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OBJECTIVE: To develop enzyme-activatable Förster resonance energy transfer (FRET) substrate probes to detect matrix metalloproteinase 12 (MMP-12) and MMP-13 activities in vivo in mouse models of inflammatory arthritis. METHODS: Peptidic FRET probes activated by MMP-12 and MMP-13 were reverse designed from inhibitors selected from a phosphinic peptide inhibitor library. Selectivity of the probes was demonstrated in vitro using MMP-1, MMP-2, MMP-3, MMP-12, and MMP-13. In vivo activation of the probes was tested in the zymosan-induced mouse model of inflammation, and probe specificity was evaluated by the MMP inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to monitor these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. RESULTS: The MMP-12 and MMP-13 activity probes (MMP12ap and MMP13ap, respectively) discriminated between the activities of the 2 enzymes. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85, P < 0.0001). MMP13ap activation increased gradually after disease onset and correlated with disease severity over a longer period of 15 days (r = 0.58, P < 0.0001). CONCLUSION: We generated two selective FRET probes that can be used to monitor MMP-12 and MMP-13 activities in live animals. MMP12ap follows the initial stage of inflammation in CIA, while MMP13ap follows the progression of the disease. The specificity of these probes is useful in monitoring the efficacy of MMP inhibitors.

Objective. To develop enzyme activatable Förster resonance energy transfer (FRET) substrate probes to detect MMP-12 and MMP-13 activities in vivo in mouse models of inflammatory arthritis Methods. Peptidic FRET probes activated by MMP-12 and MMP-13 were reverse designed from inhibitors selected from a peptide phosphinic inhibitor library. Selectivity of the probes was demonstrated in vitro using MMP-1, MMP-2, MMP-3, MMP-12, and MMP-13. In vivo activation of the probe was tested in the zymosan-induced mouse model of inflammation and probe specificity was evaluated by the metalloprotease inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to follow these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. Results. The MMP-12- and MMP-13-activity probes (MMP12ap and MMP13ap, respectively) discriminated between the two enzymatic activities. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85; p

The products of a wide variety of organic reactions were rapidly identified by their masses through a combination of thin layer chromatography (TLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Crude mixtures of peptides and glycopeptides, complex carbohydrate reactions, and a classical organic reaction were analysed using the following standard protocol. The components of the reaction mixtures were first separated on the TLC plate, scraped off, extracted and analysed by MALDI-TOFMS. The technique used is easy and applicable to most organic reactions, becoming a very powerful technique in reaction optimization once composition and identity of TLC spots have been established by MS. Moreover, the TLC/MALDI-TOFMS method was used to identify low molecular weight compounds with masses within the matrix region (100-500 u), a goal which is normally difficult to achieve. We successfully detected low molecular weight compounds by suppression of the matrix peaks using a relatively low matrix:analyte ratio (15:1 or lower). Doping both matrix and analyte solutions with [Cs]+ ions resulted in suppression of both [Na]+ and [K]+ peaks, thus enhancing the spectral signals and making identification of low molecular weight compounds more facile.

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

Recently, the C-mannosylation of a specific tryptophan residue in RNase 2 from human urine has been reported [Hofsteenge, J., et al. (1994) Biochemistry 33, 13524-13530; de Beer, T., et al. (1995) Biochemistry 34, 11785-11789]. In those studies, identification of this unusual modification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C2-alpha-mannosyltryptophan [(C2-Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivatives during Edman degradation. In this paper, we have (1) excluded the possibility that (C2-Man-)Trp arose artificially under the acidic conditions previously employed for protein and peptide isolation and analysis, by maintaining the pH >5 throughout these procedures, (2) demonstrated the occurrence of (C2-Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C2-Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from erythrocytes, and (4) found also that high-molecular mass isoforms of urinary RNase 2 are C-mannosylated. These observations firmly establish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to the well-known N- and O-glycosylations. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl residue is in a different conformation than in the glycopeptide or denatured protein, due to protein-carbohydrate interactions