Catalytic activity of human ADAM33.
ADAM33 (a disintegrin and metalloproteinase) is an asthma susceptibility gene recently identified through a genetic study of asthmatic families (van Eerdewegh et al. (2002) Nature 418, 426-430). In order to characterize the catalytic properties of ADAM33, the metalloproteinase domain of human ADAM33 was expressed in Drosophila S2 cells and purified. The N-terminal sequence of the purified metalloproteinase was exclusively (204)EARR, indicating utilization of one of three furin recognition sites. Of many synthetic peptides tested as potential substrates, four peptides derived from beta-amyloid precursor protein (APP), Kit-ligand-1 (KL-1), tumor necrosis factor-related activation-induced cytokine, and insulin B chain were cleaved by ADAM33; mutation at the catalytic site, E346A, inactivated catalytic activity. Cleavage of APP occurred at His(14)/Gln(15), not at the alpha-secretase site and was inefficient (k(cat)/K(m) (1.6 +/- 0.3) x 10(2) m(-1) s(-1)). Cleavage of a juxtamembrane KL-1 peptide occurred at a site used physiologically with a similar efficiency. Mutagenesis of KL-1 peptide substrate indicated that the P3, P2, P1, and P3' residues were critical for activity. In a transfected cell-based sheddase assay, ADAM33 functioned as a negative regulator of APP shedding and mediated some constitutive shedding of KL-1, which was not regulated by phorbol 12-myristate 13-acetate activation. ADAM33 activity was sensitive to several hydroxamate inhibitors (IK682, K(i) = 23 +/- 7 nm) and to tissue inhibitors of metalloproteinase (TIMPs). Activity was inhibited moderately by TIMP-3 and TIMP-4 and weakly inhibited by TIMP-2 but not by TIMP-1, a profile distinct from other ADAMs. The identification of ADAM33 peptide substrates, cellular activity, and a distinct inhibitor profile provide the basis for further functional studies of ADAM33.
Zou
Jun
J
Department of Allergy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. jun.zou@spcorp.com
Zhu
Feng
F
Liu
Jianjun
J
Wang
Wenyan
W
Zhang
Rumin
R
Garlisi
Charles G
CG
Liu
Yan-Hui
YH
Wang
Shihong
S
Shah
Himanshu
H
Wan
Yuntao
Y
Umland
Shelby P
SP
D051722:ADAM Proteins; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D045683:Furin / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019089:Stem Cell Factor / Q000378:metabolism; D013379:Substrate Specificity; D053221:TNF-Related Apoptosis-Inducing Ligand; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D051017:Apoptosis Regulatory Proteins; D007328:Insulin; D008562:Membrane Glycoproteins; D010455:Peptides; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D045683:Furin; D051722:ADAM Proteins; C450441:ADAM33 protein, human; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine