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Anup Kumar Misra - One of the best experts on this subject based on the ideXlab platform.

Barbara Imperiali - One of the best experts on this subject based on the ideXlab platform.

  • Chemoenzymatic Assembly of Bacterial Glycoconjugates for Site-Specific Orthogonal Labeling
    Journal of the American Chemical Society, 2015
    Co-Authors: Vinita Lukose, Garrett E. Whitworth, Ziqiang Guan, Barbara Imperiali
    Abstract:

    The cell surfaces of bacteria are replete with diverse glycoconjugates that play pivotal roles in determining how bacteria interact with the environment and the hosts that they colonize. Studies to advance our understanding of these interactions rely on the availability of chemically defined glycoconjugates that can be selectively modified under orthogonal reaction conditions to serve as discrete ligands to probe biological interactions, in displayed arrays and as imaging agents. Herein, enzymes in the N-linked protein glycosylation (Pgl) pathway of Campylobacter jejuni are evaluated for their tolerance for azide-modified UDP-sugar substrates, including derivatives of 2,4-diacetamidobacillosamine and N-acetylgalactosamine. In vitro analyses reveal that chemoenzymatic approaches are useful for the preparation of undecaprenol diphosphate-linked glycans and glycopeptides with site-specific introduction of azide functionality for orthogonal labeling at three specific sites in the Heptasaccharide glycan. The u...

  • Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway.
    Biochemistry, 2006
    Co-Authors: Kerney Jebrell Glover, Eranthie Weerapana, Mark M. Chen, Barbara Imperiali
    Abstract:

    Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C. jejuni, a Heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc-α1,4-GalNAc-α1,4-(Glcβ1,3)-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-α1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif. In this study, we have cloned, overexpressed in Escherichia coli, and purified PglC, the glycosyl-1-phosphate transferase responsible for the first step in the biosynthesis of the undecaprenyl-linked Heptasaccharide donor. In addition, we report the first synthetic route to uridine 5‘-diphosphobacillosamine. Using the uridine 5‘-diphosphobacillosamine and undecaprenyl phosphate, we demonstrate the ability of PglC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PglC does not accept uridine 5‘-diphospho-...

  • chemoenzymatic synthesis of glycopeptides with pglb a bacterial oligosaccharyl transferase from campylobacter jejuni
    Chemistry & Biology, 2005
    Co-Authors: Kerney Jebrell Glover, Eranthie Weerapana, Shin Numao, Barbara Imperiali
    Abstract:

    The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PglB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PglB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked Heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GalNAc-α1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PglB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PglB to transfer a wide variety of saccharides. With the demonstration of PglB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.

  • in vitro assembly of the undecaprenylpyrophosphate linked Heptasaccharide for prokaryotic n linked glycosylation
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Kerney Jebrell Glover, Eranthie Weerapana, Barbara Imperiali
    Abstract:

    Campylobacter jejuni has a general N-linked glycosylation pathway (encoded by the pgl gene cluster), which culminates in the transfer of a Heptasaccharide: GalNAc-α1,4-GalNAc-α1,4-(Glcβ1,3)-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac [where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] from a membrane-anchored undecaprenylpyrophosphate (Und-PP)-linked donor to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Herein we report, the cloning, overexpression, and purification of four of the glycosyltransferases (PglA, PglH, PglI, and PglJ) responsible for the biosynthesis of the Und-PP-linked Heptasaccharide. Starting with chemically synthesized Und-PP-linked Bac and various combinations of enzymes, we have deduced the precise functions of these glycosyltransferases. PglA and PglJ add the first two GalNAc residues on to the isoprenoid-linked Bac carrier, respectively. Elongation of the trisaccharide with PglH results in a hexasaccharide revealing the polymerase activity of PglH. The final branching glucose is then added by PglI, which prefers native lipids for optimal activity. The sequential activities of the glycosyl transferases in the pathway can be reconstituted in vitro. This pathway represents an ideal venue for investigating the integrated functions of a series of enzymatic processes that occur at a membrane interface.

Fanzuo Kong - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis of a Heptasaccharide fragment of the O-deacetylated GXM of C. neoformans serotype C.
    Carbohydrate research, 2005
    Co-Authors: Wei Zhao, Fanzuo Kong
    Abstract:

    Beta-D-Xylp-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)][beta-D-Xylp-(1-->4)]-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->4)]-alpha-D-Manp, the fragment of the exopolysaccharide from Cryptococcus neoformans serovar C, was synthesized as its methyl glycoside. Thus, chloroacetylation of allyl 3-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranoside (1) followed by debenzylidenation and selective 6-O-benzoylation afforded allyl 2-O-chloroacetyl-3-O-acetyl-6-O-benzoyl-alpha-D-mannopyranoside (4). Glycosylation of 4 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate (5) furnished the beta-(1-->4)-linked disaccharide 6. Dechloroacetylation gave the disaccharide acceptor 7 and subsequent coupling with 5 produced the trisaccharide 8. Deacetylation of 8 gave the trisaccharide acceptor 9 and subsequent coupling with a disaccharide 10 produced the pentasaccharide 11. Reiteration of deallylation and trichloroacetimidate formation from 11 yielded the pentasaccharide donor 12. Coupling of a disaccharide acceptor 13 with 12 afforded the Heptasaccharide 14. Subsequent deprotection gave the heptaoside 16, while selective 2-O-deacetylation of 14 gave the Heptasaccharide acceptor 15. Condensation of 15 with glucopyranosyluronate imidate 17 did not yield the expected octaoside, instead, an orthoester product 18 was obtained. Rearrangement of 18 did not give the target octaoside; but produced 15. Meanwhile, there was no reaction between 15 and the glycosyl bromide donor 19.

