Building meaningful models of glycoproteins

To the Editor:

The building of crystallographic models of proteins is guided by understanding of the primary structure of proteins and by well-established and rigorously applied stereochemical principles. However, a cursory survey of Protein Data Bank entries containing oligosaccharides suggests that of the order of one-third of entries contain significant errors in carbohydrate stereochemistry, nomenclature or even consistency with the electron density maps. Many of the stereochemical errors can be detected by reference to conformational studies of glycans^1,2 and to publicly available resources (http://www.glycosciences.de/tools/). However, these errors also indicate that there is a wide discrepancy in the sophistication of building and validation tools available for protein and carbohydrate models.

An example of the difficulties that can be encountered when building crystallographic models of glycoproteins is the recent model proposed by Szakonyi et al.³ of the Epstein-Barr virus major envelope glycoprotein, EBV gp350. EBV gp350 was expressed in Spodoptera frugiperda Sf9 cells, and Szakonyi et al.³ report that they observed electron density corresponding to the oligosaccharide chains of fourteen N-linked glycosylation sites. The crystallization of such a heavily glycosylated glycoprotein is a notable achievement. However, the proposed model contains not only systematic errors in carbohydrate stereochemistry, presumably resulting from inadequate parameter files, but also hitherto unreported motifs in the primary structures of the glycans.

The previously undescribed glycosidic linkages and motifs that Szakonyi et al.³ propose include Man-(1→3)-GlcNAc and GlcNAc-(1→3)-GlcNAc linkages (of indeterminate anomericity) within the trimannosyl core, hybrid-type glycans containing a terminal Man-(1→3)-GlcNAc linkage on the 3-antennae, and β-galactosyl motifs capping oligomannose-type glycans. We suggest that, in the absence of supporting evidence, electron density at 3.5-Å resolution should not be used to support linkages incompatible with the known biosynthetic routes of N-glycan processing. One of the advantages of the expression of EBV gp350 in Sf9 cells is the restricted range of carbohydrate structures that are likely to be observed. Such information can guide model building. Sf9 cell glycosylation is dominated by oligomannose-type (Man_5–9GlcNAc₂) and paucimannose-type glycans (Man_2–3GlcNAc₂ with or without core fucose)⁴. The highly unusual Gal₂Man₇GlcNAc₂ glycans proposed by Szakonyi et al.³ could conceivably correspond to the underprocessed α-glucosylated oligomannose glycans derived from the highly conserved starting structure of glycosylation, Glc₃Man₉GlcNAc₂ (ref. 2), but these are rarely observed on mature secreted glycoproteins⁵. Finally, the building of such large glycans seems to be inadequately supported by the deposited structure factor data.

In the wake of advances in the production and crystallization of glycoproteins, we believe there should now be a wider discussion about the application and control of refinement standards applied to glycosylation. This requires more robust validation procedures for newly reported structures as well as a systematic evaluation of those previously described (scrutiny from which the authors of this letter are not exempt). We suggest that carbohydrate-specific building and validation tools, capable of guiding the construction of biologically relevant and stereochemically accurate models, should be integrated into popular crystallographic software. Rigorous treatment of the structural biology of glycosylation can only enhance the structural analysis of glycoproteins and our understanding of their functions.