title: Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli.
abstract: Campylobacter jejuni is a human enteropathogenic bacterium possessing an N-glycosylation system. In this work, a protein glycosylation (pgl) operon conferring prokaryotic N-glycosylation in C. jejuni JCM 2013 was cloned and identified. Fourteen open reading frames (ORFs) were found in the pgl operon. The operon organization was similar to that of C. jejuni NCTC 11168, with 98% and 99% identities in overall nucleotide sequence and amino acid sequence, respectively. The pgl operon was heterologously co-expressed with model protein CmeA in the Escherichia coli BL21 ΔwaaL mutant. The immuno- and lectin-blotting analysis indicated the protein glycosylation on the recombinant CmeA. In addition, to analyze the glycan composition, the recombinant CmeA was purified and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. The mass spectrometry analysis showed the presence of the N-acetylhexosamine residue at the reducing end but not the predicted di-N-acetylbacillosamine (diNAcBac) residue. Further glycan structural study using the conventional fluorophore-labeling method revealed the GalNAcα-GalNAcα-(Hex-)HexNAc-HexNAc-HexNAc-HexNAc structure. Transcriptional analysis showed that UDP-diNAcBac synthases and diNAcBac transferase are transcribed but might not function in the constructed system. In conclusion, a pgl operon from C. jejuni JCM 2013 successfully functioned in E. coli, resulting in the observed prokaryotic glycosylation.
authors.affiliation: International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Akkaraphol
authors.initials: A
authors.lastname: Srichaisupakit
authors.affiliation: International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Ohashi
authors.affiliation: International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address: fujiyama@icb.osaka-u.ac.jp.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D016123:Campylobacter jejuni / Q000235:genetics* / Q000378:metabolism; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000117:Acetylglucosamine
keywords: Campylobacter jejuni; Escherichia coli; N-Glycan analysis; Prokaryotic glycosylation; Protein expression