title: Catalytic properties of ADAM12 and its domain deletion mutants.
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.
authors: Department of Biomedical Sciences and Biotech Research & Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark. jonas@pai.ku.dk
authors: Jonas
authors: J
authors: Jacobsen
authors: Robert
authors: R
authors: Visse
authors: Hans Peter
authors: HP
authors: Sørensen
authors: Jan J
authors: JJ
authors: Enghild
authors: Keith
authors: K
authors: Brew
authors: Ulla M
authors: UM
authors: Wewer
authors: Hideaki
authors: H
authors: Nagase
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000072199:ADAM12 Protein; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D008670:Metals; D050505:Mutant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D014168:Transferrin; D012965:Sodium Chloride; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human; D002118:Calcium