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Unusual transglycosylation activity of Flavobacterium meningosepticum endoglycosidases enables convergent chemoenzymatic synthesis of core fucosylated complex N-glycopeptides.

ABSTRACT: Structurally well defined, homogeneous glycopeptides and glycoproteins are indispensable tools for functional glycomics studies. By screening of various endo-?-N-acetylglucosaminidases through the use of appropriate synthetic donor and acceptor substrates, we have found that the Flavobacterium meningosepticum endo-?-N-acetyl-glucosaminidases (GH family 18), including Endo-F2 and Endo-F3, were able to glycosylate ?-1,6-fucosylated GlcNAc derivative to provide natural, core-fucosylated complex-type N-glycopeptides. The Endo-F2 and Endo-F3 were efficient for transferring both sialylated and asia-lylated glycans and were highly specific for an ?-1,6-fucosylated GlcNAc-peptide as acceptor for transglycosylation, showing only marginal activity with non-fucosylated GlcNAc-peptides. In contrast, we found that the commonly used endoglycosidases such as Endo-A and Endo-M, which belong to GH family 85, were unable to take ?-1,6-fucosyl-GlcNAc derivative as acceptors for transglycosylation. The novel activity of Endo-F2 and Endo-F3 was successfully applied for a highly convergent chemoenzymatic synthesis of a full-sized CD52 glycopeptide antigen carrying both terminal sialic acid and core fucose. This is the first report on endoglycosidases that are able to glycosylate ?-1,6-fucosylated GlcNAc derivatives to form natural core-fucosylated glycopeptides.

SUBMITTER: Huang W

PROVIDER: S-EPMC3449086 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Unusual transglycosylation activity of Flavobacterium meningosepticum endoglycosidases enables convergent chemoenzymatic synthesis of core fucosylated complex N-glycopeptides.

Huang Wei W Li Jie J Wang Lai-Xi LX

Chembiochem : a European journal of chemical biology 20110304 6

Structurally well defined, homogeneous glycopeptides and glycoproteins are indispensable tools for functional glycomics studies. By screening of various endo-β-N-acetylglucosaminidases through the use of appropriate synthetic donor and acceptor substrates, we have found that the Flavobacterium meningosepticum endo-β-N-acetyl-glucosaminidases (GH family 18), including Endo-F2 and Endo-F3, were able to glycosylate α-1,6-fucosylated GlcNAc derivative to provide natural, core-fucosylated complex-typ ...[more]

PMID: 21374780

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Project description:BackgroundLactic acid bacteria are a family of "generally regarded as safe" organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields.ResultsTo expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4-2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding.ConclusionsOverall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

Dataset Information

Unusual transglycosylation activity of Flavobacterium meningosepticum endoglycosidases enables convergent chemoenzymatic synthesis of core fucosylated complex N-glycopeptides.

Publications

Unusual transglycosylation activity of Flavobacterium meningosepticum endoglycosidases enables convergent chemoenzymatic synthesis of core fucosylated complex N-glycopeptides.

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