Revisiting the role of glycosylation in the structure of human IgG Fc

M. Jack Borrok, Sang Taek Jung, Tae Hyun Kang, Arthur F. Monzingo, George Georgiou

Research output: Contribution to journalArticle

89 Citations (Scopus)

Abstract

Binding of the Fc domain of Immunoglobulin G (IgG) to Fcγ receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fcγ receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an "open" Fc conformation recognized by Fcγ receptors. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 Å resolution and observed significant disorder in the C'E loop, a region critical for Fcγ receptor binding, as well as a decrease in distance between the CH2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between CH2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (Rg) by small-angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger Rg than glycosylated Fc, suggesting a more open C H2 orientation under these conditions. Moreover, the Rg of aglycosylated Fc was reduced by mutations at the CH2-CH3 interface (E382V/M428I), which confer highly selective binding to FcγRI and novel biological activities.

Original languageEnglish
Pages (from-to)1596-1602
Number of pages7
JournalACS Chemical Biology
Volume7
Issue number9
DOIs
Publication statusPublished - 2012 Sep 21
Externally publishedYes

Fingerprint

Glycosylation
Fc Receptors
Immunoglobulin G
Antibody-Dependent Cell Cytotoxicity
Cytotoxicity
Polysaccharides
Conformations
Immunoglobulin Fc Fragments
Antibodies
Bioactivity
X ray scattering
Escherichia coli
Leukocytes
X-Rays
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

Revisiting the role of glycosylation in the structure of human IgG Fc. / Borrok, M. Jack; Jung, Sang Taek; Kang, Tae Hyun; Monzingo, Arthur F.; Georgiou, George.

In: ACS Chemical Biology, Vol. 7, No. 9, 21.09.2012, p. 1596-1602.

Research output: Contribution to journalArticle

Borrok, MJ, Jung, ST, Kang, TH, Monzingo, AF & Georgiou, G 2012, 'Revisiting the role of glycosylation in the structure of human IgG Fc', ACS Chemical Biology, vol. 7, no. 9, pp. 1596-1602. https://doi.org/10.1021/cb300130k
Borrok, M. Jack ; Jung, Sang Taek ; Kang, Tae Hyun ; Monzingo, Arthur F. ; Georgiou, George. / Revisiting the role of glycosylation in the structure of human IgG Fc. In: ACS Chemical Biology. 2012 ; Vol. 7, No. 9. pp. 1596-1602.
@article{64dad76781db4ee786cd4a2a7a5dfedc,
title = "Revisiting the role of glycosylation in the structure of human IgG Fc",
abstract = "Binding of the Fc domain of Immunoglobulin G (IgG) to Fcγ receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fcγ receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an {"}open{"} Fc conformation recognized by Fcγ receptors. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 {\AA} resolution and observed significant disorder in the C'E loop, a region critical for Fcγ receptor binding, as well as a decrease in distance between the CH2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between CH2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (Rg) by small-angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger Rg than glycosylated Fc, suggesting a more open C H2 orientation under these conditions. Moreover, the Rg of aglycosylated Fc was reduced by mutations at the CH2-CH3 interface (E382V/M428I), which confer highly selective binding to FcγRI and novel biological activities.",
author = "Borrok, {M. Jack} and Jung, {Sang Taek} and Kang, {Tae Hyun} and Monzingo, {Arthur F.} and George Georgiou",
year = "2012",
month = "9",
day = "21",
doi = "10.1021/cb300130k",
language = "English",
volume = "7",
pages = "1596--1602",
journal = "ACS Chemical Biology",
issn = "1554-8929",
publisher = "American Chemical Society",
number = "9",

}

TY - JOUR

T1 - Revisiting the role of glycosylation in the structure of human IgG Fc

AU - Borrok, M. Jack

AU - Jung, Sang Taek

AU - Kang, Tae Hyun

AU - Monzingo, Arthur F.

AU - Georgiou, George

PY - 2012/9/21

Y1 - 2012/9/21

N2 - Binding of the Fc domain of Immunoglobulin G (IgG) to Fcγ receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fcγ receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an "open" Fc conformation recognized by Fcγ receptors. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 Å resolution and observed significant disorder in the C'E loop, a region critical for Fcγ receptor binding, as well as a decrease in distance between the CH2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between CH2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (Rg) by small-angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger Rg than glycosylated Fc, suggesting a more open C H2 orientation under these conditions. Moreover, the Rg of aglycosylated Fc was reduced by mutations at the CH2-CH3 interface (E382V/M428I), which confer highly selective binding to FcγRI and novel biological activities.

AB - Binding of the Fc domain of Immunoglobulin G (IgG) to Fcγ receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fcγ receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an "open" Fc conformation recognized by Fcγ receptors. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 Å resolution and observed significant disorder in the C'E loop, a region critical for Fcγ receptor binding, as well as a decrease in distance between the CH2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between CH2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (Rg) by small-angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger Rg than glycosylated Fc, suggesting a more open C H2 orientation under these conditions. Moreover, the Rg of aglycosylated Fc was reduced by mutations at the CH2-CH3 interface (E382V/M428I), which confer highly selective binding to FcγRI and novel biological activities.

UR - http://www.scopus.com/inward/record.url?scp=84867836578&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84867836578&partnerID=8YFLogxK

U2 - 10.1021/cb300130k

DO - 10.1021/cb300130k

M3 - Article

C2 - 22747430

AN - SCOPUS:84867836578

VL - 7

SP - 1596

EP - 1602

JO - ACS Chemical Biology

JF - ACS Chemical Biology

SN - 1554-8929

IS - 9

ER -