Autolytic Processing at Glu586-Ser587within the Cysteine-rich Domain of Human Adamalysin 19/Disintegrin-Metalloproteinase 19 Is Necessary for Its Proteolytic Activity

Abstract

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, A23187, 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 ␣2-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.

ADAM10, the Rate-limiting Protease of Regulated Intramembrane Proteolysis of Notch and Other Proteins, Is Processed by ADAMS-9, ADAMS-15, and the γ-Secretase

“…We observed a decrease in ADAM10 CTF generation in the ADAM9-and ADAM9/15-deficient mouse brain samples relative to WT control samples, demonstrating in vivo correlation of the in vitro cell culture studies conducted in MEF and COS cell lines. Interestingly, the reduction in ADAM10 CTF generation was less prominent in tissues such as the liver and lung, 9 which suggests the presence of additional tissue-specific ADAM10 sheddases. Apparently, the deficiency of ADAM10 shedding did not result in the accumulation of full-length ADAM10, indicating a tight control of ADAM10 holoprotein levels in the cell.…”

“…A VP16-Gal4 sequence (50) was subcloned into mADAM10 cDNA after introduction of an HpaI restriction site in the ADAM10 C terminus via site-directed mutagenesis (Stratagene) at positions G745V,H746N. A mADAM10 construct lacking the ectodomain (containing a signal peptide sequence (amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] joined to amino acids 669 -749) was FLAG-tagged (CTTGTCATCGTCGTC-CTTGTAGTC) before the stop codon at the C terminus. The PCR product was ligated into a pcDNA3.1 vector (ADAM10⌬E-flag).…”

“…A second group (ADAMs 2,7,11,18,22,23,and 29) is characterized by an inactive protease domain, and they seem to be mainly important for cell adhesion and fusion. A large third group of ADAMs displays a broad expression profile and has demonstrated (ADAMs 8,9,10,12,17,19, and 28) or predicted (ADAMs 15,20,21,30, and 33) proteolytic activity. These proteases play a major role in the ectodomain shedding of proteins involved in paracrine signaling, cell adhesion, and intracellular signaling (reviewed in Refs.…”

Catalytic Activity of Human ADAM33

“…For instance, TIMP-3 was found to inhibit ADAM12 (27), ADAM17 (34), and ADAM19 (16), whereas TIMP-1 and TIMP-3 inhibited ADAM10 (35). The TIMPs are secreted proteins but may be found at the cell surface in association with membranebound proteins.…”

“…However, only some of these ADAMs were demonstrated experimentally to possess catalytic activity, including ADAM8 (6, 7), ADAM9 (8), ADAM10 (9), ADAM12 (10), ADAM15 (11,12), ADAM17 (13,14), ADAM19 (15)(16)(17), ADAM28 (18), and ADAM33 (19). For only a few ADAMs, a physiological substrate has been identified (3).…”