PMID- 24092836
OWN - NLM
STAT- MEDLINE
DCOM- 20140721
LR  - 20151119
IS  - 1460-2423 (Electronic)
IS  - 0959-6658 (Linking)
VI  - 24
IP  - 1
DP  - 2014 Jan
TI  - Evaluating the role of conserved amino acids in bacterial 
      O-oligosaccharyltransferases by in vivo, in vitro and limited proteolysis assays.
PG  - 39-50
LID - 10.1093/glycob/cwt087 [doi]
AB  - Bacterial O-Oligosaccharyltransferases (O-OTases) constitute a growing family of 
      enzymes that catalyze the transfer of a glycan from a lipid carrier to protein 
      acceptors. O-OTases are inner membrane proteins that display limited sequence 
      similarity, except for the Wzy_C signature domain also present in a predicted 
      periplasmic loop of the WaaL ligase, the enzyme responsible for transferring the 
      O antigen to the lipid A core. The mechanism of O-OTase-dependent glycosylation 
      is poorly understood. In this work, conserved amino acid residues in the O-OTases 
      were replaced with alanine in PglL, the O-OTase of Neisseria meningitidis. The 
      activity of wild-type PglL and its mutant derivatives were analyzed in vivo in 
      engineered Escherichia coli cells, and in in vitro assays. We identified two 
      additional sites of pilin glycosylated exclusively by PglL in E. coli. Both sites 
      are modified with phosphoglycerol (PG) by different enzymes in Neisseria 
      gonorrhoeae and Neisseria meningitidis. Limited proteolysis experiments revealed 
      a conformational change that is triggered upon interaction of the C-terminal 
      region of PglL with the lipid-linked oligosaccharide (LLO) substrate. These 
      experiments showed that Q178 and Y405 are required for optimal function, whereas 
      H349 is essential for activity and plays a critical role in the interaction with 
      LLO. The equivalent His residue is also essential for WaaL activity, which 
      suggests a common mechanism for both enzymes, and supports the hypothesis that 
      O-glycosylation and lipopolysaccharide (LPS) synthesis are evolutionarily 
      related. These results contribute to the elucidation of the mechanism of 
      O-OTases, which are promising targets for novel antibiotics and present an 
      enormous potential for glycoengineering novel vaccines and therapeutics.
FAU - Musumeci, Matias A
AU  - Musumeci MA
AD  - Department of Biological Sciences, Alberta Glycomics Centre, University of 
      Alberta, CW405 Biological Sciences Building, Edmonton, AB, Canada T6G 2E9.
FAU - Faridmoayer, Amirreza
AU  - Faridmoayer A
FAU - Watanabe, Yasuharu
AU  - Watanabe Y
FAU - Feldman, Mario F
AU  - Feldman MF
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20131002
PL  - England
TA  - Glycobiology
JT  - Glycobiology
JID - 9104124
RN  - 0 (Bacterial Proteins)
RN  - 0 (Lipopolysaccharides)
RN  - 0 (Membrane Proteins)
RN  - EC 2.4.1.- (Hexosyltransferases)
RN  - EC 2.4.99.18 (dolichyl-diphosphooligosaccharide - protein glycotransferase)
SB  - IM
MH  - Bacterial Proteins/*chemistry/genetics/metabolism
MH  - Glycosylation
MH  - Gram-Negative Bacteria/*enzymology/genetics
MH  - Hexosyltransferases/*chemistry/genetics/metabolism
MH  - Lipopolysaccharides/biosynthesis/chemistry/genetics
MH  - Membrane Proteins/*chemistry/genetics/metabolism
MH  - *Sequence Analysis, Protein
OTO - NOTNLM
OT  - O-linked glycosylation
OT  - O-oligosaccharyltransferases
OT  - bacteria
OT  - limited proteolysis
EDAT- 2013/10/05 06:00
MHDA- 2014/07/22 06:00
CRDT- 2013/10/05 06:00
PHST- 2013/10/05 06:00 [entrez]
PHST- 2013/10/05 06:00 [pubmed]
PHST- 2014/07/22 06:00 [medline]
AID - cwt087 [pii]
AID - 10.1093/glycob/cwt087 [doi]
PST - ppublish
SO  - Glycobiology. 2014 Jan;24(1):39-50. doi: 10.1093/glycob/cwt087. Epub 2013 Oct 2.