Exosites in Hypervariable Loops of ADAMTS Spacer Domains control Substrate Recognition and Proteolysis
2019
ADAMTS(A
Disintegrin-like and Metalloproteinase domain with
Thrombospondintype 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating
proteoglycansincluding
versicanand
aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant
ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major
versicancleavage fragment was used to determine, for the first time, kinetic constants for
versicanproteolysis.
ADAMTS-5 (kcat/Km 35 × 105 M−1 s−1) is a more potent (~18-fold) versicanase than
ADAMTS-4 (kcat/Km 1.86 × 105 M−1 sec−1), whereas
ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of
ADAMTS-5 19-fold and that of
ADAMTS-4 167-fold. Co-deletion of the
ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of
ADAMTS-5 (residues 739–744 and 837–844) and
ADAMTS-4 (residues 717–724 and 788–795) with those of
ADAMTS-13, which does not cleave
proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of
versican. The hypervariable loops of
ADAMTS-5 are shown to be important also for its
aggrecanaseactivity. Together with previous work on
ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of
ADAMTSproteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.
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