Catalytic properties of ADAM19.
ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.
Chesneau
Valérie
V
Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Becherer
J David
JD
Zheng
Yufang
Y
Erdjument-Bromage
Hediye
H
Tempst
Paul
P
Blobel
Carl P
CP
D051722:ADAM Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis; D002460:Cell Line; D002522:Chlorocebus aethiops; D019483:Disintegrins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases; D045726:Metalloproteases; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D019483:Disintegrins; D008565:Membrane Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; D045726:Metalloproteases; D051722:ADAM Proteins; C411902:ADAM19 protein, human; C096116:Adam19 protein, mouse; D008666:Metalloendopeptidases