The carbohydrate chains that modify proteins play key roles in a variety of physiological and pathological functions, such as intercellular communications, viral infections and immune reactions [1]. These biological processes involve molecular recognition mediated by carbohydrate–protein and carbohydrate–carbohydrate interactions, which can be potential therapeutic targets [2,3,4,5]. In addition to these extracellular events, accumulating evidence has indicated that high mannose-type oligosaccharides displayed on proteins are actively involved in glycoprotein fate determination processes in cells, such as folding, translocation and degradation, through interactions with a series of intracellular lectins [6,7,8]. In the endoplasmic reticulum (ER), a nascent polypeptide chain is covalently linked to the triantennary oligosaccharide, Glc₃Man₉GlcNAc₂ (G3M9), which is subsequently trimmed by glucosidases and mannosidases to result in a variety of high mannose-type oligosaccharides. These processing intermediates exhibit specific glycotopes that are recognized by intracellular lectins functioning as molecular chaperones and cargo receptors and by those involved in ER-associated protein degradation. Namely, the glycotopes as fate determinants of glycoproteins are embedded in the triantennary structure of G3M9 and exposed with trimming by the ER enzymes.

The recently emerged crystal structures of the intracellular lectins complexed with oligosaccharides have provided atomic pictures demonstrating the mechanisms by which glycotopes are recognized by the lectins [8]. In general, however, the oligosaccharides have significant degrees of internal motions [9]. For the quantitative evaluation of the energetics of carbohydrate–lectin interactions, it is essential to understand the conformational dynamics of the oligosaccharides in their free forms, as well as in their lectin-bound forms.

NMR spectroscopy is undoubtedly a powerful method to provide atomic information on the conformations, dynamics and interactions of oligosaccharides in solution. However, NMR studies of oligosaccharide structures remain challenging compared to those of proteins, because of the poor spectral resolutions resulting from the small variations in the functional groups and the lack of a recombinant technique to produce homogenous glycoforms in sufficient quantities [10]. To overcome these difficulties, we have recently developed a methodology for the overexpression of a homogeneous high mannose-type oligosaccharide with ¹³C labeling with a genetically engineered yeast strain [11].

In the ER of yeast, G3M9 is trimmed by glycosidases I and II, resulting in the Man₉GlcNAc₂ (M9) glycoform, which is then converted to the Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc glycoform (abbreviated as M8B) by the removal of the terminal mannose residue (D2) of the central branch by the action of ER α-mannosidase [12] (Figure 1). Subsequently, glycoproteins exhibiting the M8B glycan are transported from the ER to the Golgi, where the glycan is hypermannosylated by α1-2-, α1-3- and α1-6-mannosyl transferases and phosphorylated by mannosylphosphate transferase [12]. Therefore, the M8B glycoform could exclusively accumulate in yeast by knocking out the genes encoding α1-3- and α1-6-mannosyl transferases (termed MNN1 and OCH1, respectively), along with MNN4, which encodes a positive regulator of mannosylphosphate transferase [13,14]. By metabolic labeling with this engineered yeast strain, we have successfully prepared high yields of uniformly or site-selectively ¹³C-labeled M8B oligosaccharides of suitable homogeneity [11].

In the present study, we extended this methodology to the production of isotopically labeled M9 oligosaccharide. By comparing the NMR spectral data of ¹³C-labeled M8B and M9 collected at higher magnetic fields beyond 900 MHz of the proton observation frequency, the conformational impact of the removal of the mannose residue was examined at an atomic level.

In the present study, we successfully extended the yeast-engineering methodology to produce ¹³C-labeled M9 for detailed NMR analyses. By inspecting the ¹³C-edited NOE data, the interactions between the outer branches and the inner core were identified in M8B and M9, the former of which, however, exhibited the foldback conformation more extensively.

