Date on Master's Thesis/Doctoral Dissertation

5-2019

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Oral Health and Rehabilitation

Degree Program

Interdisciplinary Studies (Individualized Degree), PhD

Committee Chair

Potempa, Jan

Committee Member

Lamont, Rich

Committee Member

Graham, James

Committee Member

Demuth, Don

Committee Member

Scott, David

Author's Keywords

gingipains; T9SS; QC; glutaminyl cyclase

Abstract

Background: Porphyromonas gingivalis, a major pathogen associated with chronic periodontitis, secretes variety of proteins, majority of which begins with glutamine. Several of these proteins were found with pyroglutamate (pGlu) at N-terminus suggesting the presence of this posttranslational modification pathway in P.gingivalis. The observation that N-terminal glutamine is over-represented as the first amino acid after signal peptide cleavage, and subsequent confirmation of pGlu formation on the nascent protein via mass spectrometry, led us to conclude that an enzyme must be present as the executor of this reaction. Hypothesis: PG2157 is a glutaminyl cyclase and is responsible for the cyclization of N-terminal glutamine residues. Methods: A homology search was used to identify a gene (PG2157) encoding a protein homologous to human glutaminyl cyclase (QC) in the P. gingivalis genome. The gene was cloned, expressed in E. coli and recombinant PgQC purified. The protein was crystalized, and structure determined by molecular replacement. The rPgQC activity was characterized with respect to pH, ionic strength, optimum substrate specificity, and sensitivity to inhibition by an array of non-specific and specific inhibitors. Finally, subcellular localization of PgQC in P. gingivalis was determined. Results: PgQC specificity is restricted for N-terminal glutamine. The enzyme converts this residue to pGlu with kcat/Km at 1.34 s-1. The reaction was fastest at low ionic strength and at pH around 8.0. The activity was inhibited by o-phenanthroline (≥100µM) and EDTA (≥100mM EDTA). Cu2+ and Zn2+ at ≥100nM exerted ≥90% inhibition. The activity was also significantly affected by cysteamine, imidazole, and reduced glutathione. In bacterial cells PgQC was found associated with the inner membrane as a lipoprotein facing the periplasm. The crystalline structure of PgQC showed strong similarity to human QC on the atomic level. Nevertheless, an inhibitor specific for human QC had a limited effect on the PgQC activity. Conclusions: PgQC is an enzyme resembling mammalian QC and it is responsible for pyroglutamination of proteins secreted by the T9SS of P. gingivalis. This activity is likely essential for bacterium viability since all attempts to produce a viable PgQC knockout failed. Taking into account that also T. forsythia and P. intermedia possess similar enzymes and the frequency of the Q value of Bacteriodetes it is likely that similar post-translatonal modification plays a pivotal role in protein secretion by these periodontal pathogens. Therefore, inhibition of bacterial QC may represent a novel approach to treat periodontal diseases.

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