The Bacteroidetes Q-Rule: Pyroglutamate in Signal Peptidase I Substrates

Abstract

Bacteroidetes feature prominently in the human microbiome, as major colonizers of the gut and clinically relevant pathogens elsewhere. Here, we reveal a new Bacteroidetes specific feature in the otherwise widely conserved Sec/SPI (Sec translocase/signal peptidase I) pathway. In Bacteroidetes, but not the entire FCB group or related phyla, signal peptide cleavage exposes N-terminal glutamine residues in most SPI substrates. Reanalysis of published mass spectrometry data for five Bacteroidetes species shows that the newly exposed glutamines are cyclized to pyroglutamate (also termed 5-oxoproline) residues. Using the dental pathogen Porphyromonas gingivalis as a model, we identify the PG2157 (also called PG_RS09565, Q7MT37) as the glutaminyl cyclase in this species, and map the catalytic activity to the periplasmic face of the inner membrane. Genetic manipulations that alter the glutamine residue immediately after the signal peptide in the pre-pro-forms of the gingipains affect the extracellular proteolytic activity of RgpA, but not RgpB and Kgp. Glutamine statistics, mass spectrometry data and the mutagenesis results show that the N-terminal glutamine residues or their pyroglutamate cyclization products do not act as generic sorting signals.

Expression of human and Porphyromonas gingivalis glutaminyl cyclases in periodontitis and rheumatoid arthritis–A pilot study

“…, gingipains are translocated from periplasm via outer membrane by type IX secretion system (T9SS), where a modification of the enzymes occurs (de Diego et al, 2016). The very recent report mentioned before (Bochtler et al, 2018) found N-terminal pGlu in 27 proteins, and it is speculated that PgQC pyroglutaminates N-terminal of majority of proteins secreted by P. gingivalis. All these proteins with N-terminal pGlu are putative substrates for signal peptidase I, an enzyme catalysing release of proteins from the membrane.…”

“…gingivalis QC have been published (Bochtler et al, 2018). Using several fractions of cell extracts, QC activity and presence of QC (determined by Western blotting of fractions) was localized at the inner membrane only (Bochtler et al, 2018). Our FRIL electron microscopy photographs confirm a location of the enzyme in the periplasm.…”

“…All these proteins with N-terminal pGlu are putative substrates for signal peptidase I, an enzyme catalysing release of proteins from the membrane. However, only for RgpA but not for RgpB and Kgp a role for glutaminyl cyclization in T9SS could be assumed when using different P. gingivalis W83 mutants (Bochtler et al, 2018). The exact function of P. gingivalis QC should be a topic in further research.…”

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis

“…The glutaminyl cyclase (QC) from P. gingivalis ( Pg QC), identified recently using sophisticated proteomic analyses ( 21 ), represents such an attractive target. QCs belong to the family of aminoacyltransferases and catalyze the cyclization of N-terminal glutamine/glutamate residues of peptides and proteins with concomitant release of ammonia/water ( Fig.…”

“…Further analyses indicated the presence of an N-terminal lipid anchor ( 37 ), consistent with the authors’ demonstration that Pg QC localizes to the inner periplasmatic membrane, which in turn is supported by freeze-fracture replica immunolabeling (FRIL) electron microscopy ( 38 ). It is thought that the enzyme catalyzes the cyclization of N-terminal glutamine residues of periplasmic, outer membrane integrated, and extracellular proteins after their translocation into the periplasm and subsequent removal of the signal peptides by the SP I signal peptidase ( 21 , 39 , 40 ), which could stabilize substrate proteins by protecting them against proteolytic degradation by periplasmatic and host cell aminopeptidases.…”