Journal
EXPERIMENTAL CELL RESEARCH
Volume 298, Issue 1, Pages 285-295Publisher
ELSEVIER INC
DOI: 10.1016/j.yexcr.2004.04.022
Keywords
A disintegrin and metalloproteinase 19; meltrin beta; ectodomain shedding; proteolytic processing; enzyme activity regulation; disulfide bonds; site-directed mutagenesis; structure-function analyses; reducing agent; pH effect
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Funding
- NCI NIH HHS [CA78646] Funding Source: Medline
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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 Glu(586)-Ser(587), 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 hADAM 19 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. Cys(605), Cys(633), Cys(639), and Cys(643) 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 Lys(543)-Val(544) 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 hADAM 19 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. (C) 2004 Elsevier Inc. All rights reserved.
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