TY  - THES
AB  - Adamalysin 19 (a disintegrin and metalloproteinase 19, ADAM19, or meltrin [beta]) is a plasma membrane metalloproteinase. In this dissertation, I revealed that hADAM19 zymogen contains two functional furin recognition sites (RXK/RR), R197PRR200 and R200MKR203, between its pro- and catalytic-domain by protein N-terminal sequencing, specific furin inhibitors, mutagenesis, and furin-deficient mammalian cell lines. R200MKR203 is the primary cleavage site, but R197PRR200 is an alternative site for activation when the primary site is abolished in hADAM19. Co-localization between furin and hADAM19 is also identified in the ER/Golgi complex and/or the trans-Golgi network. Furthermore, hADAM19 is processed at E586-S587 within cysteine-rich domain by an autolytic mechanism and the sizes of the side chains of the residues at both the E586 and S587 are critical for this processing. This proceesig is necessary for hADAM19 to exert its proteolytic activities in vitro as illustrated by mutagenesis, such as both E586 to D586 and S587 to T587. Based on this processing, I also developed a flourescamine assay to determine the activity of soluble hADAM19 by using a peptide substrate of AcRPLESNV, which is identical to the sequence encompassing the processing site. Interestingly, the autolytic processing at E586-S587 occurs intramolecularly, not intermolecularly, producing an N-terminal fragment associated with its C-terminal fragment through disulfide bonds. The cysteine residues at C605, C633, and C643 within the residual cysteine-rich domain of the C-terminal fragment are partially responsible for the covalent association of the C-fragment with the N-terminal fragment. A new processing site at K543-V544 was identified in soluble mutants when these cysteine residues were individually mutated to serine residues. These soluble mutated proteins were properly folded and secreted, but were less stable and more easily degraded compared with the soluble wild type, as illustrated by trypsin digestion and inactive mutant studies, suggesting that the proteolytic activity and the stability of soluble hADAM19 may be regulated by the formation of disulfide bonds that include C605, C633, C639, and C643. Moreover, the new processing site resulted from autolytic cleavage and was shown to be independent of the processing at E586-S587 by utilizing the double mutations E586 to D586 or E346 to A346 in soluble representative mutants. Correspondingly, shed fragments are detectable in the media from MDCK cells stably expressing the full length mutant with C633 to S633, but neither C643 to S643 nor the wild type. Finally, the processing or shedding of hADAM19 is unaffected by Ilomastat (GM6001), but is obviously enhanced by phorbol-12 myristate 13-acetate, a protein kinase C activator; however, A23187, a calcium ionphore, or calmodulin antagonists, such as calmidazolium (CLM), W7, and trifluoperazine (TFP) blocks of inhibits this processing or shedding, indicating the same complex signal pathways may be involved in both processing and shedding of hADAM19.
DA  - 2003
KW  - ,  , ADAM19 , Activation , Processing , Mutagenesis
LA  - eng
PY  - 2003
TI  - Intracellular activation and processing of human disintegrin and metalloproteinase 19 (hADAM19)
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-2810
Y2  - 2024-11-22T03:41:14
ER  -