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Impact of pro-domain stability of matrix metalloproteinase-8 on the outcome of sepsis

Introduction

Animal studies suggest matrix metalloproteinase-8 (MMP8) (neutrophil collagenase) impairs neutrophil (PMN) recruitment in inflammation; in humans, MMP8 has been associated with inflammation. We hypothesized that septic patients with single nucleotide polymorphisms (SNPs) in the MMP8 promoter region will have a survival advantage, and this advantage is due to differences in MMP8 enzymatic activity and not MMP8 levels.

Methods

We examined data from patients with CAP-associated sepsis (GenIMS), analyzed three functional SNPs (rs3765620, rs1940475, rs11225395) in 1,567 Caucasians and tested associations with 60-day and 90-day mortality and severe sepsis incidence. We simulated functional MMP8 SNPs using anisotropic network modeling. Modeling suggested pro-domain structural stability affecting zymogen activation. Based upon the predictions, we then studied zymogen activation using bioluminescent resonance energy transfer (BRET). We generated recombinant pro-MMP8 with a pro-domain tag of luciferase and carboxy terminus tag of green fluorescent protein. BRET signal was generated when luciferase-cleaved substrate produced a photon transferring energy to the GFP acceptor. GFP in turn emitted a green light signal when the donor/acceptor pairs were spatially close. Upon MMP activation, pro-domain is cleaved causing a loss in BRET signal.

Results

The rs1940475 genotype causing an amino acid mutation in the pro-domain was significantly associated with 90-day mortality (AA: 8.5%, AG: 11.1%, GG: 14.7%, P = 0.007). Cumulative incidence showed that the A allele was associated with better 90-day survival. Computer simulation of the mutation suggests a delayed activation. BRET assay confirmed that pro-domain mutation of MMP8 (K87E) rendered it less amenable to activation.

Conclusions

Our results suggest altering the structural stability of the inhibitory MMP8 pro-domain impacts enzyme activation. Therapeutics targeting pro-domain could be used to modulate MMP function and control downstream inflammatory processes in sepsis.

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Correspondence to J McLaughlin.

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McLaughlin, J., Rella, J., Bakan, A. et al. Impact of pro-domain stability of matrix metalloproteinase-8 on the outcome of sepsis. Crit Care 15, P278 (2011). https://doi.org/10.1186/cc9698

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Keywords

  • Rs1940475 Genotype
  • Amino Acid Mutation
  • MMP8 Promoter
  • Zymogen Activation
  • Bioluminescent Resonance Energy Transfer