N-Linked Glycosylation of Folded Proteins by the Bacterial Oligosaccharyltransferase

N-linked protein glycosylation is found in all domains of life. In eukaryotes, it is the most abundant protein modification of secretory and membrane proteins, and the process is coupled to protein translocation and folding. We found that in bacteria, N-glycosylation can occur independently of the protein translocation machinery. In an in vitro assay, bacterial oligosaccharyltransferase glycosylated a folded endogenous substrate protein with high efficiency and folded bovine ribonuclease A with low efficiency. Unfolding the eukaryotic substrate greatly increased glycosylation. We propose that in the bacterial system, glycosylation sites are located in flexible parts of folded proteins, whereas the eukaryotic cotranslational glycosylation evolved to a mechanism presenting the substrate in a flexible form before folding.

[1]  Matthias Müller,et al.  Co-translocation of a Periplasmic Enzyme Complex by a Hitchhiker Mechanism through the Bacterial Tat Pathway* , 1999, The Journal of Biological Chemistry.

[2]  M. Aebi,et al.  Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[3]  G. Hoops,et al.  A comparison of proteins and peptides as substrates for microsomal and solubilized oligosaccharyltransferase. , 1994, Bioorganic & medicinal chemistry.

[4]  Simon J North,et al.  N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. , 2002, Science.

[5]  W. Lennarz,et al.  The molecular basis of coupling of translocation and N-glycosylation. , 2006, Trends in biochemical sciences.

[6]  B. Imperiali,et al.  Asparagine-linked glycosylation: specificity and function of oligosaccharyl transferase. , 1995, Bioorganic & medicinal chemistry.

[7]  G. von Heijne,et al.  A Nascent Secretory Protein 5 Traverse the Ribosome/Endoplasmic Reticulum Translocase Complex as an Extended Chain (*) , 1996, The Journal of Biological Chemistry.

[8]  J. Ravetch,et al.  Anti-Inflammatory Activity of Immunoglobulin G Resulting from Fc Sialylation , 2006, Science.

[9]  G. Caponigro,et al.  Green fluorescent protein as a scaffold for intracellular presentation of peptides. , 1998, Nucleic acids research.

[10]  J. Weiner,et al.  A Novel and Ubiquitous System for Membrane Targeting and Secretion of Cofactor-Containing Proteins , 1998, Cell.

[11]  N. Sharon,et al.  Lectins--proteins with a sweet tooth: functions in cell recognition. , 1995, Essays in biochemistry.

[12]  B. Imperiali,et al.  Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni. , 2005, Chemistry & biology.

[13]  C. Kristensen,et al.  Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII. , 2005, Glycobiology.

[14]  A. Feinstein,et al.  The conformational effects of N-glycosylation on the tailpiece from serum IgM. , 1991, European journal of biochemistry.

[15]  A. Helenius,et al.  Role of ribosome and translocon complex during folding of influenza hemagglutinin in the endoplasmic reticulum of living cells. , 2000, Molecular biology of the cell.

[16]  D. Kelleher,et al.  An evolving view of the eukaryotic oligosaccharyltransferase. , 2006, Glycobiology.

[17]  Markus Aebi,et al.  Definition of the bacterial N‐glycosylation site consensus sequence , 2006, The EMBO journal.

[18]  R. Dwek,et al.  Glycosylation and the immune system. , 2001, Science.

[19]  R. Daniel,et al.  Export of active green fluorescent protein to the periplasm by the twin‐arginine translocase (Tat) pathway in Escherichia coli , 2001, Molecular microbiology.

[20]  A. Helenius,et al.  Roles of N-linked glycans in the endoplasmic reticulum. , 2004, Annual review of biochemistry.

[21]  A. Helenius,et al.  Recognition of local glycoprotein misfolding by the ER folding sensor UDP-glucose:glycoprotein glucosyltransferase , 2000, Nature Structural Biology.

[22]  C. Szymanski,et al.  Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni * , 2002, The Journal of Biological Chemistry.