DOI: 10.1073/pnas.0507311102. <i>In vitro</i> assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation

In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation

10.1073/pnas.0507311102

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

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.

Bibliography

Glover, K. J., Weerapana, E., & Imperiali, B. (2005). In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation. Proceedings of the National Academy of Sciences, 102(40), 14255â14259.

Authors 3

Kerney Jebrell Glover (first)
Eranthie Weerapana (additional)
Barbara Imperiali (additional)

References 15 Referenced 117

10.1046/j.1365-2958.1999.01415.x
10.1038/nrmicro1100
10.1099/mic.0.26721-0
10.1093/glycob/cwi019
10.1074/jbc.M206114200
10.1099/00221287-144-8-2049
10.1126/science.298.5599.1790
10.1073/pnas.54.1.235
10.1016/S0021-9258(18)92003-X
10.1111/j.1365-2958.2005.04519.x
10.1016/S0966-842X(03)00079-9
10.1016/j.carres.2003.07.009
Weerapana E. Glover K. J. Chen M. M. & Imperiali B. J. Am. Chem. Soc. in press.
10.1021/jo0100345
10.1016/S0304-4165(98)00127-5

Dates

Type	When
Created	19 years, 11 months ago (Sept. 26, 2005, 8:35 p.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 2:03 p.m.)
Indexed	1 week, 1 day ago (Aug. 26, 2025, 3:01 a.m.)
Issued	19 years, 11 months ago (Sept. 26, 2005)
Published	19 years, 11 months ago (Sept. 26, 2005)
Published Online	19 years, 11 months ago (Sept. 26, 2005)
Published Print	19 years, 10 months ago (Oct. 4, 2005)

Funders 0

None

BibTeX

@article{Glover_2005, title={In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation}, volume={102}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.0507311102}, DOI={10.1073/pnas.0507311102}, number={40}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Glover, Kerney Jebrell and Weerapana, Eranthie and Imperiali, Barbara}, year={2005}, month=sep, pages={14255–14259} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        8,
        26
      ]
    ],
    "date-time": "2025-08-26T07:01:19Z",
    "timestamp": 1756191679798
  },
  "reference-count": 15,
  "publisher": "Proceedings of the National Academy of Sciences",
  "issue": "40",
  "content-domain": {
    "domain": [
      "www.pnas.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        2005,
        10,
        4
      ]
    ]
  },
  "abstract": "<jats:p>\n            <jats:italic>Campylobacter jejuni</jats:italic>\n            has a general N-linked glycosylation pathway (encoded by the\n            <jats:italic>pgl</jats:italic>\n            gene cluster), which culminates in the transfer of a heptasaccharide: GalNAc-\u03b11,4-GalNAc-\u03b11,4-(Glc\u03b21,3)-GalNAc-\u03b11,4-GalNAc-\u03b11,4-GalNAc-\u03b11,3-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\n            <jats:italic>in vitro</jats:italic>\n            . This pathway represents an ideal venue for investigating the integrated functions of a series of enzymatic processes that occur at a membrane interface.\n          </jats:p>",
  "DOI": "10.1073/pnas.0507311102",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2005,
        9,
        27
      ]
    ],
    "date-time": "2005-09-27T00:35:00Z",
    "timestamp": 1127781300000
  },
  "page": "14255-14259",
  "update-policy": "http://dx.doi.org/10.1073/pnas.cm10313",
  "source": "Crossref",
  "is-referenced-by-count": 117,
  "title": "<i>In vitro</i>\n            assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation",
  "prefix": "10.1073",
  "volume": "102",
  "author": [
    {
      "given": "Kerney Jebrell",
      "family": "Glover",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Departments of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139"
        }
      ]
    },
    {
      "given": "Eranthie",
      "family": "Weerapana",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Departments of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139"
        }
      ]
    },
    {
      "given": "Barbara",
      "family": "Imperiali",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Departments of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139"
        }
      ]
    }
  ],
  "member": "341",
  "published-online": {
    "date-parts": [
      [
        2005,
        9,
        26
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_3_2_1_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1046/j.1365-2958.1999.01415.x"
    },
    {
      "key": "e_1_3_2_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nrmicro1100"
    },
    {
      "key": "e_1_3_2_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1099/mic.0.26721-0"
    },
    {
      "key": "e_1_3_2_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/glycob/cwi019"
    },
    {
      "key": "e_1_3_2_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.M206114200"
    },
    {
      "key": "e_1_3_2_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1099/00221287-144-8-2049"
    },
    {
      "key": "e_1_3_2_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.298.5599.1790"
    },
    {
      "key": "e_1_3_2_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.54.1.235"
    },
    {
      "key": "e_1_3_2_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(18)92003-X"
    },
    {
      "key": "e_1_3_2_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1365-2958.2005.04519.x"
    },
    {
      "key": "e_1_3_2_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0966-842X(03)00079-9"
    },
    {
      "key": "e_1_3_2_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.carres.2003.07.009"
    },
    {
      "key": "e_1_3_2_13_2",
      "unstructured": "Weerapana E. Glover K. J. Chen M. M. & Imperiali B. J. Am. Chem. Soc. in press."
    },
    {
      "key": "e_1_3_2_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/jo0100345"
    },
    {
      "key": "e_1_3_2_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0304-4165(98)00127-5"
    }
  ],
  "container-title": "Proceedings of the National Academy of Sciences",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://pnas.org/doi/pdf/10.1073/pnas.0507311102",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2022,
        4,
        12
      ]
    ],
    "date-time": "2022-04-12T18:03:15Z",
    "timestamp": 1649786595000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://pnas.org/doi/full/10.1073/pnas.0507311102"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        2005,
        9,
        26
      ]
    ]
  },
  "references-count": 15,
  "journal-issue": {
    "issue": "40",
    "published-print": {
      "date-parts": [
        [
          2005,
          10,
          4
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1073/pnas.0507311102"
  ],
  "URL": "http://dx.doi.org/10.1073/pnas.0507311102",
  "relation": {},
  "ISSN": [
    "0027-8424",
    "1091-6490"
  ],
  "subject": [],
  "container-title-short": "Proc. Natl. Acad. Sci. U.S.A.",
  "published": {
    "date-parts": [
      [
        2005,
        9,
        26
      ]
    ]
  },
  "assertion": [
    {
      "value": "2005-07-05",
      "order": 0,
      "name": "received",
      "label": "Received",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "2005-09-26",
      "order": 2,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}