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Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-N-acetylglucosamine labeling of immunoglobulin G1.

Barb AW - Biochemistry (2014)

Bottom Line: However, widely applicable methods do not yet exist.Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[(13)C,(15)N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry.Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

View Article: PubMed Central - PubMed

Affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.

ABSTRACT
Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure-activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [(13)C,(15)N]-N-acetylglucosamine (GlcNAc). UDP-[(13)C,(15)N]GlcNAc was synthesized enzymatically in a one-pot reaction from [(13)C]glucose and [(15)N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[(13)C,(15)N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [(13)C,(15)N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

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1H–13C HSQC spectra of IgG1Fc witha Man5 N-glycan following addition of [13C,15N]GlcNAc. (A) A 2D 1H–13C HSQC spectrum of the *N-Man5 N-glycan followingEndoF1-catalyzed hydrolysis is shown as gray contours. Blue contoursshow the positions of peaks from IgG1 Fc bearing a *N-Man5 N-glycan. Arrows indicate the direction of peak movementbecause of interactions with the Fc polypeptide. Peak labels thatcorrespond to a figure of β-linked GlcNAc are shown (inset)and refer to the carbon position of the 1H–13C peak. 1JC–C couplings are not resolved because of the limited resolution inthe 13C dimension. (B) 1D 13C-observe NMR spectrumof *N-Man5 Fc. 1JC–C values are indicated. (C) 2D 1H–15N HSQC spectra before and after N-glycanhydrolysis with the same colors used in panel A.
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fig5: 1H–13C HSQC spectra of IgG1Fc witha Man5 N-glycan following addition of [13C,15N]GlcNAc. (A) A 2D 1H–13C HSQC spectrum of the *N-Man5 N-glycan followingEndoF1-catalyzed hydrolysis is shown as gray contours. Blue contoursshow the positions of peaks from IgG1 Fc bearing a *N-Man5 N-glycan. Arrows indicate the direction of peak movementbecause of interactions with the Fc polypeptide. Peak labels thatcorrespond to a figure of β-linked GlcNAc are shown (inset)and refer to the carbon position of the 1H–13C peak. 1JC–C couplings are not resolved because of the limited resolution inthe 13C dimension. (B) 1D 13C-observe NMR spectrumof *N-Man5 Fc. 1JC–C values are indicated. (C) 2D 1H–15N HSQC spectra before and after N-glycanhydrolysis with the same colors used in panel A.

Mentions: NMR spectra of IgG1 Fc following Gnt1-catalyzedremodeling of the Man5 glycan using UDP-[13C,15N]GlcNAc revealed peaks for each 1H–13C and 1H–15N moiety (Figure 5). The peaks were relatively intense and narrow,considering the glycans are part of the ∼55 kDa Fc. This propertyindicates the presence of significant GlcNAc motion relative to thepolypeptide domains and is consistent with similar measurements ofgalactose and sialic acid residues on the 3–4–5 branchof the N-glycan [as opposed to the 3′–4′–5′branch (Figure 1)].12,25,341JC–C couplings from a one-dimensional (1D) 13C-observe experimentagreed with the resonance assignments based on an assignment of β-GlcNAc(Figure 5B). Similar spectra were observedat 25, 37, and 50 °C, and peak positions in duplicate sampleswere reproduced.


Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-N-acetylglucosamine labeling of immunoglobulin G1.

Barb AW - Biochemistry (2014)

1H–13C HSQC spectra of IgG1Fc witha Man5 N-glycan following addition of [13C,15N]GlcNAc. (A) A 2D 1H–13C HSQC spectrum of the *N-Man5 N-glycan followingEndoF1-catalyzed hydrolysis is shown as gray contours. Blue contoursshow the positions of peaks from IgG1 Fc bearing a *N-Man5 N-glycan. Arrows indicate the direction of peak movementbecause of interactions with the Fc polypeptide. Peak labels thatcorrespond to a figure of β-linked GlcNAc are shown (inset)and refer to the carbon position of the 1H–13C peak. 1JC–C couplings are not resolved because of the limited resolution inthe 13C dimension. (B) 1D 13C-observe NMR spectrumof *N-Man5 Fc. 1JC–C values are indicated. (C) 2D 1H–15N HSQC spectra before and after N-glycanhydrolysis with the same colors used in panel A.
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fig5: 1H–13C HSQC spectra of IgG1Fc witha Man5 N-glycan following addition of [13C,15N]GlcNAc. (A) A 2D 1H–13C HSQC spectrum of the *N-Man5 N-glycan followingEndoF1-catalyzed hydrolysis is shown as gray contours. Blue contoursshow the positions of peaks from IgG1 Fc bearing a *N-Man5 N-glycan. Arrows indicate the direction of peak movementbecause of interactions with the Fc polypeptide. Peak labels thatcorrespond to a figure of β-linked GlcNAc are shown (inset)and refer to the carbon position of the 1H–13C peak. 1JC–C couplings are not resolved because of the limited resolution inthe 13C dimension. (B) 1D 13C-observe NMR spectrumof *N-Man5 Fc. 1JC–C values are indicated. (C) 2D 1H–15N HSQC spectra before and after N-glycanhydrolysis with the same colors used in panel A.
Mentions: NMR spectra of IgG1 Fc following Gnt1-catalyzedremodeling of the Man5 glycan using UDP-[13C,15N]GlcNAc revealed peaks for each 1H–13C and 1H–15N moiety (Figure 5). The peaks were relatively intense and narrow,considering the glycans are part of the ∼55 kDa Fc. This propertyindicates the presence of significant GlcNAc motion relative to thepolypeptide domains and is consistent with similar measurements ofgalactose and sialic acid residues on the 3–4–5 branchof the N-glycan [as opposed to the 3′–4′–5′branch (Figure 1)].12,25,341JC–C couplings from a one-dimensional (1D) 13C-observe experimentagreed with the resonance assignments based on an assignment of β-GlcNAc(Figure 5B). Similar spectra were observedat 25, 37, and 50 °C, and peak positions in duplicate sampleswere reproduced.

Bottom Line: However, widely applicable methods do not yet exist.Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[(13)C,(15)N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry.Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

View Article: PubMed Central - PubMed

Affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.

ABSTRACT
Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure-activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [(13)C,(15)N]-N-acetylglucosamine (GlcNAc). UDP-[(13)C,(15)N]GlcNAc was synthesized enzymatically in a one-pot reaction from [(13)C]glucose and [(15)N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[(13)C,(15)N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [(13)C,(15)N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

Show MeSH