Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences.
The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the -2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the -2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native 'QYNST' sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.
Anne A
AA
Ollis
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.
Yi
Y
Chai
Department of Microbiology, Cornell University, Ithaca, NY 14853 USA.
Aravind
A
Natarajan
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.|Comparative Biomedical Sciences, Cornell University, Ithaca, NY 14853 USA.
Emily
E
Perregaux
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.
Thapakorn
T
Jaroentomeechai
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.|Comparative Biomedical Sciences, Cornell University, Ithaca, NY 14853 USA.
Cassandra
C
Guarino
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.
Jessica
J
Smith
Proteomics and Mass Spectrometry Core Facility, Cornell University, Ithaca, New York 14853.
Sheng
S
Zhang
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.|Department of Microbiology, Cornell University, Ithaca, NY 14853 USA.|Comparative Biomedical Sciences, Cornell University, Ithaca, NY 14853 USA.
Matthew P
MP
DeLisa
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019295:Computational Biology; D057225:Data Mining; D016680:Genome, Bacterial; D023281:Genomics; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D010802:Phylogeny; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013549:Sweetening Agents / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D006023:Glycoproteins; D007141:Immunoglobulin Fc Fragments; D008565:Membrane Proteins; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; D013549:Sweetening Agents; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase