Evidence for disulfide involvement in the regulation of intramolecular autolytic processing by human adamalysin19/ADAM19.
Human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19) is activated by furin-mediated cleavage of the prodomain followed by an autolytic processing within the cysteine-rich domain at Glu586-Ser587, which occurs intramolecularly, producing an NH2 terminal fragment (N-fragment) associated with its COOH-terminal fragment (C-fragment), most likely through disulfide bonds. When stable Madin-Darby canine kidney (MDCK) transfectants overexpressing soluble hADAM19 were treated with dithiothreitol (DTT) or with media at pH 6.5, 7.5, or 8.5, the secretion and folding of the enzyme were not affected. Autolytic processing was blocked by DTT and pH 6.5 media, which favor disulfide reduction, but was increased by pH 8.5 media, which promotes disulfide formation. Cys605, Cys633, Cys639, and Cys643 of the C-fragment appear to be partially responsible for the covalent association between the C-fragment and the N-fragment. A new autolytic processing site at Lys543-Val544 was identified in soluble mutants when these cysteine residues were individually mutated to serine residues. Shed fragments were also detectable in the media from MDCK cells stably expressing the full-length Cys633Ser mutant. Ilomastat/GM6001 inhibited hADAM19 with an IC50 of 447 nM, but scarcely affected the shedding process. The cysteine-rich domain likely forms disulfide bonds to regulate the autolytic processing and shedding of hADAM19.
Kang
Tiebang
T
Department of Chemistry and Biochemistry, Florida State University, Tallahassee 32306-4390, USA.
Tschesche
Harald
H
Amy Sang
Qing-Xiang
QX
D051722:ADAM Proteins; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000502:physiology; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D019483:Disintegrins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology*; D004285:Dogs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D012694:Serine / Q000378:metabolism
D004151:Dipeptides; D019483:Disintegrins; D004220:Disulfides; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D012694:Serine; D051722:ADAM Proteins; C411902:ADAM19 protein, human; D008666:Metalloendopeptidases; D003545:Cysteine; D004229:Dithiothreitol