N-glycan microheterogeneity regulates interactions of plasma proteins

Abstract

Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein-protein, or protein-small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin-hemoglobin (Hp-Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp-Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein-drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.

Keywords: glycoprotein; mass spectrometry; protein interactions.

Fig. 1.

Native MS analysis of asialo-AGP. (A) Native mass spectrum of asialo-AGP. AGP structure with five highly branched N-glycans at Asn15, Asn38, Asn54, Asn75, and Asn85 are shown. The monosaccharide residues are labeled according to the Consortium for Functional Glycomics guidance (blue for GlcNAc, yellow for Gal, and green for Man). (B) A zero-charged native mass spectrum of asialo-AGP is constructed using UniDec. Each peak is assigned to the corresponding glycan composition (SI Appendix, Table S1). Peaks with the same hexose (Hex) and N-acetylhexosamine (HexNAc) numbers are labeled P0 to P9. The number of fucose residues assigned to each peak (Fuc₀ to Fuc₄) is denoted with colored arrows. The presence of an “uncapped” GlcNAc residue on AGP is denoted with a red asterisk (*). This may arise either from incomplete processing of AGP, escaping from the N-glycosylation biosynthesis pathway, or from degradation AGP in vivo and/or in vitro.

Fig. 2.

Resolving the microheterogeneity of asialo-Hp. (A) Native mass spectrum of asialo-Hp. Hp structure with four biantennary N-glycans at Asn180, Asn203, Asn207, and Asn237 at each Hp β-subunit is shown. (B) A zero-charged native mass spectrum of asialo-Hp. Each peak is assigned to the corresponding glycan composition (SI Appendix, Table S2). Peaks with the same Hex and HexNAc numbers are labeled P0 to P9. The number of fucose residues assigned to each peak (Fuc₀ to Fuc₄) is denoted with colored arrows.

Fig. 3.

Native MS analysis of asialo-AGP and warfarin binding. (A) Native mass spectra of asialo-AGP in 1% DMSO and asialo-AGP with 50 µM warfarin in 1% DMSO. Warfarin binding to asialo-AGP results in a mass shift of 308.33 Da. Unfucosylated P3F0/P4F0 and monofucosylated P3F1/P4F1 are labeled as blue and red peaks, respectively. Warfarin-bound asialo-AGP peaks do not overlap with apo-asialo-AGP peaks. (B) Plots of the warfarin concentration to the relative abundances of warfarin-bound unfucosylated (P1F0 to P8F0) and monofucosylated (P1F1 to P8F1) glycoproteoforms. The abundance of each peak was normalized to the total abundance of P4F0 form (P4F0 unbound and bound peaks). Dissociation constants for each glycoproteoform were calculated by a one-site specific binding model. Error bars represent the SE of three replicate experiments. (C) Bar graph of dissociation constants of unfucosylated (P1 to P8) and monofucosylated (P1 to P8) glycoproteoforms show that fucosylation and N-glycan branching elevate dissociation constant of asialo-AGP to warfarin. (D) AGP structure with warfarin docking to the hydrophobic cavity. N-glycans on Asn38 and Asn75 are close to the hydrophobic cavity for drug binding.

Fig. 4.

Native MS analysis of asialo-Hp and Hb interactions. (A) Hp–Hb complex structure. The N-glycans on Asn180 and Asn203 are proximal to the Hp–Hb interaction interface. Native mass spectra of asialo-Hp and asialo-Hp–Hb complex (SI Appendix, Fig. S6) are deconvoluted to zero-charged spectra (B and C) using UniDec. The unfucosylated (blue) and monofucosylated (red) peak series of fully glycosylated Hp and the corresponding peak series of Hp–Hb complex with the same glycan compositions are assigned and labeled as P1 to P7. Their relative abundances are extracted and normalized. The ratios of their relative abundances are plotted as bar graph (D). Error bars represent the SE of three replicate experiments. (E) The spectra of gas-phase dissociated Hp–Hb complexes from which one βHb has been removed.

Fig. 5.

The schematic illustration of glycosylation regulation of glycoprotein–drug/protein interactions. N-glycan branching and fucosylation may introduce steric hindrance to the warfarin binding pocket inhibiting AGP–drug binding. N-glycan branching attenuates Hp–Hb binding affinity. On the contrary, fucosylation stabilizes the Hp–Hb complex.