In their pioneering works [17,18], Vliegenthart and coworkers implied the existence of a foldback conformation in the M9 derivative lacking the innermost GlcNAc residue, based on the anomerization effects of reducing terminal GlcNAc2 on the anomeric proton chemical shifts of ManD2 and ManA, observed as their peak splitting. A more pronounced peak splitting was reported for ManA in the M8B derivative without GlcNAc1, suggesting that the removal of ManD2 exposes ManA, thereby allowing its interaction with the core GlcNAc2 residue [19]. These data are qualitatively consistent with the present NOE data obtained with the PA derivatives of M8B and M9.

The foldback conformation was also identified in the M8B glycan attached to ribonuclease B by observing a long-range NOE between ManA-H₁ and GlcNAc2-Ac, which was also observed in the present study [20]. Similarly, the heterogeneous high mannose-type N-glycans[(Man)_nGlcNAc₂, n = 5–8] displayed on a soluble form of human CD2 (shCD2) expressed in CHO cells showed long-range NOE connectivities between ManA-H₁/H₂ and GlcNAc2-Ac, which, however, were not observed when ManD2 was present [21,22]. Here, we did not detect the NOE between ManA-H₂ and GlcNAc2-Ac in M8B (Figure 5), and this was probably because the NOE observed in shCD2 originated from its major glycoforms, such as M7 (40%) and M6 (34%) [21]. On the other hand, the undetectability of the long-range ManA-GlcNAc NOEs in the presence of ManD2 was either because of the low abundance of the D2-containing glycoform (6%) or a conformational difference [21]. We detected no NOE between ManD2 and GlcNAc2 in M9 (Figure 4a), supporting a gross conformational rearrangement upon the removal of the terminal D2 residue.

The conformational rigidity of M9 has been proposed by inspecting NMR-derived local conformational restraints, such as interglycosidic NOEs and scalar couplings [23,24] and MD simulations in explicit water [25], while in vacuo MD simulations have suggested considerable variabilities of the glycosidic angles of M9 [26]. In particular, 1-ns MD simulations in water showed that M9 exhibited conformational fluctuation within a restricted conformational space with maintenance of an extended overall conformation in which several strongly coordinated water molecules mediated interbranch hydrogen bonds [25]. Consistently, our ¹³C-edited NOE data identified NOE connectivities among the outer branches of M9 (Figure 5 and Table S2). However, it should be noted that the present study also indicated intramolecular interactions between the outer branch and core of M9 associated with several long-range NOE connectivities (Figure 5), which could not be interpreted solely based on the ensemble of extended conformations. Because NMR parameters, including NOE, provide an average picture of a dynamic conformational ensemble, the long-range NOE connectivities are supposed to reflect transient, minor and foldback conformations assumed by M9, which might be barely detected in the MD simulation that is conducted for a limited period. Such conformational variabilities of M9, as well as of M8B, might be significantly associated with their affinities and specificities for the intracellular lectins and glycan-trimming enzymes.

The method of ¹³C labeling combined with high-field NMR spectroscopy has now opened new opportunities for detailed conformational analyses of oligosaccharides. In this study, we successfully observed extensive datasets of long-range NOE connectivities in M8B and M9, which will enable experimental validation of computational simulations for the characterization of dynamic conformational ensembles of these oligosaccharides. To improve the accuracy of the validation, additional experimental data are necessary as sources of long-range geometric information. In previous studies, we demonstrated that pseudocontact shifts observed by paramagnetic lanthanide tagging provide long distance information for validating the conformational ensemble of oligosaccharides derived from MD simulations [37,38]. Paramagnetic relaxation enhancement and residual dipolar coupling are also useful for characterizing the conformational dynamics of oligosaccharides [39,40]. The stable-isotope-labeling technique in conjunction with higher magnetic fields will offer a technical basis to facilitate the collection of these NMR datasets for the elucidation of the conformational dynamics of the high mannose-type oligosaccharides in their free and lectin-bound forms.