15 bacterial n-glycosylation

What discovery challenged the belief that N-linked glycosylation was exclusive to eukaryotes?

Identification of an N-glycosylation gene cluster in Campylobacter jejuni

What gene encodes the bacterial oligosaccharyltransferase?

pglB

Which eukaryotic protein is homologous to PglB?

STT3

What percentage sequence identity does PglB share with STT3?

Approximately 20%

What does the conserved motif in PglB and STT3 indicate?

A critical catalytic role conserved across evolution

Which Campylobacter protein was used to demonstrate bacterial glycosylation?

AcrA

How does AcrA migrate in a pglB knockout strain?

At a lower molecular weight

What causes the higher molecular weight form of AcrA?

PglB-dependent glycosylation

Why are three AcrA bands observed when pgl genes are reintroduced?

They represent zero

What happens when conserved residues in the WWD motif of PglB are mutated?

Glycosylation is abolished

What experiment showed the system works in E. coli?

Expression of the pgl locus causing AcrA glycosylation

Why is trypsin used before mass spectrometry?

To digest proteins into predictable peptides

Which residues does trypsin cleave after?

Lysine and arginine

Why are glycosidic bonds useful in MS/MS analysis?

They fragment more easily than peptide bonds

Which protein was used for glycan structural analysis?

Peb3

What type of glycan is attached in Campylobacter N-glycosylation?

A heptasaccharide

Where does bacterial N-glycosylation occur?

The periplasm

Is bacterial N-glycosylation co-translational or post-translational?

Post-translational

What lipid carrier is used in bacteria for N-glycan assembly?

Undecaprenyl phosphate

Which protein flips the glycan across the membrane?

PglK

What is the bacterial N-glycosylation sequon?

D/E-X-N-X-S/T

What is the eukaryotic N-glycosylation sequon?

N-X-S/T

What is the major significance of this discovery?

It enables bacterial glycoengineering and recombinant glycoprotein production

what is one major biological significance of Campylobacter N-glycosylation?

rare in bacteria and raises evolutionary questions about why only certain genera have it

What did discovery of PglB help identify in eukaryotes?

STT3 is the catalytic oligosaccharyltransferase subunit

What did bacterial N-glycosylation enable structurally?

X-ray structures that clarified N-OST catalytic mechanisms