Frances Willenbrock

A novel coumarin‐labelled peptide for sensitive continuous assays of the matrix metalloproteinases

(7‐methoxycoumarin‐4‐yl)Acetyl‐Pro‐Leu‐Gly‐Leu‐(3‐[2,4‐dinitrophenyl]‐l‐2,3‐diaminopropionyl)‐Ala‐Arg‐NH2 (Mca‐Pro‐Leu‐Gly‐Leu‐Dpa‐Ala‐Arg‐NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly flourescent 7‐methoxycoumarin group is efficiently quenched by energy transfer to the 2,4‐dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3,4,24,23) cleaves the substrate at the Gly‐Leu bond with a 190‐fold increase in fluorescence (λcm 328 nm, λcm 393 nm). In assays of the human matrix metalloproteinases, Mca‐Pro‐Leu‐Gly‐Leu‐Dpa‐Ala‐Arg‐NH2 is about 50 to 100 times more sensitive than dinitrophenyl‐Pro‐Leu‐Gly‐Leu‐Trp‐Ala‐d‐Arg‐NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (k cal/K m) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.

Regulation of Matrix Metalloproteinase Activitya

Matrix metalloproteinases (MMPs) are thought to initiate the degradation of the extracellular matrix during the remodeling of connective tissues. A clear understanding of the mechanisms governing the regulation of their activity during normal physiological processes should give further insights into the uncontrolled remodeling occurring in degradative pathologies. Regulation of the MMPs occurs at the level of gene expression, with precise spatial and temporal compartmentalization of both synthesis and secretion by resident cells as well as by those cells invading the tissue. Extracellularly, MMPs are further regulated by the extent of processing of the proform to an active enzyme and by the relative production of the specific inhibitors of MMPs, the tissue inhibitors of metalloproteinases (TIMPs). Furthermore, the potential for association of MMPs with the cell surface or extracellular matrix components further constrains their relationship with substrates, activators and inhibitors, acting as a further regulator of MMP activity. We initiated a program of study of the MMPs and TIMPs to ascertain the relation between their structure and their function, with particular emphasis on the mechanisms of biological regulation. Recombinant wild-type proteins and specific mutants, including deletion and site mutations, have been prepared using a mammalian expression system.’ Aspects of our findings, which illustrate the fundamental importance of the domain structure of MMPs and TIMPs in their biology, are presented here.

Human Tissue Inhibitor of Metalloproteinases 3 Interacts with Both the N- and C-terminal Domains of Gelatinases A and B REGULATION BY POLYANIONS

We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of stromelysin revealed that residues 568–631 of gelatinase A were required for rapid association with TIMP-3. The N-terminal domain of gelatinase B alone also showed slower association with TIMP-3, again implying significant C-domain interactions. The isolation of complexes between TIMP-3 and progelatinases A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proenzymes. We analyzed the effect of various polyanions on the inhibitory activity of TIMP-3 in our soluble assay. The association rate was increased by dextran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matrix, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity.

The Specificity of TIMP-2 for Matrix Metalloproteinases Can Be Modified by Single Amino Acid Mutations

Residues 1–127 of human TIMP-2 (N-TIMP-2), comprising three of the disulfide-bonded loops of the TIMP-2 molecule, is a discrete protein domain that folds independently of the C-terminal domain. This domain has been shown to be necessary and sufficient for metalloproteinase inhibition and contains the major sites of interaction with the catalytic N-terminal domain of active matrix metalloproteinases (MMPs). Residues identified as being involved in the interaction with MMPs by NMR chemical shift perturbation studies and TIMP/MMP crystal structures have been altered by site-directed mutagenesis. We show, by measurement of association rates and apparent inhibition constants, that the specificity of these N-TIMP-2 mutants for a range of MMPs can be altered by single site mutations in either the TIMP “ridge” (Cys1–Cys3 and Ser68–Cys72) or the flexible AB loop (Ser31–Ile41). This work demonstrates that it is possible to engineer TIMPs with altered specificity and suggests that this form of protein engineering may be useful in the treatment of diseases such as arthritis and cancer where the selective inhibition of key MMPs is desirable.

Analysis of the Interaction of TIMP‐2 and MMPs: Engineering the Changes

The t issue i nhibitors of m etallo p roteinases (TIMPs) regulate the activation and proteolytic activity of the matrix metalloproteinases (MMPs). An imbalance in the relative concentrations of MMP and TIMP has been observed in many degradative diseases. 1 Four members of the family (TIMPs 1–4) have been identified and cloned from a number of species. The TIMPs share 40% sequence similarity with considerably higher structural similarity. The TIMPs consist of a six-loop structure formed by 12 conserved cysteine residues. The first three loops form the N-terminal domain, which is highly conserved and has been shown to bind directly into the active site of MMPs. The C-terminal regions, composed of the final three loops, are more divergent and may be responsible for the selectivity of inhibition and binding efficiency of TIMPs to MMPs. We have initiated a study examining interactions of the N-terminal domain of TIMP-2 (N–TIMP-2) with MMPs, using isolated N-terminal domains expressed in E. coli . The rationale for this study was initially based on the NMR solution structure of N–TIMP-2, 2 which showed that the core of the N–TIMP-2 protein is a closed fivestranded β -barrel homologous to the oligosaccharide/oligonucleotide–binding protein family. Further information has been obtained from the crystal structure of the catalytic domain of MMP3–TIMP-1 complex, 3 NMR chemical shift perturbation studies on the interaction of MMP3 and N–TIMP-2, 4 and most recently the crystal structure of MT1-MMP and TIMP-2. 5 This body of work has identified three major regions of the N–TIMP-2 protein as being important in the interaction with MMPs: Cys 1 Ser 4 , and Ala 70 -Gly 73 , which together form a “ridge” on the surface of the TIMP and interact directly with the active site cleft of MMP-3; and Glu 28 -Lys 41 , which is at the apex of the AB loop forming a β -hairpin. We have made site-directed mutants in two of these regions and investigated the effect on the association rate with several MMPs.