title: Autoactivation of human ADAM8: a novel pre-processing step is required for catalytic activity.
abstract: Members of the ADAM (a disintegrin and metalloproteinase) family of proteins possess a multidomain architecture which permits functionalities as adhesion molecules, signalling intermediates and proteolytic enzymes. ADAM8 is found on immune cells and is induced by multiple pro-inflammatory stimuli suggesting a role in inflammation. Here we describe an activation mechanism for recombinant human ADAM8 that is independent from classical PC (pro-protein convertase)-mediated activation. N-terminal sequencing revealed that, unlike other ADAMs, ADAM8 undergoes pre-processing at Glu(158), which fractures the Pro (pro-segment)-domain before terminal activation takes place to remove the putative cysteine switch (Cys(167)). ADAM8 lacking the DIS (disintegrin) and/or CR (cysteine-rich) and EGF (epidermal growth factor) domains displayed impaired ability to complete this event. Thus pre-processing of the Pro-domain is co-ordinated by DIS and CR/EGF domains. Furthermore, by placing an EK (enterokinase) recognition motif between the Pro- and catalytic domains of multiple constructs, we were able to artificially remove the pro-segment prior to pre-processing. In the absence of pre-processing of the Pro-domain a marked decrease in specific activity was observed with the autoactivated enzyme, suggesting that the Pro-domain continued to associate and inhibit active enzyme. Thus, pre-processing of the Pro-domain of human ADAM8 is important for enzyme maturation by preventing re-association of the pro-segment with the catalytic domain. Given the observed necessity of DIS and CR/EGF for pre-processing, we conclude that these domains are crucial for the proper activation and maturation of human ADAM8.
authors.affiliation: Pfizer Global Research and Development, Chesterfield, MO 63017, USA.
authors.forename: Troii
authors.initials: T
authors.lastname: Hall
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Leone
authors.forename: Joseph F
authors.initials: JF
authors.lastname: Wiese
authors.forename: David W
authors.initials: DW
authors.lastname: Griggs
authors.forename: Lyle E
authors.initials: LE
authors.lastname: Pegg
authors.forename: Adele M
authors.initials: AM
authors.lastname: Pauley
authors.forename: Alfredo G
authors.initials: AG
authors.lastname: Tomasselli
authors.forename: Marc D
authors.initials: MD
authors.lastname: Zack
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D004789:Enzyme Activation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D051722:ADAM Proteins; C068490:ADAM8 protein, human