How and why plants and human N-glycans are different: Insight from molecular dynamics into the “glycoblocks” architecture of complex carbohydrates

Abstract

N-glycosylation is one of the most abundant and diverse post-translational modifications of proteins, implicated in protein folding and structural stability, and mediating interactions with receptors and with the environment. All N-glycans share a common core from which linear or branched arms stem from, with functionalization specific to different species and to the cells' health and disease state. This diversity generates a rich collection of structures, all diversely able to trigger molecular cascades and to activate pathways, which also include adverse immunogenic responses. These events are inherently linked to the N-glycans 3D architecture and dynamics, which remains for the large part unresolved and undetected because of intrinsic structural disorder. In this work we use molecular dynamics (MD) simulations to provide insight into N-glycans 3D structure by analysing the effects of a set of very specific modifications found in plants and invertebrate N-glycans, which are immunogenic in humans. We also compare these structural motifs and combine them with mammalian Nglycans motifs to devise strategies for the control of the N-glycan 3D structure through sequence. Our results suggest that the N-glycans architecture can be described in terms of the local spatial environment of groups of monosaccharides. We define these "glycoblocks" as self-contained 3D units, uniquely identified by the nature of the residues they comprise, their linkages and structural/dynamic features. This alternative description of glycans 3D architecture can potentially lead to an easier prediction of sequence-to-structure relationships in complex carbohydrates, with important implications in glycoengineering design.

The oligomannose N-glycans 3D architecture and its response to the FcγRIIIa structural landscape

“…We used conventional MD simulations, run in parallel for 500 ns from nine uncorrelated starting points 16,17 , to characterize the 3D structure and dynamics of human oligomannose Nglycans, when unlinked, see Man5 is the simplest oligomannose found in vertebrates and the substrate of GlcNAc transferase I (GnTI), responsible to start the N-glycan complex functionalization in the Golgi 6 . As found for complex biantennary N-glycans 16,17 the Man5 chitobiose core and the following Manβ(1-4)-GlcNAc linkage are rigid with only one conformation significantly occupied, see Figure 2 and Table S.1, while the (1-3) arm adopts an outstretched conformation with flexibility in a range of 40° around the psi torsion angle, see Figure 2 and Table 1. The Man5 (1-6) arm has a relatively more complex dynamics, hinging around the preferential 'open' conformation 16,17 , populated at 82%, where the Manα(1-3)-Man branch can be orientated towards the front of the page and the Manα(1-6)-Man branch towards the back of the page, or vice versa.…”

“…As an example, we have shown how the sequence (and branching) of complex N-glycans determines the 3D structure, which in turn drives their recognition 16,17 . In this work we extend our dataset of free (unlinked) N-glycans structures to the vertebrate oligomannose type, where, as shown in Figure 1, the common pentasaccharide unit, i.e.…”

“…Indeed, conformational sampling through conventional and/or enhanced molecular dynamics (MD) schemes allows us to characterize the dynamic behaviour of different glycoforms at the atomistic level of details. Within this context, for the past few years our lab contributed to the knowledge of N-glycans dynamics by providing information on their 3D architecture and relative flexibility from extensive MD-based conformational sampling 16, 17 . As an example, we have shown how the sequence (and branching) of complex N-glycans determines the 3D structure, which in turn drives their recognition 16, 17 .…”

“…Within this context, for the past few years our lab contributed to the knowledge of N-glycans dynamics by providing information on their 3D architecture and relative flexibility from extensive MD-based conformational sampling 16, 17 . As an example, we have shown how the sequence (and branching) of complex N-glycans determines the 3D structure, which in turn drives their recognition 16, 17 . In this work we extend our dataset of free (unlinked) N-glycans structures to the vertebrate oligomannose type, where, as shown in Figure 1 , the common pentasaccharide unit, i.e.…”

“…N-glycan coloured according to the SNFG convention.DiscussionIn this work we analysed the 3D structure and dynamics of human oligomannose N-glycans, from Man5 to Man9, when free (unlinked) in solution and also determined how the effect of FcγRIIIa (CD16a) surface landscape modulates their structural equilibria. Despite similarities with complex N-glycans16,17 , both in terms of the core chitobiose rigidity and of the relatively low degree of flexibility of the (1-3) arm, oligomannoses have a very unique architecture, which changes with the progressive functionalization of the arms. More specifically, Man5 shows a clear propensity for an 'open' structure, where the (1-6) arm is outstretched orientating the two branches on either side of the (1-3) arm, see Figures 2 and Small variations of the open structure, determined by two accessible values of the (1-6) arm omega torsion are also populated, see Figure 3, and of the additional degrees of freedom of both (1-3/6) branches, which closely reflect the dynamics of the arms, see Table 1.…”