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

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Critical Care201115 (Suppl 1) :P278

https://doi.org/10.1186/cc9698

  • Published:

Keywords

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

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.

Authors’ Affiliations

(1)
University of Pittsburgh, PA, USA
(2)
University of Vienna, Austria
(3)
University of Tulane, New Orleans, LA, USA

Copyright

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