Functional differences of the catalytic and non-catalytic domains in human ADAMTS-4 and ADAMTS-5 in aggrecanolytic activity

ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase2) are multidomain metalloproteinases belonging to the ADAMTS family. We have previously reported that human ADAMTS-5 has much higher aggrecanolytic activity than human ADAMTS-4. To investigate the different proteolytic activity of the two enzymes, we generated a series of chimeras by exchanging various non-catalytic domains of the two proteinases. We found that the catalytic domain of ADAMTS-5 has higher intrinsic catalytic ability than that of ADAMTS-4. The studies also demonstrated that the non-catalytic domains of ADAMTS-5 are more effective modifiers than those of ADAMTS-4, making both catalytic domains more active against aggrecan, an Escherichia coli-expressed interglobular domain of aggrecan and fibromodulin. Addition of the C-terminal thrombospondin type I motif of ADAMTS-5 to the C terminus of ADAMTS-4 increased the activity of ADAMTS-4 against aggrecan and fibromodulin severalfold. In contrast to previous reports (Kashiwagi, M., Enghild, J. J., Gendron, C., Hughes, C., Caterson, B., Itoh, Y., and Nagase, H. ( 2004) J. Biol. Chem. 279, 10109-10119 and Gao, G., Plaas, A., Thompson, V. P., Jin, S., Zuo, F., and Sandy, J. D. ( 2004) J. Biol. Chem. 279, 10042 10051), our detailed investigation of the role of the C-terminal spacer domain of ADAMTS-4 indicated that full-length ADAMTS-4 is similar to 20-times more active against aggrecan than its spacer domain deletion mutant, even at the Glu(373)-Ala(374) site of the interglobular domain. This discrepancy is most likely due to selective inhibition of full-length ADAMTS-4 by heparin, particularly for cleavage at the Glu(373)-Ala(374) bond. However, removal of the spacer domain from ADAMTS-4 greatly enhanced more general proteolytic activity against non-aggrecan substrates, e. g. E. coli-expressed interglobular domain, fibromodulin, and carboxymethylated transferrin.