Intramolecular Glycan–Protein Interactions in Glycoproteins

C H A P T E R

E I G H T E E N

Intramolecular Glycan–Protein Interactions in Glycoproteins Adam W. Barb,* Andrew J. Borgert,† Mian Liu,* George Barany,‡ and David Live* Contents 365 367 374 382 382

1. Introduction 2. O-Linked Glycoproteins 3. N-Linked Glycoproteins Acknowledgments References

Abstract Glycoproteins are a major class of glycoconjugates displaying a variety of mutual interactions between glycan and protein moieties that ultimately affect molecular organization. Modulation of the pendant glycan structures is important in tuning the functions of glycoproteins. Here we discuss structural aspects and some of the challenges to studying intramolecular interactions between carbohydrate and protein elements in several forms of O-linked as well as Nlinked glycoproteins. These illustrate the importance of the relationship of context to function in protein glycosylation.

1. Introduction The challenges of glycomics are formidable, considering the diversity of oligosaccharides that arise from the large number of constituent determinants (Cummings, 2009). In addition, carbohydrates are often found in combination with other molecular structures, further elaborating the complexity (van Kooyk and Rabinovich, 2008). Glycoproteins may represent the most diverse category of glycoconjugates, particularly in light of the estimate that over half of all mammalian proteins carry glycans (Apweiler * Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA Center for Magnetic Resonance Research, University of Minnesota, Minneapolis, Minnesota, USA Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA

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Methods in Enzymology, Volume 478 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)78018-6

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et al., 1999). Further, the glycans associated with a specific protein may vary over time with physiological states of cells and tissues (Ohtsubo and Marth, 2006). The intricate processes of posttranslational glycosylation in both assembly and remodeling of the glycans also introduce a degree of microheterogeneity in natural mature glycoproteins even when these are isolated from a single source (Rich and Withers, 2009). The complex mixtures obtained from natural sources have presented difficulties in studies of glycoproteins and in understanding the intramolecular relationships between carbohydrate and protein moieties in glycoproteins. Such associations do, however, have an impact on the distinct properties and functions of a glycoprotein, particularly their recognition, and therefore on functional glycomics. In this chapter, we address aspects of these intramolecular relationships. The increasing interest in conformational aspects of glycopeptides and glycoproteins is reflected in a recent review on the subject (Meyer and Moller, 2007). For the most part, the glycosylation modifications of proteins fall into two classes, N-linked, where the glycan is joined to the protein through the side chain amide of an Asn residue, or O-linked, using the hydroxyl of a Ser or Thr residue (Varki et al., 2009). The N-linked glycosylation occurs cotranslationally through the transfer of a preassembled common oligosaccharide core to the side chain Asn nitrogen, in the consensus sequence Asn-Xaa-Ser/Thr (where Xaa is any residue except Pro) on the growing polypeptide chain, connected through a b-GlcNAc residue. Thus, the residues closest to the linkage site are constant, even though the more distal residues can vary. O-linked glycans are assembled in a stepwise manner on the protein, and are more varied in protein sequence context, in linking glycan, and in glycan composition. Mucin glycosylation is initiated with an a-O-GalNAc (Ten Hagen et al., 2003), although the importance of Oglycans based on other linkages, such as a-O-Man (Barresi and Campbell, 2006; Chai et al., 1999) and modifications of a single b-O-GlcNAc (Whelan and Hart, 2006), has been recognized recently. Proteoglycans are also a significant class of O-linked glycoproteins, characterized by long carbohydrate chains linked to the protein backbone through a xylose, with the proportion of long carbohydrate polymers overwhelming the protein. Due to the absence of significant three-dimensional structural data for this latter class, this will not be discussed here. The significant chemical structural differences of the two major types of linkages impact the intramolecular interactions between glycan and protein components. For N-linked glycans, the point of linkage is three bonds removed from the polypeptide backbone. Additionally, the b stereochemistry of the glycosidic linkage directs the glycan away from the backbone, decreasing the contact between glycan and protein components in the immediate vicinity of the modification. Sites of N-linked glycosylation tend to be widely dispersed. In contrast, the linkage point for O-linked

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glycans is closer to the backbone, only two bonds removed, and for those with a-glycosidic linkages, the initiating residue lies proximal to the peptide backbone, facilitating intimate interactions. Additionally, for mucin-like Olinked glycosylation there are often numerous and neighboring sites of modification, which can amplify the effects of the glycan–polypeptide intramolecular backbone interactions. The recognized high-resolution structure determination techniques for identifying intramolecular interactions are crystallography and nuclear magnetic resonance (NMR). Generally, glycoyslation is considered detrimental to protein crystallization (Lee et al., 2009), and often efforts are made to remove or remodel glycans to either eliminate this concern, or, particularly in the case of N-linked structures, to minimize the heterogeneity by trimming back the glycans (Lee et al., 2009; Rich and Withers, 2009). For mucins, the high density of glycosylation would only further compound this, and may explain the lack of crystallographic data on these molecules. Solution state NMR, though, has proven to be an effective tool in examining carbohydrate structures on glycoproteins (Meyer and Moller, 2007). The method also offers a distinct advantage in directly accessing the dynamics of the structures which reflect both the intramolecular and intermolecular interactions of glycoproteins.

2. O-Linked Glycoproteins The significance of intramolecular interactions in affecting the properties of native mucin O-linked glycoproteins at a global scale was realized early on from a variety of biophysical studies. Extended structures were visualized in electron micrographs of glycosylated mucin domains (Rose et al., 1984). Comparison of glycosylated and deglycosylated mucins using NMR (Gerken and Jentoft, 1987; Gerken et al., 1989) and light scattering techniques (Shogren et al., 1989) demonstrated the organizational consequences of the a-O-GalNAc modification, insofar as an extended and more rigid organization was observed. While the structural properties of mucin domains differ from those of globular proteins, they do present ordered structures that dictate the dispositions of their glycans. Intramolecular interactions are responsible for this organization, with significant implications for recognition of their glycans. This affects cellular signaling through cellsurface glycoproteins, with CD43 and CD45 being two examples (Garner and Baum, 2008). Studies on natural material have provided insights into the larger scale conformational aspects of mucins. However, the intrinsic natural high molecular weight and microheterogeneity render such material problematic for high-resolution structural analysis that would enable elucidation of the detailed intramolecular interactions giving rise to the conformational features. An exception to this is the highly regular fish antifreeze

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mucin glycoprotein (AFGP), which has a repeating triad of amino acids, predominantly AAT with some PAT, and with the T residues glycosylated. It has been possible to isolate a fraction of modest molecular weight from natural AFGP preparations, making its study tractable (Bush and Feeney, 1986; Lane et al., 1998). Given the general complications associated with accessing a broader range of structures from natural material, peptide synthesis methodology has emerged as an attractive and important alternative (Buskas et al., 2006). This has permitted the preparation of glycopeptides, particularly those bearing short glycans, with a wide variety of defined amino acid sequences and patterns of glycosylation. The extension of conventional peptide synthesis methodology to O-linked glycopeptides requires additional considerations in preparing glycosylated building blocks before assembly (Buskas et al., 2006), and in the deprotection of the sugar hydroxyls at the final stages of synthesis. These considerations have been successfully addressed (e.g., Liu et al., 2005, 2008), and recent advances employing microwave-assisted solid-phase peptide synthesis techniques are further enhancing glycopeptide synthesis efficiency (Matsushita et al., 2006). With the findings that the initial S- or T-linked GalNAc residues dominate the intramolecular interactions organizing mucin glycopeptides (Coltart et al., 2002), as discussed below, this synthetically most accessible and simplest mucin form is an effective model for investigating the intramolecular interactions and the core glycopeptide scaffold. While the minimal S/T-a-O-GalNAc element, or Tn antigen, is not normally revealed in humans, it is found on cell surfaces of tumor cells associated with aberrantly glycosylated mucins, and is correlated with a poor clinical prognosis. This has generated interest in the Tn glycopeptides (Springer, 1997). Mucin structures with more complex glycans have been successfully prepared using chemical and/or enzymatic approaches for elaborating the carbohydrates (Matsushita et al., 2006; Tarp et al., 2007). Although many conformational questions regarding mucin motifs can be addressed with modest sized fragments, application of native chemical ligation (NCL) methods has offered significant advances for exploring larger segments (Kan and Danishefsky, 2009; Payne and Wong, 2010). In considering a glycopeptide-based strategy for studying mucin glycoproteins, the potential absence of native tertiary interactions in studying short segments is a concern. Mucins, however, adopt extended conformations which preclude long-range tertiary interactions with sequentially remote regions in the native glycoprotein. Therefore, the same local interactions dominate the conformations of the motifs, both as parts of the native structures and as isolated short segments of these glycoproteins. Thus, mucin glycopeptides are expected to provide realistic structural models for components of the native glycoprotein. As elaborated below, these short segments display the features that are consistent with

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those of the global arrangement of glycosylated mucins. Thus, glycopeptides are valuable tools for studying O-linked structures. NMR studies on AFGP provided interatomic distance information from a number of nuclear Overhauser effect (NOE) interactions and bond angles based on proton J couplings. These, along with conformational energy calculations, provided a model for the glycosylated AT*AA sequence in the AFGP, revealing an extended and stabilized structure for the motif consistent with the larger organization of mucins (Lane et al., 1998). The AFGP studies also provided evidence for the existence of a hydrogen bond between the GalNAc amide and the carbonyl of the Thr residue to which it is attached (Mimura et al., 1992). Extension to examples with clusters of immediately adjacent sites of glycosylation, based on synthetic segments of mucins from glycophorin (Schuster et al., 1999) and MUC7 (Naganagowda et al., 1999), have also been reported. In these latter cases, a twisted extended structure of the backbone for the glycosylated forms was found, with an indication that the peptide backbone structure showed characteristics of a polyproline II helix. Combined synthetic and NMR efforts facilitated a systematic analysis of a series of glycopeptide constructs based on an Nterminal motif from the cell surface glycoprotein CD43, S*T*T*AV, where the asterisks denote glycosylation (Coltart et al., 2002). Three constructs were prepared with conventional mucin a-linked glycans of increasing complexity, GalNAca (Tn), Galb1-3GalNAca (T), and Galb1-3 (Neu5Aca2,6)GalNAca (ST). The ST glycan has been associated with CD43 in acute myelogenous leukemia (Fukuda et al., 1986). For the glycopeptide core, consisting of the GalNAc residues and the peptide, numerous NMR NOE contacts, including between the proximal sugar and peptide, were observed. These provided internuclear distance relationships (Coltart et al., 2002). The distances, along with J coupling parameters that relate to bond torsion angle, provided an extensive set of constraints for structure calculations. Key features, largely invariant with the size of the attached glycan, are immediately evident from these parameters. The HN to Ha 3J couplings are large, supporting an extended backbone structure. The numerous NOE interactions between the proximal GalNAc and the peptide backbone show interactions for this sugar, while there are a lack of NOE interactions between the peripheral sugar residues and the core glycopeptide, indicating that they are not in intimate contact with the peptide. Using the NMR constraints, structural refinement calculations were carried out with the Xplor program (Schwieters et al., 2006), and resulting coordinates for structures fitting the experimental constraints are available from the Protein Data Bank database (http://www.rcsb.org/pdb) entry 1kyj. The tightly clustered family of accepted structures is consistent with an extended, stable, and well-ordered structure. An important feature of Thr residues with a-O-GalNAc attached is the small 3J coupling between the Thr Ha and Hb. This limits the range of allowed torsion angles between

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these C–H bonds to values either close to 90
or 270
, with the former confirmed by NOEs. The structures showed a spacial relationship for the Nacetyl amide of the three glycosylated residues with both the glycosidic and carbonyl oxygens of their respective amino acid residues that indicate hydrogen bonding interactions. The existence of such an interaction is independently supported by the slow exchange rates for the N-acetyl amide protons (Coltart et al., 2002), also seen by others (Lane et al., 1998). The structure also reveals hydrophobic interactions between the N-acetyl methyl groups and side chain methyl groups on the iþ2 reside that would aid in promoting and propagating the extended structure down the polypeptide chain. In the absence of glycosylation, NMR results for the naked peptide itself suggest considerable conformational flexibility, typical of short peptides in general. NMR relaxation parameters, NOE, T1, T1r, and T2, are linked directly to molecular dynamics (Cavanaugh et al., 2007; Ishima and Torchia, 2000), and these have been used to verify the conclusions above by 13C relaxation measurements of the tri-Tn-S*T*T*AV glycopeptide listed in Table 18.1 (D. Live, unpublished results). The S2-order parameters extracted from these measurements were derived with the extended model free analysis using the program Modelfree (Mandel et al., 1995). S2 has a maximum value of 1 when there is no local motion, and values of 0.6 can be related to angular variations of only 30
(Ishima and Torchia, 2000), indicating that at most there is only a limited range of segmental motion for this glycopeptide core, in keeping with a well-defined conformation. Such effects have been observed in mucin glycopeptides by others (Grinstead et al., 2002). These data provide direct evidence that the glycosylated core, comprising the three glycosylated amino acids and their attached GalNAcs, largely behave as a single conformational entity. The existence of well-defined structure has important implications, since this establishes the relative orientations of the glycans: a feature which is relevant to recognition of mucin motifs on specific glycoproteins. Table 18.1 13C NMR relaxation parameters, T1 (s), T2 (s), and NOE measured at natural abundance using proton detected 2D 1H-13C experiments (Cavanaugh et al., 2007), and the derived order parameter S2 for sites in the (Tn)3 S*T*T*AV moleculea

T1 T2 NOE S2 a

S1a

T2a

T3a

A4a

T2b

T3b

S1GC1

T2GC1

T3GC1

0.41 0.25 1.53 0.69

0.36 0.20 1.35 0.83

0.37 0.21 1.36 0.81

0.43 0.27 1.56 0.61

0.37 0.16 1.29 0.88

0.36 0.19 1.32 0.90

0.41 0.26 1.54 0.67

0.40 0.21 1.39 0.76

0.41 0.24 1.43 0.71

S1GC1, T2GC1, and T3GC1 refer to the anomeric carbon sites on the GalNAc residues associated with the respective amino acid residues. Measured at 18.8 T, 800 MHz 1H frequency.

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Additionally, NMR residual dipolar couplings (RDCs) (de Alba and Tjandra, 2002) for this construct have been determined (D. Live and A. Borgert, unpublished results) in a weakly aligning cetylpyridinium bromide (CPBr)/hexanol/sodium bromide liquid crystalline medium (Barrientos et al., 2000). These provided additional structural constraints, independent of the earlier data, and were consistent with the original structure. Further refinement of the structure with these values resulted in only minor structural adjustments (Fig. 18.1). The consistency of these data with the earlier structure, and the sensitivity of the RDCs to motion over a broad range of frequencies, further support the contention that this glycopeptide does not undergo major conformational fluctuations. The effects of variation in local density of glycosylation on conformation have been explored in a MUC2-derived sequence, PTTTPLK, which is known to be a substrate for polypeptide GalNAc transferases (Takeuchi et al., 2002). Constructs were synthesized (Liu et al., 2005) with all permutations of the pattern of glycosylation by a-O-GalNAc on the threonine residues, and NMR studies were carried out. The structures of these molecules were computed from NOE and J coupling NMR experimental restraints (A. Borgert, M. Liu, G. Barany, and D. Live, unpublished results). For those with two or three substitutions, RDC values in didodecyl/ dihexyl-phosphatidylcholine media (Ottiger and Bax, 1999) were determined and used as well. In this triplet motif, the organization around the respective individually glycosylated Thr residues are largely unchanged relative to the peptide backbone, as neighboring sites are glycosylated (Fig. 18.2). With this increasing density of glycosylation, the extent of segmental motion becomes more restricted, reflecting the increased carbohydrate–peptide interactions. Interestingly, the core structure with all three Thr residues glycosylated overlays the analogous glycosylated S*T*T* segment of the earlier construct quite well, with preservation of the associated interactions, suggesting a consistent triplet structural motif and the relative lack of sensitivity to Ser versus Thr residue at the N-terminal position. T3

S1 T2

Figure 18.1 Overlay of the closest to the average of the families of structures of the S*T*T* segment of (Tn)3 S*T*T*AV determined without RDC constraints, sticks, and with RDC constraints included, lines.

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PT*TTPLK

PT*TPLK

PTTT*PLK

PT*T*T*PLK

Figure 18.2 The closest to the average for the family of structures for each of the three PTTTPLK constructs with a single site of a-O-GalNAc glycosylation, and the one with all three sites glycosylated.

There have been several structural studies on peptides and mucin glycopeptides from the tandem repeat of MUC1 (Dziadek et al., 2006; Grinstead et al., 2002; Kirnarsky et al., 2000). This glycoprotein has been the focus of attention since it is overexpressed with aberrant glycosylation, particularly the Tn epitope, in tumor cells (Dziadek et al., 2006). The most detailed study is an NMR analysis of a glycosylated form of the full tandem repeat element GSTAPPAHGVTSAPDTRPAP with a single ST epitope at Thr11, as recently reported (Dziadek et al., 2006). The NOE contacts and 3J coupling parameters, as well as structural features in the vicinity of the single glycoyslation site, are quite consistent with those previously published findings for glycosylated amino acids in the clustered S*T*T*AV structure (Coltart et al., 2002). These include contacts between the N-acetyl methyl group and the iþ2 Ala Me, and a 2.5 A˚ distance between the N-acetyl N and the Thr11 carbonyl oxygen, supporting a direct intramolecular interaction through a hydrogen bond. A number of mucin structures reported are consistent with direct hydrogen bonding between a GalNAc amide and the peptide backbone, for example, AFGP (Mimura et al., 1992), MUC1 glycopeptide (Dziadek et al., 2006), MUC2 glycopeptides, and S*T*T*AV (Coltart et al., 2002) glycopeptide. However, on the basis of NMR and molecular dynamics simulations of isolated C- and N-capped a-O-GalNAc-Ser and Thr amino acids and dipeptides composed of Ser and Thr residues, questions have been raised about their existence in other contexts (Corzana et al., 2006b, 2007,

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2009). In these instances, solvent-mediated hydrogen bonds have been proposed. The orientations and interatomic distances of the hydrogen bonding functional groups found in the larger glycopeptides would seem to preclude insertion of a water molecule, as do the reduced amide exchange rates observed in the larger systems. While direct hydrogen bonding may not be the case in all instances of larger glycopeptides, it would appear that the amino acid residues immediately adjacent to the glycosylation sites can play important roles both in restricting the local conformation and on local solvation. Thus, very small models may be of too limited size to accurately reflect all of the interactions between carbohydrate and peptide components in more native-like mucin systems. The differential response to a versus b stereochemistry at GalNAc glycosidic linkages to the amino acid can be deduced directly from comparison of the (a-T)3 S*T*T*AV and the (b-T)3 S*T*T*AV constructs (Coltart et al., 2002), and provides further evidence for the role of specific interactions on the mechanism of structural stabilization. The reduced number of glycan to peptide NOEs, the 3J coupling values, and poorer dispersion of the peptide amide chemical shifts in the case of the b-linkage relative to the a-linkage, as well as reduced amide proton exchange lifetimes, suggest diminished interactions between the carbohydrate and peptide components. These parameters are further consistent with dynamic averaging of multiple conformational states. The increased conformational lability is consistent with the fact that the b-linkage redirects the GalNAc residue away from the peptide, largely disrupting the interactions among functional groups of the two moieties found in the a-linked forms. While only the a-linkage occurs in conventional mucins, an analogous b-O-linked GlcNAc modification occurs naturally as a sparsely distributed transient regulatory modification on some cytosolic proteins (Whelan and Hart, 2006). Investigation of N- and C-terminal capped Ser/Thr-b-O-GlcNAc models are consistent with the sugar residue being oriented away from the backbone, and with increased flexibility (Corzana et al., 2006a). This modification has also been studied on a synthetic b-O-GlcNAc glycopeptide from RNA polymerase II, where a site of b-O-GlcNAc attachment has been identified (Simanek et al., 1998). NOE interactions with the peptide backbone are lacking here, consistent with the GlcNAc projecting away from the peptide portion. However, it was found that glycosylation did induce a propensity for a turn in the backbone. Analysis showed clustering into one of two major families. Together, these are consistent with greater molecular flexibility and a more exposed carbohydrate. This GlcNAc modification on two related peptide sequences has been examined in complex with an MHC molecule by crystallography (Glithero et al., 1999). In support of glycopeptide flexibility, the crystal structure for one of the glycopeptides in the complex shows evidence that two different rotamers in the GlcNAc glycosidic linkage can occur. Thus, whereas the

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a-linkage positions functional groups to promote conformational and chemical stability of mucin glycopeptides, nature has employed the less constrained b-linkage in a more labile regulatory application that can modulate phosphorylation. Another O-linked modification for which glycan–polypeptide interactions have ramifications is that of a-O-mannose-linked glycans. Recently, this has been identified in mammalian glycoproteins, with a-dystroglycan being the only well-characterized example (Barresi and Campbell, 2006). There is evidence for other glycoproteins modified in this way as well (Chai et al., 1999). This modification has taken on considerable importance, since aberrations in the O-Man-linked glycan have been related to forms of muscular dystrophy. Electron micrographs show the central mucin-like region of this glycoprotein is extended (Brancaccio et al., 1995), as in conventional mucins with a-O-GalNAcs. This posed the question of whether an a-O-Man modification can support an extended arrangement, and if so, through what mechanism. To investigate this, a-O-Man glycopeptides based on a-dystroglycan sequences were synthesized and studied by NMR (Liu et al., 2008). The resulting structures show considerable disorder, relative to the same sequence with a-O-GalNAc, particularly in the arrangement of the pendant sugars (A. Borgert, M. Liu, G. Barany, and D. Live, unpublished results). This can be rationalized in the context of the earlier findings that specific sugar functional groups, notably the N-acetyl modification, and their location are important for interactions that stabilize extended structures. In their absence, glycosylation should not lead to the extended arrangement. While emphasis has been placed on the presence of the a-O-Man-linked glycans because of their identification with biological function, investigations of the central mucin-like region of a-dystroglycan (Sasaki et al., 1998) have noted that there are also a number of coexisting a-OGalNAc-linked glycans in this region. From the inability of the a-O-Man modifications to stabilize the observed extended structure, it appears that the intramolecular interactions involving these a-O-GalNAc-linked glycans are important in imparting the structural stability to the extended arrangement. This would be important for its mechanical function in tissue organization, as well as for the appropriate presentation of the O-Man glycans to receptors.

3. N-Linked Glycoproteins Implicit in one of the functions of N-linked glycans, the participation in protein folding quality control (Varki et al., 2009), is intramolecular interaction between the glycan component and the polypeptide chain. As with O-linked species, N-linked glycoproteins display microheterogeneity, a feature that similarly complicates their study. In view of the significance of

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tertiary interactions in globular glycoproteins with N-linked glycosylation, the rationale for studying isolated glycopeptide segments as models in these latter cases is less well founded. Rather, to accurately describe conformational properties, there is the more demanding requirement for the full glycoprotein, and particularly, for homogeneous glycoprotein samples. This has been approached by both chemical and biological strategies. From a purely chemical perspective, NCL strategies have been extended to the assembly of defined synthetic fragments, both peptide and glycopeptide, into full glycoproteins. This has shown impressive results and considerable promise (Kan and Danishefsky, 2009; Payne and Wong, 2010; Yamamoto et al., 2008). A variation on this theme involves bacterial expression of a protein segment that is then linked, using NCL, to a synthetic glycopeptide, completing the glycoprotein (Piontek et al., 2009). Chemoenzymatic synthesis has been used as well (Wang, 2008). Biological approaches have used wild-type cells, or those with modifications in the glycosylation machinery, often combined with in vitro enzymatic remodeling of the product glycoproteins (Chang et al., 2007; Lee et al., 2009; Li et al., 2001; Lustbader et al., 1996; Rich and Withers, 2009; Schwarz et al., 2010; Slynko et al., 2009), to achieve the final preparations. In the biosynthetic approach, it is necessary to consider that the pendant glycans installed can be organism specific, which can affect the detailed characteristics of the product, as well as choice of expression system. An additional factor in the prospects for success of the various approaches is that, in the absence of some or all of the attached glycoforms, the underlying protein, or even the partially glycosylated form, may not properly fold or may aggregate (Lee et al., 2009). For Chinese hamster ovary (CHO) cells, there are a variety of available glycosylation mutants (North et al., 2010) that can aid in producing more homogeneous glycoproteins on their own, or provide material that is more readily remodeled. Human embryonic kidney (HEK) cells offer promise in expressing glycoproteins bearing human glycoforms (Lee et al., 2009). The relative ease in working with yeast cultures has made them, and in particular Pichia pastoris, popular organisms for producing glycoproteins (Rich and Withers, 2009). The high mannose glycans it installs can be trimmed to the level of the first N-acetylglucosamine residue by treatment with endoglycosidase H; the GlcNAc-Asn site then serves as a site for enzymatically reattaching a desired glycoform using methods recently reported (Rich and Withers, 2009; Wang, 2008). For glycoproteins isolated directly from their natural sources, complex-type glycans may also be trimmed in a similar manner with endoglycosidase D or S (Allhorn et al., 2008; Yamaguchi et al., 2006). Exposed complex-type glycans may sometimes be completely removed from native glycoproteins using PNGase F (Plummer and Tarentino, 1991). Heterogeneity of mature glycoproteins may be dramatically reduced by sequentially trimming the glycan termini using a host of

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obligate exoglycosidases, including a neuraminidase (Clostridium perfringens), a b1-4-galactosidase (Bacteroides fragilis), an N-acetylglucosaminidase (Xanthomonas manihotis), and an a1-3-mannosidase (X. manihotis; New England Biolabs). Following digestion with a1-3 mannosidase, an a1-6 mannosidase (X. manihotis) will remove the a1-6-linked mannose residue. It is important to note that this enzyme does not act on branched substrates; therefore, the a1-3-linked mannose residue must be removed first (New England Biolabs; A.W. Barb and J.H. Prestegard, unpublished data). Glycosidases are commonly inhibited by steric interactions with the polypeptide. This is particularly so with endoglycosidases which are often ineffective towards glycans buried or confined by protein tertiary structure (Blanchard et al., 2008). Steric effects appear to be less of an obstacle for exoglycosidase-catalyzed digestions. Incomplete or heterogeneous glycan termini may be remodeled using glycosyltransferases and their corresponding sugar nucleotide substrates. This strategy has been employed to incorporate NMR-active isotopes into mammalian-expressed glycoproteins (Barb et al., 2009; Macnaughtan et al., 2008; Yamaguchi et al., 1998). Commonly used enzymes are the human a2-3 and a2-6 sialyl-transferases and the bovine b1-4 galactosyltransferase (Chung et al., 2006; Krapp et al., 2003; Raju et al., 2001; Scallon et al., 2007). In most cases, these reactions may be run to completion with each glycan terminus modified. There have been reports of incomplete sialylation of the occluded N-glycans of the IgG Fc fragment discussed below (Barb et al., 2009; Kobata, 2008; Raju et al., 2001); some of this effect is attributable to the slow sialylation of the a1-6Man-linked branch by the human a2-6 sialyl-transferase (Barb et al., 2009). Glycans on intact glycoproteins (Fig. 18.3A), whether as originally isolated or after remodeling, can be characterized by matrix-assisted laser desorption ionization (MALDI)-MS or electrospray ionization (ESI)-MS methods (Gong et al., 2009). However, minor glycoforms may be poorly resolved by MS. For proteins with multiple glycosylation sites, deconvoluting the contributions from different glycans and peak overlap can be challenging. As an alternative approach, liberated, permethylated glycans can be analyzed with MALDI-MS (Anumula and Taylor, 1992). These techniques do not perform well in distinguishing configurational isomers, which generally require releasing glycans followed by derivatization and HPLC-based coelution (Holland et al., 2002; Rice, 2000; Takahashi et al., 1995). A 1D NMR-based approach has been described that can quickly differentiate isomers, but this is limited with heterogeneous samples and is fundamentally insensitive (Vliegenthart et al., 1983). Because of the dynamic features of N-glycans, even when crystallography of glycoproteins with extended oligosaccharides is successful, the electron density for portions of the carbohydrate may be unresolved. Furthermore, even if their electron density is observed, identification of

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A

a1-2 a1-2

a1-2

a1-3

B

a1-3

b1-4

a1-6

a1-2

a1-6 b1-4

a1-6

a2-3 b1-4

b1-2

a1-3

a1-3 b1-4 a1-6

High mannose

a1-6 b1-4

b – GlcNAc

Hybrid

a1-6

a2-3 b1-4

OH H4 H6 O HO

a1-3

NAc

Asn H1

a1-6

b1-4

a1-6

b–GlcNAc

N H3

b1-2

H6′ O H2 H5

b1-4

Complex

Figure 18.3 Panel (A): Types of carbohydrate structures and common linkages that are N-linked to glycoproteins. Note the a1-6 and a1-3-linked mannose residues which also specify the terminal branches of the complex-type biantennary glycan. Gray squares represent N-acetylglucosamine (GlcNAc), dark gray circles mannose, open triangles fucose, open circles galactose, and black diamonds N-acetylneuraminic acid residues. Panel (B): Structure of a core b-GlcNAc residue depicted as the first residue of an N-glycan with a linkage to the asparagine residue and a linkage to the second bGlcNAc residue of the chitobiose core. Note the hydrophobic surface of this residue formed by the H1, H3, H5, H6, and H60 protons.

the actual sugars represented by a particular region of electron density may be uncertain (Chen et al., 2005). Unlike for amino acids, information from other sources may be insufficient to unequivocally know the glycan sequence. It has been noted that, historically, there are a number of errors in the carbohydrate portion of X-ray determined glycoprotein structures deposited in the PDB, although efforts are being made to correct these (Lutteke, 2009). The dynamics of the N-linked glycans puts NMR at a significant advantage in monitoring the characteristics of oligosaccharide chain conformation and interactions, not only because it is better suited for describing dynamics, but also because the experiments are done in solution. Hence, effects of crystal packing on the glycans, which largely extend from the protein surface, do not bias the results. NMR also provides explicit assignment of each sugar residue for which shift assignments can be made. While the addition of carbohydrate adds complexity to the molecules, crosspeaks from the carbohydrate components in 2D or 3D 1H–13C maps fall in regions where they can readily be resolved from those of the protein component (de Beer et al., 1994). NMR studies of these generally large glycoprotein systems can benefit from the incorporation of 13C and 15N labeling that has proven so valuable in protein structural studies. The labeling can be done either uniformly, segmentally, or on a residue (either amino acid or sugar) specific basis, using P. pastoris, CHO (Lustbader et al., 1996), HEK (Liu et al., 2007), and bacterial expression systems.

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A survey of glycoprotein structures has found that N-linked glycans occur in a wide variety of protein secondary structural elements and surface geometries (Petrescu et al., 2006). Linked Asn residues have been observed in several topologies, ranging from the bottoms of deeply recessed concave surfaces to steeply convex surfaces (Petrescu et al., 2004). These observations argue against the presence of a dominant single strong and specific interaction between proximal residues and the proteins, and are consistent with the initial sugar residues being oriented largely out of the way of the protein backbone and interacting comparatively weakly with it. The most frequently observed contacts of the N-glycan with the polypeptide are to the first N-acetylglucosamine residue, and are variable. This residue is often found near a hydrophobic surface or pocket with the H1– H3–H5 face of the residue (Fig. 18.3B) lying against it. The N-acetyl methyl can also make hydrophobic contacts with the surface by occupying a shallow pocket. Indeed, aromatic and proline residues are enriched around N-X-T/S sequons (Petrescu et al., 2004), and are most likely to interact with the GlcNAc, as observed by X-ray crystallography. The N78 glycan of the a-subunit of human chorionic gonadotropin (pdb 1hd4) typifies this arrangement, where the H1–H3–H5 face is against proline and valine residues, and the methyl is in a pocket formed by valine, isoleucine, and alanine residues (Erbel et al., 2000). Hydrogen bonds to the carbonyl oxygen of the N-acetyl are also observed, as in the Epstein-Barr virus major envelope glycoprotein gp350 (pdb 2h6o) at N195 where the oxygen atom is in position to form a hydrogen bond with a backbone NH of G289 (Szakonyi et al., 2006). In many cases, however, there are no clear contacts between the first residue and the polypeptide. Similar interactions are observed, though less frequently, for the second N-acetylglucosamine residue in the glycan. In theory, polar contacts with any sugar hydrogen bond donor or acceptor may occur, although most observed interactions involve the C6 OH and the N-acetyl carbonyl oxygen atom. For example, the O6 from the second N-acetylglucosamine residue of the IgA Fc N263 glycan (pdb 1ow0) is in position to form a hydrogen bond to the side chain oxygens from D255 (Herr et al., 2003). Sugar ring proton hydrophobic contacts are likewise observed, including N-acetyl methyl–hydrophobic interactions as exemplified by the human FcaRI structure (pdb 1ow0) with the methyl from the second N-acetylglucosamine on the N58 glycan packed between side chains from Glu and Tyr residues (Herr et al., 2003). The dearth of coordinates beyond the first two GlcNAc residues indicates that there are few stable interactions between the distal carbohydrate residues and the polypeptide. There are notable exceptions with extensive glycan coordinates in the Protein Data Bank, although it is important to note that even when glycans are observed in X-ray crystallography, structures may be influenced by crystal packing (Chen et al., 2005), and may not

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represent the ensemble of solution conformations available to the N-glycan. Where coordinates are available, glycans do not typically interact tightly with the polypeptide. Unlike high affinity interactions, N-glycan–polypeptide interactions lack a high degree of surface complementarity, buried hydrophobic surfaces, and extensive hydrogen bonding. NMR studies on some N-linked glycosylation sites show NOE contacts between the proximal sugars and the Asn side chain (Erbel et al., 2000; Slynko et al., 2009; Wyss et al., 1995), as well as those of neighboring side chains, indicating proximity of this sugar residue to the amino acid side chains. This can vary even among sites in a single glycoprotein. For human chorionic gonadotropin, glycan–protein NOEs for the glycan at the N52 site are largely lacking, but are present at the N78 site (Erbel et al., 2000; Weller et al., 1996). It is also noted in these glycoproteins that the mobility of the GlcNAc is more restricted than the peripheral sugar residues, implying interactions of some nature. In CD2, even the first GlcNAc has several NOE contacts with the K61 side chain, four residues removed from the glycosylation site, and has a stabilizing effect on protein attributed to defusing the locally high concentration of positive charges on the protein surface (Hanson et al., 2009; Wyss et al., 1995) particularly associated with K61. Nonetheless, using wild-type and K61A mutants of CD2, it has been shown that, independent of surface charge, the presence of the first sugar is an important contributor to protein folding and stability (Hanson et al., 2009). Indeed, trimming the N-glycan to the first N-acetylglucosamine residue is a strategy used for structural biology applications that often maintains protein stability, while substantively reducing conformational and configurational heterogeneity (Chang et al., 2007). Although both hydrophobic and polar distal residue–polypeptide interactions are observed, hydrophobic interactions probably dominate the stabilizing and enhanced folding benefits of N-glycans. It is notable that many carbohydrate residues have defined hydrophobic faces, as mentioned above. b-Galactose and b-mannose residues are examples where the H1– H3–H4–H5–H6–H60 (Fig. 18.3) and H1–H2–H3–H5–H6–H60 faces, respectively, are markedly hydrophobic in nature. It is likely that these faces cover hydrophobic polypeptide surfaces, although these may not always be obvious due to the highly dynamic nature of the distal portion of N-glycans. This is demonstrated in erythropoietin (EPO), a glycosylated peptidic growth factor, where glycosylation shields hydrophobic patches that are otherwise accessible to promote aggregation (Cheetham et al., 1998; Narhi et al., 1991; Toyoda et al., 2000, 2002). In a structure of the human Zn-a2-glycoprotein (pdb 1zag), the hydrophobic face of the b-mannose residue is proximal to a Tyr side chain (Sanchez et al., 1999). The H3ax–H4– H6 face of N-acetylneuraminic acid has also been observed in contact with hydrophobic residues, and was observed packed against a Trp side chain in the structure of fibrinogen (pdb 3ghg). These results are unusual for

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resolving coordinates for a large glycan with seemingly few conformationstabilizing contacts (Kollman et al., 2009). The Fc fragment of immunoglobulin G (IgG) has extensive distal glycan–polypeptide interactions which affect Fc receptor binding and directly impact human disease. IgG is a 150 kDa protein with two antigen binding Fab fragments and one Fc fragment (Fig. 18.4). The Fc fragment mediates interactions between IgG and immune system components, including the Fcg receptors, and the C1q component of complement, among others (Roitt et al., 2001). Even separated from the Fab fragments, the Fc fragment maintains its full strength in these interactions. The heavy chain of IgG has one conserved N-glycosylation site at N297, with a complex-type, biantennary glycan that generally varies in the amount of terminal galactose when purified from the serum of healthy individuals (Arnold et al., 2007). In rheumatoid arthritis patients, the amount of terminal galactose is inversely proportional to the severity of the disease (Parekh et al., 1985). X-ray diffraction-derived structural models of the Fc fragment are unusual in that nearly the entirety of the glycan is resolved and occupies the space between the two Cg2 domains (pdb 1fc1; Deisenhofer, 1981). The position of these glycans in the polypeptide interstitial region likely accounts for the observed regular structure. The residues on the a1-6Manlinked branch of the glycan are stabilized through hydrophobic and polar interactions with the surface of the Cg2 domain. The H1–H3–H5 face of the first GlcNAc residue makes some hydrophobic contacts with V264, and the N-acetyl carbonyl oxygen atom hydrogen bonds to the D265 side chain. H4, H6, and H60 of the second residue make hydrophobic contacts, and the

Fab

Fab Light chain

Heavy chain Asn 297

Hinge Cg2

Cg2

Fc Cg3

Cg3

Figure 18.4 Schematic of IgG showing the Fc fragment, the position of the conserved N-glycan, and the relative orientations of the Cg2 and Cg3 domains.

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N-acetyl carbonyl oxygen atom is in position to hydrogen bond with the R301 side chain NH. As is commonly observed for N-glycans with a core fucose residue, no contacts between the fucose and the protein surface are observed in these crystal structures. The b-mannose residue makes extensive contacts with its H1–H2–H3–H5 face to F241. From this point, the a1-3Man branch of the glycan extends into the dimer interface, away from the polypeptide surface, and the a1-6Man branch follows the contour of the Cg2 domain. The a1-6Man N-acetylglucosamine residue makes extensive contacts through its H1–H3–H5 face to F243. The K246 amine is in position to bind O4 of GlcNAc and the anomeric oxygen of galactose on the a1-6Man branch. An NMR study of the Fc-conjugated glycan revealed a broader 13C line width for the anomeric carbon of galactose on the a1-6Man-linked branch, when compared to the same carbon on the a1-3Man-linked branch (Yamaguchi et al., 1998). This offers evidence in support of the hypothesis that the line widths from the a1-6Man-linked branch are broadened due to a more intimate interaction with the protein surface, restricting its motion relative to the a1-3Man-linked branch. Mutating residues along this binding interface, including F241, F243, V264, D265, and R301, dramatically increased the amount of galactose and sialic acid containing termini (Lund et al., 1996) and indicated a greater accessibility of the glycan termini to the galactosyl- and sialyl-transferases in the Golgi. The a1-3Man-linked branch residues interact with each other across the dimer interface primarily through hydrophobic interactions (Deisenhofer, 1981). The composition of the N-glycans is intimately linked to Fc structure and function. The distance between the two Cg2 domains decreases upon truncation of the glycan (Krapp et al., 2003), and backbone amide chemical shift changes at the Cg2/Cg3 interface indicate a structural rearrangement consistent with this type of movement (Yamaguchi et al., 2006). Based on the position of the glycans, it is likely that the size of the Fc glycan is a principle determinant in the spacing of the two Cg2 domains. The IgG Fc glycan composition also affects binding to the C1q component of complement (Leatherbarrow et al., 1985) and the low affinity (low mM) Fc receptors, FcgRII and FcgRIII, in contrast to the Fca receptor–IgA Fc interaction which is insensitive to glycan composition or absence of sugar (Gomes et al., 2008). Deglycosylated IgG Fc shows no measurable affinity towards either FcgRIIb or FcgRIII (Mimura et al., 2001; Yamaguchi et al., 2006). Fc with the glycans truncated to the mannose residues shows restored binding affinity for both receptors, when compared to deglycosylated forms. The GlcNAc and galactose residues slightly affect these interactions, though differently for FcgRII and FcgRIII. It is interesting to note that sialylation decreased the strength of the Fc–FcgRIIb/FcgRIII interaction roughly 10fold (Kaneko et al., 2006). One explanation is that the Fc glycans, residing between the heavy chain monomers, tune the interaction with the FcgRs

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through bulk effects alone. Based on the structure of the Fc–FcgRIII complex, the glycans do not bind to the receptor directly (Radaev et al., 2001). However, one can speculate that specific a1-6Man-branch–Fc polypeptide interactions, rather than bulk effects, are critical for proper IgG Fc structure, dynamics, and function.

ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of General Medical Science GM066148 (D.L.) and the National Institutes of Arthritis and Musculoskeletal Diseases AR056055 (D.L.), and a fellowship from the National Institutes of Arthritis and Musculoskeletal Diseases F32AR058084 (A.B.).

REFERENCES Allhorn, M., Olin, A. I., Nimmerjahn, F., and Collin, M. (2008). Human IgG/Fc gamma R interactions are modulated by streptococcal IgG glycan hydrolysis. PLoS ONE 3, e1413. Anumula, K. R., and Taylor, P. B. (1992). A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. Anal. Biochem. 203, 101–108. Apweiler, R., Hermjakob, H., and Sharon, N. (1999). On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta Gen. Subj. 1473, 4–8. Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., and Dwek, R. A. (2007). The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21–50. Barb, A. W., Brady, E. K., and Prestegard, J. H. (2009). Branch-specific sialylation of IgG-Fc glycans by ST6Gal 1. Biochemistry 48, 9705–9707. Barresi, R., and Campbell, K. P. (2006). Dystroglycan: From biosynthesis to pathogenesis of human disease. J. Cell Sci. 119, 199–207. Barrientos, L. G., Dolan, C., and Gronenborn, A. M. (2000). Characterization of surfactant liquid crystal phases suitable for molecular alignment and measurement of dipolar couplings. J. Biomol. NMR 16, 329–337. Blanchard, V., Frank, M., Leeflang, B. R., Boelens, R., and Kamerling, J. P. (2008). The structural basis of the difference in sensitivity for PNGase F in the de-N-glycosylation of the native bovine pancreatic ribonucleases B and BS. Biochemistry 47, 3435–3446. Brancaccio, A., Schulthess, T., Gesemann, M., and Engel, J. (1995). Electron-microscopic evidence for a mucin-like region in chick muscle alpha-dystroglycan. FEBS Lett. 368, 139–142. Bush, C. A., and Feeney, R. E. (1986). Conformation of the glycotripeptide repeating unit of antifreeze glycoprotein of polar fish as determined from the fully assigned proton NMR-spectrum. Int. J. Pept. Protein Res. 28, 386–397. Buskas, T., Ingale, S., and Boons, G. J. (2006). Glycopeptides as versatile tools for glycobiology. Glycobiology 16, 113R–136R. Cavanaugh, J., Fairbrother, W. J., Palmer, A. G., and Skelton, N. J. (2007). Protein NMR Spectroscopy. Elsevier Academic Press, Amsterdam. Chai, W. G., Yuen, C. T., Kogelberg, H., Carruthers, R. A., Margolis, R. U., Feizi, T., and Lawson, A. M. (1999). High prevalence of 2-mono- and 2, 6-di-substituted manol-

Intramolecular Interactions

383

terminating sequences among O-glycans released from brain glycopeptides by reductive alkaline hydrolysis. Eur. J. Biochem. 263, 879–888. Chang, V. T., Crispin, M., Aricescu, A. R., Harvey, D. J., Nettleship, J. E., Fennelly, J. A., Yu, C., Boles, K. S., Evans, E. J., Stuart, D. I., Dwek, R. A., Jones, E. Y., et al. (2007). Glycoprotein structural genomics: Solving the glycosylation problem. Structure 15, 267–273. Cheetham, J. C., Smith, D. M., Aoki, K. H., Stevenson, J. L., Hoeffel, T. J., Syed, R. S., Egrie, J., and Harvey, T. S. (1998). NMR structure of human erythropoietin and a comparison with its receptor bound conformation. Nat. Struct. Biol. 5, 861–866. Chen, B., Vogan, E. M., Gong, H. Y., Skehel, J. J., Wiley, D. C., and Harrison, S. C. (2005). Determining the structure of an unliganded and fully glycosylated SIV gp120 envelope glycoprotein. Structure 13, 197–211. Chung, S., Joo, H., Jang, K., Lee, H., Lee, S., and Kim, B. (2006). Galactosylation and sialylation of terminal glycan residues of human immunoglobulin G using bacterial glycosyltransferases with in situ regeneration of sugar-nucleotides. Enzyme Microb. Technol. 39, 60–66. Coltart, D. M., Royyuru, A. K., Williams, L. J., Glunz, P. W., Sames, D., Kuduk, S. D., Schwarz, J. B., Chen, X. T., Danishefsky, S. J., and Live, D. H. (2002). Principles of mucin architecture: Structural studies on synthetic glycopeptides bearing clustered mono-, di-, tri-, and hexasaccharide glycodomains. J. Am. Chem. Soc. 124, 9833–9844. Corzana, F., Busto, J. H., Engelsen, S. B., Jimenez-Barbero, J., Asensio, J. L., Peregrina, J. M., and Avenoza, A. (2006a). Effect of beta-O-glucosylation on L-ser and L-thr diamides: A bias toward alpha-helical conformations. Chem. Eur. J. 12, 7864–7871. Corzana, F., Busto, J. H., Jimenez-Oses, G., Asensio, J. L., Jimenez-Barbero, J., Peregrina, J. M., and Avenoza, A. (2006b). New insights into alpha-GalNAc-ser motif: Influence of hydrogen bonding versus solvent interactions on the preferred conformation. J. Am. Chem. Soc. 128, 14640–14648. Corzana, F., Busto, J. H., Jimenez-Oses, G., de Luis, M. G., Asensio, J. L., JimenezBarbero, J., Peregrina, J. M., and Avenoza, A. (2007). Serine versus threonine glycosylation: The methyl group causes a drastic alteration on the carbohydrate orientation and on the surrounding water shell. J. Am. Chem. Soc. 129, 9458–9467. Corzana, F., Busto, J. H., de Luis, M. G., Jimenez-Barbero, J., Avenoza, A., and Peregrina, J. M. (2009). The nature and sequence of the amino acid aglycone strongly modulates the conformation and dynamics effects of Tn antigen’s clusters. Chem. Eur. J. 15, 3863–3874. Cummings, R. D. (2009). The repertoire of glycan determinants in the human glycome. Mol. Biosyst. 5, 1087–1104. de Alba, E., and Tjandra, N. (2002). NMR dipolar couplings for the structure determination of biopolymers in solution. Prog. Nucl. Magn. Reson. Spectrosc. 40, 175–197. de Beer, T., van Zuylen, C. E. M., Hard, K., Boelens, R., Kaptein, R., Kamerling, J. P., and Vliegenthart, J. F. G. (1994). Rapid and simple approach for the NMR resonance assignment of the carbohydrate chains of an intact glycoprotein—application of gradientenhanced natural-abundance H-1-C-13 HSQC and HSQC-TOCSY to the alpha-subunit of human chorionic-gonadotropin. FEBS Lett. 348, 1–6. Deisenhofer, J. (1981). Crystallographic refinement and atomic models of a human fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A˚ resolution. Biochemistry 20, 2361–2370. Dziadek, S., Griesinger, C., Kunz, H., and Reinscheid, U. M. (2006). Synthesis and structural model of an alpha(2, 6)-sialyl-T glycosylated MUC1 eicosapeptide under physiological conditions. Chem. Eur. J. 12, 4981–4993. Erbel, P. J. A., Karimi-Nejad, Y., van Kuik, J. A., Boelens, R., Kamerling, J. P., and Vliegenthart, J. F. G. (2000). Effects of the N-linked glycans on the 3D structure of the free alpha-subunit of human chorionic gonadotropin. Biochemistry 39, 6012–6021.

384

Adam W. Barb et al.

Fukuda, M., Carlsson, S. R., Klock, J. C., and Dell, A. (1986). Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemiacells, and acute myelogenous leukemia-cells. J. Biol. Chem. 261, 2796–2806. Garner, O. B., and Baum, L. G. (2008). Galectin-glycan lattices regulate cell-surface glycoprotein organization and signaling. Biochem. Soc. Trans. 36, 1472–1477. Gerken, T. A., and Jentoft, N. (1987). Structure and dynamics of porcine submaxillary mucin as determined by natural abundance C-13 NMR-spectroscopy. Biochemistry 26, 4689–4699. Gerken, T. A., Butenhof, K. J., and Shogren, R. (1989). Effects of glycosylation on the conformation and dynamics of O-linked glycoproteins—C-13 NMR-studies of ovine submaxillary mucin. Biochemistry 28, 5536–5543. Glithero, A., Tormo, J., Haurum, J. S., Arsequell, G., Valencia, G., Edwards, J., Springer, S., Townsend, A., Pao, Y. L., Wormald, M., Dwek, R. A., Jones, E. Y., et al. (1999). Crystal structures of two H-2D(b)/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity. Immunity 10, 63–74. Gomes, M. M., Wall, S. B., Takahashi, K., Novak, J., Renfrow, M. B., and Herr, A. B. (2008). Analysis of IgA1 N-glycosylation and its contribution to Fc alpha RI binding. Biochemistry 47, 11285–11299. Gong, B., Cukan, M., Fisher, R., Li, H., Stadheim, T. A., and Gerngross, T. (2009). Characterization of N-linked glycosylation on recombinant glycoproteins produced in Pichia pastoris using ESI-MS and MALDI-TOF. Meth. Mol. Biol. 534, 213–223. Grinstead, J. S., Koganty, R. R., Krantz, M. J., Longenecker, B. M., and Campbell, A. P. (2002). Effect of glycosylation on muc1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions. Biochemistry 41, 9946–9961. Hanson, S. R., Culyba, E. K., Hsu, T. L., Wong, C. H., Kelly, J. W., and Powers, E. T. (2009). The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability. Proc. Natl. Acad. Sci. USA 106, 3131–3136. Herr, A. B., Ballister, E. R., and Bjorkman, P. J. (2003). Insights into IgA-mediated immune responses from the crystal structures of human Fc alpha RI and its complex with IgA1-Fc. Nature 423, 614–620. Holland, M., Takada, K., Okumoto, T., Takahashi, N., Kato, K., Adu, D., Ben-Smith, A., Harper, L., Savage, C. O., and Jefferis, R. (2002). Hypogalactosylation of serum IgG in patients with ANCA-associated systemic vasculitis. Clin. Exp. Immunol. 129, 183–190. Ishima, R., and Torchia, D. A. (2000). Protein dynamics from NMR. Nat. Struct. Biol. 7, 740–743. Kan, C., and Danishefsky, S. J. (2009). Recent departures in the synthesis of peptides and glycopeptides. Tetrahedron 65, 9047–9065. Kaneko, Y., Nimmerjahn, F., and Ravetch, J. V. (2006). Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670–673. Kirnarsky, L., Prakash, O., Vogen, S. M., Nomoto, M., Hollingsworth, M. A., and Sherman, S. (2000). Structural effects of O-glycosylation on a 15-residue peptide from the mucin (MUC1) core protein. Biochemistry 39, 12076–12082. Kobata, A. (2008). The N-linked sugar chains of human immunoglobulin G: Their unique pattern, and their functional roles. Biochim. Biophys. Acta Gen. Subj. 1780, 472–478. Kollman, J. M., Pandi, L., Sawaya, M. R., Riley, M., and Doolittle, R. F. (2009). Crystal structure of human fibrinogen. Biochemistry 48, 3877–3886. Krapp, S., Mimura, Y., Jefferis, R., Huber, R., and Sondermann, P. (2003). Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J. Mol. Biol. 325, 979–989. Lane, A. N., Hays, L. M., Feeney, R. E., Crowe, L. M., and Crowe, J. H. (1998). Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from antarctic cod. Protein Sci. 7, 1555–1563.

Intramolecular Interactions

385

Leatherbarrow, R. J., Rademacher, T. W., Dwek, R. A., Woof, J. M., Clark, A., Burton, D. R., Richardson, N., and Feinstein, A. (1985). Effector functions of a monoclonal aglycosylated mouse IgG2a: Binding and activation of complement component C1 and interaction with human monocyte Fc receptor. Mol. Immunol. 22, 407–415. Lee, J. E., Fusco, M. L., and Saphire, E. O. (2009). An efficient platform for screening expression and crystallization of glycoproteins produced in human cells. Nat. Protoc. 4, 592–604. Li, P. Z., Go, X. G., Arellano, R. O., and Renugopalakrishnan, V. (2001). Glycosylated and phosphorylated proteins—expression in yeast and oocytes of xenopus: Prospects and challenges—relevance to expression of thermostable proteins. Protein Expr. Purif. 22, 369–380. Liu, M., Barany, G., and Live, D. (2005). Parallel solid-phase synthesis of mucin-like glycopeptides. Carbohydr. Res. 340, 2111–2122. Liu, S., Venot, A., Meng, L., Tian, F., Moremen, K. W., Boons, G. J., and Prestegard, J. H. (2007). Spin-labeled analogs of CMP-NeuAc as NMR probes of the alpha-2, 6-sialyltransferase ST6Gal 1. Chem. Biol. 14, 409–418. Liu, M., Borgert, A., Barany, G., and Live, D. (2008). Conformational consequences of protein glycosylation: Preparation of O-mannosyl serine and threonine building blocks, and their incorporation into glycopeptide sequences derived from alpha-dystroglycan. Biopolymers 90, 358–368. Lund, J., Takahashi, N., Pound, J. D., Goodall, M., and Jefferis, R. (1996). Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc gamma receptor i and influence the synthesis of its oligosaccharide chains. J. Immunol. 157, 4963–4969. Lustbader, J. W., Birken, S., Pollak, S., Pound, A., Chait, B. T., Mirza, U. A., Ramnarain, S., Canfield, R. E., and Brown, J. M. (1996). Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures. J. Biomol. NMR 7, 295–304. Lutteke, T. (2009). Analysis and validation of carbohydrate three-dimensional structures. Acta Crystallogr. D Biol. Crystallogr. 65, 156–168. Macnaughtan, M. A., Tian, F., Liu, S., Meng, L., Park, S., Azadi, P., Moremen, K. W., and Prestegard, J. H. (2008). C-13-sialic acid labeling of glycans on glycoproteins using ST6Gal 1. J. Am. Chem. Soc. 130, 11864–11865. Mandel, A. M., Akke, M., and Palmer, A. G. (1995). Backbone dynamics of Escherichia-coli ribonuclease H1—correlations with structure and function in an active enzyme. J. Mol. Biol. 246, 144–163. Matsushita, T., Hinou, H., Fumoto, M., Kurogochi, M., Fujitani, N., Shimizu, H., and Nishimura, S. I. (2006). Construction of highly glycosylated mucin-type glycopeptides based on microwave-assisted solid-phase syntheses and enzymatic modifications. J. Org. Chem. 71, 3051–3063. Meyer, B., and Moller, H. (2007). Conformation of glycopeptides and glycoproteins. Top. Curr. Chem. 267, 187–251. Mimura, Y., Yamamoto, Y., Inoue, Y., and Chujo, R. (1992). NMR-study of interaction between sugar and peptide moieties in mucin-type model glycopeptides. Int. J. Biol. Macromol. 14, 242–248. Mimura, Y., Sondermann, P., Ghirlando, R., Lund, J., Young, S. P., Goodall, M., and Jefferis, R. (2001). Role of oligosaccharide residues of IgG1-Fc in Fc gamma RIIb binding. J. Biol. Chem. 276, 45539–45547. Naganagowda, G. A., Gururaja, T. L., Satyanarayana, J., and Levine, M. J. (1999). NMR analysis of human salivary mucin (MUC7) derived O-linked model glycopeptides: Comparison of structural features and carbohydrate-peptide interactions. J. Pept. Res. 54, 290–310.

