Abstract
Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower affinity (9-44 nM). However, TIMP-1 is a much weaker inhibitor. N-TIMP-3 variants that lack MMP inhibitory activity but retained the ability to inhibit ADAM17/TACE failed to inhibit ADAM12. These results indicate unique enzymatic properties of ADAM12 among the members of the ADAM family of metalloproteinases.