  • synthesis of Heptasaccharide and nonasaccharide analogues of the lentinan repeating unit
    Carbohydrate Research, 2005
    Co-Authors: Guangbin Yang, Fanzuo Kong
    Abstract:

    Abstract The allyl glycoside β- d -Glc p -(1→3)-β- d -Glc p -(1→3)-[β- d -Glc p -(1→6)]-β- d -Glc p -(1→3)-β- d -Glc p -(1→3)-[β- d -Glc p -(1→6)]-α- d -Glc p ( 18 ) and the acetonyl glycoside of β- d -Glc p -(1→3)-[β- d -Glc p -(1→6)]-β- d -Glc p -(1→3)-β- d -Glc p -(1→3)-[β- d -Glc p -(1→6)]-β- d -Glc p -(1→3)-β- d -Glc p -(1→3)-[β- d -Glc p -(1→6)]-α- d -Glc p ( 28 ) were synthesized as analogues of the lentinan heptaose repeating unit. 4,6- O -Benzylidenated monosaccharide donor 3 and 4,6- O -benzylidenated tetrasaccharide acceptor 14 were used to ensure the β-linkage in the synthesis of 18 , while 4,6- O -benzylidenated disaccharide acceptor 20 , and 4,6- O -benzylidenated disaccharide donors 21 and 24 were used to ensure the β-linkage in the synthesis of 28 .

  • synthesis of two Heptasaccharide analogues of the lentinan repeating unit
    Carbohydrate Research, 2003
    Co-Authors: Wei Zhao, Guangbin Yang, Fanzuo Kong
    Abstract:

    Abstract β- d -Glc p -(1→3)-β- d -Glc p -(1→3)-β- d -Glc p -(1→3)-β- d -Glc p -(1→3)-[β- d -Glc p -(1→3)-β- d -Glc p -(1→6)]-β- d -Glc p ( 18 ) and the allyl glycoside of β- d -Glc p -(1→3)-[β- d -Glc p -(1→6)]-β- d -Glc p -(1→3)-β- d -Glc p -(1→3)-β- d -Glc p -(1→3)[-β- d -Glc p -(1→6)]-α- d -Glc p ( 29 ) were synthesized as the analogues of the lentinan repeating heptaose by building the pentasaccharide backbones first, followed by attaching the side chains. 4,6- O -Benzylidenated mono- 13 or disaccharide 8 were used as the acceptor to ensure the β linkage in the synthesis of 18 , while 4,6- O -benzylidenated disaccharides 21 and 23 were used as the donor and acceptor, respectively, to ensure the β linkage in the synthesis of 29 .

  • synthesis of a mannose Heptasaccharide existing in baker s yeast saccharomyces cerevisiae x2180 1a wild type strain
    Carbohydrate Research, 2003
    Co-Authors: Youlin Zeng, Jianjun Zhang, Jun Ning, Fanzuo Kong
    Abstract:

    Abstract A mannose Heptasaccharide existing in baker's yeast, Saccharomyces cerevisiae X2180-1A wild-type strain, was effectively synthesized as its allyl glycoside via TMSOTf-promoted condensation of a disaccharide donor 13 with a pentasaccharide acceptor 12 , followed by deprotection. The pentasaccharide 12 was constructed by coupling of 2,3,4,6-tetra- O -benzoyl-α- d -mannopyranosyl-(1→3)-2,4,6-tri- O -benzoyl-α- d -mannopyranosyl-(1→2)-3,4,6-tri- O -benzoyl-α- d -mannopyranosyl-(1→2)-3,4,6-tri- O -benzoyl-α- d -mannopyranosyl trichloroacetimidate ( 9 ) with allyl 6- O -acetyl-3,4-di- O -benzoyl-α- d -mannopyranoside ( 10 ), followed by deacetylation. The tetrasaccharide 9 was obtained by coupling of 2,3,4,6-tetra- O -benzoyl-α- d -mannopyranosyl-(1→3)-2,4,6-tri- O -benzoyl-α- d -mannopyranosyl trichloroacetimidate ( 5 ) with allyl 3,4,6-tri-O-benzoyl-α- d -mannopyranosyl-(1→2)-3,4,6-tri- O -benzoyl-α- d -mannopyranoside ( 6 ), followed by deallylation and trichloroacetimidation. The disaccharides 6 and 13 were readily obtained by known methods.

Chinmoy Mukherjee - One of the best experts on this subject based on the ideXlab platform.

Rajib Panchadhayee - One of the best experts on this subject based on the ideXlab platform.