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The Development of Improved Therapeutics through a Glycan- “Designer” Approach
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
The most well-established method for recombinant glycoprotein production for vaccine development is Protein Glycan Coupling Technology (Fig. 20.1). The method uses safe E. coli strains with various mutations to optimize the level of glycosylation and reduce production of endotoxin. To reduce E. coli natural glycoprotein production and natural synthesis of toxic lipopolysaccharide the PGCT uses WaaL (O-antigen ligase) and WecA (transferase) knockout E. coli mutants. WecA enzyme catalyzes the transfer of the GlcNAc-1-phosphate from undecaprenyl-GlcNAc onto the carrier lipid moiety yielding a first lipid-linked intermediate involved in LPS synthesis. The engineered strain is able to produce a glycoconjugate with high efficiency due to a lack of competition for either the undecaprenyl-phosphate lipid carrier by WecA or the OPS II-Und-PP substrate from WaaL (Garcia-Quintanilla et al., 2014).
Helicobacter pylori employs a general protein glycosylation system for the modification of outer membrane adhesins
Published in Gut Microbes, 2022
Kai-Wen Teng, Kai-Siang Hsieh, Ji-Shiuan Hung, Chun-Jen Wang, En-Chi Liao, Pei-Chun Chen, Ying-Hsuan Lin, Deng-Chyang Wu, Chun-Hung Lin, Wen-Ching Wang, Hong-Lin Chan, Shau-Ku Huang, Mou-Chieh Kao
Gene-disrupted mutants targeting the LPS O-antigen assembly and ligation enzymes WecA, Wzk, and WaaL were generated from the wild-type strains 26695 and G27 and confirmed by LPS silver staining analysis to verify this hypothesis (Figure 3a and Supplementary Figure S4A). Compared to the tested adhesins in the wild-type strains 26695 and G27, the two-step molecular weight upshift glycosylation patterns all disappeared in the corresponding knockout mutants, suggesting that these LPS O-antigen biosynthesis-related enzymes are necessary for the posttranslational modification of the tested adhesins in H. pylori (Figure 3b-d and Supplementary Figure S4B-D). The glycans required for O-antigen biosynthesis are assembled onto and transferred by an inner membrane lipid carrier, undecaprenyl phosphate (UndP).26 The synthetic and recycling pathway of UndP has been reported to be inhibited by an antibiotic, bacitracin.26 After bacitracin treatment, the molecular size upshift patterns of the adhesins also disappeared, thus further confirming that the three O-antigen biosynthesis-related enzymes tested here indeed participate in glycan transfer for adhesin glycosylation in H. pylori (Figure 3e).
Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51
Published in Biofouling, 2018
Xiaochun Huang, Chang Chen, Chunhua Ren, Yingying Li, Yiqin Deng, Yiying Yang, Xiongqi Ding
Eight ORFs are shown to encode various glycotransferases that utilize nucleotidylated sugars to add the sugars in turn to the growing repeat unit. The proteins encoded by ORF1, ORF2, ORF3, ORF5, ORF10, ORF11 and ORF17 are homologies to RfaB, RfbG, WcaJ, WecA, RfaB, WcaA, and WbyL, respectively, while ORF8 could not be designated as being any specific protein with a known function. The deduced amino acid sequence contains conserved domains common in GTA-type glycotransferase superfamily proteins. Interestingly, both WcaJ and WecA are enzymes predicted to initiate the synthesis of polysaccharides. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P) (Hug and Feldman 2011). However, WcaJ catalyzes the first step in CA synthesis by the attachment of glucose-1-phosphate onto Und-P, yielding the intermediate undecaprenyl-pyrophosphoryl-Glu (Patel et al. 2012), while WecA transfers the GlcNAc-1-phosphate moiety onto Und-P, yielding undecaprenyl-pyrophosphoryl-GlcNAc, which is the lipid intermediate involved in the synthesis of O-antigen (Lehrer et al. 2007). In addition to glycotransferases, an acetyltransferase (WcaF) encoded by ORF12 presumably adds an acetyl group to the first fucosyl residue of the repeat unit.