Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates

Abstract

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triplehelical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triplehelical substrates at subsites P3, P1, P1′, and P10′ individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1′ subsite and N-isobutyl Gly (NLeu) in the P10′ subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772–786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7′ peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

Second Generation Triple-Helical Peptide Inhibitors of Matrix Metalloproteinases

“…Side chain deprotection and cleavage of all THPs from the resin were achieved by two treatments with H 2 O–thioanisole–EDT–TFA (5:5:2.5:87.5) for 3 h each in Ar. THPs were purified by RP-HPLC (see below) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as described99 using an Applied Biosystems Voyager DE-PRO. [M + H] + values for THPs were 4183.4 Da for HLRT (theoretical 4184.64 Da), 4195.6 for FLRT (theoretical 4194.67 Da), 4261.1 Da for VQH (theoretical 4257.63 Da), 4267.0 Da for VSW (theoretical 4265.65 Da), 4272.8 Da for IHK (theoretical 4271.72), 4318.9 Da for IYF (theoretical 4316.75 Da), and 4150.8 Da for FLDP (theoretical 4149.59 Da).…”

Wharton’s jelly-derived mesenchymal stem cells combined with praziquantel as a potential therapy for Schistosoma mansoni-induced liver fibrosis

“…A less favorable, yet still beneficial, response was obtained when WJMSCs was given at a later stage post infection (at the 16 th week). This could be attributed to the decrease in the ratio of type III/I collagen with relative increase in type I collagen, which is very resistant to collagenases, owing to its triple helical structure 55 . Moreover, the varying activity of the ECM components and their remodelling enzymes, increased stiffness, and abundance of growth factors, are all considered very important determinants of the liver microenvironment at late stages of fibrosis compared to early stages that had presumably influenced the response to treatment, especially when PZQ was given in combination.…”

Tricine as a convenient scaffold for the synthesis of C-terminally branched collagen-model peptides

“…While constructing peptoid-containing THPs using solid-phase methodology, 55 we considered incorporating tricine, N -[tris(hydroxymethyl)methyl]glycine, as a building block using a simple method that is compatible with existing Fmoc chemistry. Tricine, commonly used as a buffering agent, 56 is scarcely soluble in organic solvents.…”

“…With the exception of the manual incorporation of tricine, the synthetic route proceeded using standard Fmoc chemistry on a microwave peptide synthesizer (Liberty Blue, CEM Corporation) according to previously published protocols. 55…”

“…MS characterization of linear and branched THPs is typically achieved by digestion using trypsin and characterization of the resulting fragments. 55 The α1(I)772-786 sequence contains an Arg residue at position 780, which represents a potential trypsin cleavage site. DAE α1(I)772-786 THP and DAB α1(I)772-786 THP were digested overnight at room temperature with agarose bead-immobilized trypsin, followed by HPLC analysis (Fig.…”