title: Inactivation of N-TIMP-1 by N-terminal acetylation when expressed in bacteria.
abstract: The high-affinity binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is essential for regulation of the turnover of the extracellular matrix during development, wound healing, and progression of inflammatory diseases, such as cancer, atherosclerosis, and arthritis. Bacterially expressed N-terminal inhibitory domains of TIMPs (N-TIMPs) have been used extensively for biochemical and biophysical study of interactions with MMPs. Titration of N-TIMP-1 expressed in E. coli indicates, however, that only about 42% of the protein is active as an MMP inhibitor. The separation of inactive from fully active N-TIMP-1 has been achieved both by MMP affinity and by high-resolution cation exchange chromatography at an appropriate pH, based on a slight difference of charge. Purification by cation exchange chromatography with a Mono S column enriches the active portion of N-TIMP-1 to >95%, with K(i) of 1.5 nM for MMP-12. Mass spectra reveal that the inactive form differs from active N-TIMP-1 in being N-terminally acetylated, underscoring the importance of the free alpha-NH(2) of Cys1 for MMP inhibition. N(alpha)-acetylation of the CTCVPP sequence broadens the N-terminal sequence motifs reported to be susceptible to alpha-amino acetylation by E. coli N-acetyl transferases.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA. vandorens@missouri.edu
authors.forename: Steven R
authors.initials: SR
authors.lastname: Van Doren
authors.forename: Shuo
authors.initials: S
authors.lastname: Wei
authors.forename: Guanghua
authors.initials: G
authors.lastname: Gao
authors.forename: Beverly B
authors.initials: BB
authors.lastname: DaGue
authors.forename: Mark O
authors.initials: MO
authors.lastname: Palmier
authors.forename: Harinath
authors.initials: H
authors.lastname: Bahudhanapati
authors.forename: Keith
authors.initials: K
authors.lastname: Brew
mesh_terms: D000107:Acetylation; D020816:Amino Acid Motifs; D019881:Aminoacyltransferases / Q000378:metabolism; D000818:Animals; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006801:Humans; D053508:Matrix Metalloproteinase 12 / Q000737:chemistry* / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D061965:Matrix Metalloproteinase Inhibitors; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D019881:Aminoacyltransferases; D053508:Matrix Metalloproteinase 12