. (A) Mutant aggrecans were categorized into four groups having mutations within the IGD of bovine aggrecan. The first group of mutants (Group A) contains mutations of conserved amino acids in the 355Thr-Glu362 sequence found N-terminal to the IGD cleavage site. Second group (Group B) contains mutations at Thr and Asn residues, within the N-glycosylation motif (368AsnIleThr370) near the site of scission. The third group (Group C) contains mutations in a Thr-rich region (352Thr-Thr357) found N-terminal to the IGD cleavage site. The fourth group (Group D) consists of mutations of Arg375 and Ser377, C-terminal to the Glu373-Ala374 cleavage site. (B) Kyte-Doolittle hydropathy plots of WT and mutant aggrecan. Shaded areas indicate significant deviations from the WT hydropathy plot.

Fig. 3

Western blot analysis of fragments derived from WT and mutant recombinant aggrecan: Effects of mutation of conserved Val356, Val361, and Glu362 residues on cleavage by ADAMTS-4 (p68). (A) WT, and V356A, V361A, and E362D mutant aggrecan and ADAMTS-4-generated fragments produced at intervals up to 120 minutes. (B) WT and V356A-V361A-E362D triple mutant aggrecan were digested with ADAMTS-4 for up to 24 h. Antibody used for each Western blot is indicated at the left. Intact aggrecan and fragments generated by ADAMTS4 digestion are numbered at right as described in Miwa et al. [16]. The band labeled 4/5 are unresolved fragments having TFKEEE1666 and TAGELE1480 C-termini.

Fig. 4

Effects of non-conservative mutation of the WT 360AspValGlu362 (DVE) motif on cleavage by ADAMTS-4 (p68). The significance of the DVE sequence in the IGD was examined by producing the mutant D360L to generate LVE, the sequence preceding the more susceptible sites in the CS-2 domain. The WT (DVE) was also mutated to 360AspGlnLys362 (V361Q-E362K) and 360HisGlnLys362 (D360H-V361Q-E362K).

Fig. 5

The effect of N-linked glycosylation at Asn368 on aggrecan cleavage by ADAMTS-4 (p40) at Glu373-Ala374. (A) Mutations at Asn368 and Thr370 remove a potential N-linked site (fork symbol). In N368Q the potentially O-glycosylated Thr (line symbol) remains available. (B) Mutation at Asn368 and Thr370 both result in reduction of the molecular mass of anti-FLAG and anti-NITEGE reactive fragment following digestion with ADAMTS-4 (p40) consistent with elimination of N-linked glycosylation at Asn368.

Fig. 6

The effect of the N-linked oligosaccharide motif upon ADAMTS-4 cleavage. WT and mutant aggrecans were digested with ADAMTS-4 (p68) to compare the cleavage efficiency at the Glu373-Ala374 bond. (A) N368Q mutation, (B) T370Q mutation.

Fig. 7

The effect of a cluster of threonine residues on aggrecan cleavage by ADAMTS-4 (p68) at Glu373-Ala374. A cluster of threonines (Thr352, Thr355, and Thr357) were simultaneously mutated to glutamine or valine (A, B). WT and mutant aggrecans were digested with ADAMTS-4 (p68) to compare the cleavage efficiency at the Glu373-Ala374 bond by the appearance of anti-NITEGE reactive fragments within the time-course assay. The molecular mass of the major band in each digestion is approximately 66.5 kDa.

Fig. 8

The susceptibility of recombinant aggrecan carrying species-mimicking sequence to ADAMTS-4 (p68). (A) Species specific variation found in the P2′ and P4′ position (boldface) relative to the Glu373-Ala374 cleavage site. WT and P′-mutants were digested with (B) ADAMTS-4 (p68) or with (C) ADAMTS-5, and cleavage at Glu373-Ala374 was analyzed by Western blot analysis using anti-FLAG (M2), and anti-NITEGE antibodies. (bands numbered as in Miwa et al. [16].)

Fig. 9

Model for interactions between aggrecan IGD and ADAMTS-4 or ADAMTS-5. Initial docking of the proteinase may occur via hydrophobic interactions between exosite residues and a region of the IGD remote from the cleavage site. In the second step, the active site cleft of the exosite-bound proteinase may dock with the cleavage site of the IGD substrate. Additional interactions may occur between Ser373 and the catalytic or disintegrin-like domain. Binding to the catalytic cleft is followed by chain scission at Glu373-Ala374 and dissociation of the exosite-bound and catalytic cleft-bound polypeptides. The indicated KS-substituted Thr and Asn residues are found in cartilage-derived aggrecan, and absent from recombinant aggrecan. Residues that were mutated in this study are shown in boldface.