Autolytic processing at Glu586-Ser587 within the cysteine-rich domain of human adamalysin 19/disintegrin-metalloproteinase 19 is necessary for its proteolytic activity.
We investigated the regulation of the proteolytic activity of human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19). It was processed at Glu(586)(P1)-Ser(587)(P1') site in the cysteine-rich domain as shown by protein N-terminal sequencing. This truncation was autolytic as illustrated by its R199A/R200A or E346A mutation that prevented the zymogen activation by furin or abolished the catalytic activity. Reagents that block furin-mediated activation of pro-hADAM19, decRVKR-CMK, and brefeldin A abrogated this processing. The sizes of the side chains of the P1 and P1' residues are critical for the processing of hADAM19. The amount of processing product in the E586Q or S587A mutant with a side chain almost the same size as that in the wild type was almost equal. Conversely, very little processing was observed when the size of the side chain was changed significantly, such as in the E586A, E586G, or S587F mutants. Two mutants with presumably subtle structural distinctions from wild type hADAM19, E586D and S587T, displayed rare or little processing and had very low capacities to cleave alpha2-macroglobulin and a peptide substrate. Therefore, this processing is necessary for hADAM19 to exert its proteolytic activities. Moreover, a new peptide substrate, Ac-RPLE-SNAV, which is identical to the processing site sequence, was cleaved at the E-S bond by soluble hADAM19 containing the catalytic and disintegrin domains. This enzyme cleaved the substrate with K(m), k(cat), and k(cat)/K(m) of 2.0 mm, 2.4/min, and 1200 m(-1) min(-1), respectively, using a fluorescamine assay. Preliminary studies showed that a protein kinase C activator, phorbol 12-myristate 13-acetate, promoted the cellular processing of hADAM19; however, three calmodulin antagonists, trifluoperazine, W7, and calmidazolium, impaired this cleavage, indicating complex signal pathways may be involved in the processing.
Kang
Tiebang
T
Department of Chemistry and Biochemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee 32306-4390, USA.
Park
Hyun I
HI
Suh
Yewseok
Y
Zhao
Yun-Ge
YG
Tschesche
Harald
H
Sang
Qing-Xiang Amy
QX
D051722:ADAM Proteins; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D019483:Disintegrins; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases; D045726:Metalloproteases; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017931:DNA Primers; D019483:Disintegrins; D008565:Membrane Proteins; D009124:Muscle Proteins; D018698:Glutamic Acid; D012694:Serine; D045726:Metalloproteases; D051722:ADAM Proteins; C411902:ADAM19 protein, human; C096116:Adam19 protein, mouse; D008666:Metalloendopeptidases; D003545:Cysteine; D013755:Tetradecanoylphorbol Acetate