386

Adam W. Barb et al.

Narhi, L. O., Arakawa, T., Aoki, K. H., Elmore, R., Rohde, M. F., Boone, T., and Strickland, T. W. (1991). The effect of carbohydrate on the structure and stability of erythropoietin. J. Biol. Chem. 266, 23022–23026. North, S. J., Huang, H. H., Sundaram, S., Jang-Lee, J., Etienne, A. T., Trollope, A., Chalabi, S., Dell, A., Stanley, P., and Haslam, S. M. (2010). Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity. J. Biol. Chem. 285, 5759–5775. Ohtsubo, K., and Marth, J. D. (2006). Glycosylation in cellular mechanisms of health and disease. Cell 126, 855–867. Ottiger, M., and Bax, A. (1999). Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values. J. Biomol. NMR 13, 187–191. Parekh, R. B., Dwek, R. A., Sutton, B. J., Fernandes, D. L., Leung, A., Stanworth, D., Rademacher, T. W., Mizuochi, T., Taniguchi, T., Matsuta, K., et al. (1985). Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316, 452–457. Payne, R. J., and Wong, C. H. (2010). Advances in chemical ligation strategies for the synthesis of glycopeptides and glycoproteins. Chem. Commun. 46, 21–43. Petrescu, A. J., Milac, A. L., Petrescu, S. M., Dwek, R. A., and Wormald, M. R. (2004). Statistical analysis of the protein environment of N-glycosylation sites: Implications for occupancy, structure, and folding. Glycobiology 14, 103–114. Petrescu, A. J., Wormald, M. R., and Dwek, R. A. (2006). Structural aspects of glycomes with a focus on N-glycosylation and glycoprotein folding. Curr. Opin. Struct. Biol. 16, 600–607. Piontek, C., Silva, D. V., Heinlein, C., Pohner, C., Mezzato, S., Ring, P., Martin, A., Schmid, F. X., and Unverzagt, C. (2009). Semisynthesis of a homogeneous glycoprotein enzyme: Ribonuclease C: Part 2. Angew. Chem. Int. Ed. 48, 1941–1945. Plummer, T. H., Jr., and Tarentino, A. L. (1991). Purification of the oligosaccharidecleaving enzymes of Flavobacterium meningosepticum. Glycobiology 1, 257–263. Radaev, S., Motyka, S., Fridman, W. H., Sautes-Fridman, C., and Sun, P. D. (2001). The structure of a human type III Fc gamma receptor in complex with Fc. J. Biol. Chem. 276, 16469–16477. Raju, T. S., Briggs, J. B., Chamow, S. M., Winkler, M. E., and Jones, A. J. (2001). Glycoengineering of therapeutic glycoproteins: In vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues. Biochemistry 40, 8868–8876. Rice, K. G. (2000). Derivatization strategies for preparing N-glycan probes. Anal. Biochem. 283, 10–16. Rich, J. R., and Withers, S. G. (2009). Emerging methods for the production of homogeneous human glycoproteins. Nat. Chem. Biol. 5, 206–215. Roitt, I. M., Brostoff, J., and Male, D. K. (2001). Immunology Mosby, Edinburgh. Rose, M. C., Voter, W. A., Sage, H., Brown, C. F., and Kaufman, B. (1984). Effects of deglycosylation on the architecture of ovine submaxillary mucin glycoprotein. J. Biol. Chem. 259, 3167–3172. Sanchez, L. M., Chirino, A. J., and Bjorkman, P. (1999). Crystal structure of human ZAG, a fat-depleting factor related to MHC molecules. Science 283, 1914–1919. Sasaki, T., Yamada, H., Matsumura, K., Shimizu, T., Kobata, A., and Endo, T. (1998). Detection of O-mannosyl glycans in rabbit skeletal muscle alpha-dystroglycan. Biochim. Biophys. Acta Gen. Subj. 1425, 599–606. Scallon, B. J., Tam, S. H., McCarthy, S. G., Cai, A. N., and Raju, T. S. (2007). Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol. Immunol. 44, 1524–1534.

Intramolecular Interactions

387

Schuster, O., Klich, G., Sinnwell, V., Kranz, H., Paulsen, H., and Meyer, B. (1999). ’Wavetype’ structure of a synthetic hexaglycosylated decapeptide: A part of the extracellular domain of human glycophorin A. J. Biomol. NMR 14, 33–45. Schwarz, F., Huang, W., Li, C. S., Schulz, B. L., Lizak, C., Palumbo, A., Numao, S., Neri, D., Aebi, M., and Wang, L. X. (2010). A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation. Nat. Chem. Biol. 6, 264–266. Schwieters, C. D., Kuszewski, J. J., and Clore, G. M. (2006). Using Xplor-NIH for NMR molecular structure determination. Prog. Nucl. Magn. Reson. Spectrosc. 48, 47–62. Shogren, R., Gerken, T. A., and Jentoft, N. (1989). Role of glycosylation on the conformation and chain dimensions of O-linked glycoproteins—light-scattering-studies of ovine submaxillary mucin. Biochemistry 28, 5525–5536. Simanek, E. E., Huang, D. H., Pasternack, L., Machajewski, T. D., Seitz, O., Millar, D. S., Dyson, H. J., and Wong, C. H. (1998). Glycosylation of threonine of the repeating unit of RNA polymerase II with beta-linked N-acetylglucosame leads to a turnlike structure. J. Am. Chem. Soc. 120, 11567–11575. Slynko, V., Schubert, M., Numao, S., Kowarik, M., Aebi, M., and Allain, F. H. T. (2009). NMR structure determination of a segmentally labeled glycoprotein using in vitro glycosylation. J. Am. Chem. Soc. 131, 1274–1281. Springer, G. F. (1997). Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy. J. Mol. Med. 75, 594–602. Szakonyi, G., Klein, M. G., Hannan, J. P., Young, K. A., Ma, R. L. Z., Asokan, R., Holers, V. M., and Chen, X. J. S. (2006). Structure of the Epstein-Barr virus major envelope glycoprotein. Nat. Struct. Mol. Biol. 13, 996–1001. Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995). Threedimensional elution mapping of pyridylaminated N-linked neutral and sialyl oligosaccharides. Anal. Biochem. 226, 139–146. Takeuchi, H., Kato, K., Hassan, H., Clausen, H., and Irimura, T. (2002). O-GalNAc incorporation into a cluster acceptor site of three consecutive threonines—distinct specificity of GalNAc-transferase isoforms. Eur. J. Biochem. 269, 6173–6183. Tarp, M. A., Sorensen, A. L., Mandel, U., Paulsen, H., Burchell, J., TaylorPapadimitriou, J., and Clausen, H. (2007). Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat. Glycobiology 17, 197–209. Ten Hagen, K. G., Fritz, T. A., and Tabak, L. A. (2003). All in the family: The UDPGalNAc: Polypeptide N-acetylgalactosaminyltransferases. Glycobiology 13, 1R–16R. Toyoda, T., Itai, T., Arakawa, T., Aoki, K. H., and Yamaguchi, H. (2000). Stabilization of human recombinant erythropoietin through interactions with the highly branched Nglycans. J. Biochem. 128, 731–737. Toyoda, T., Arakawa, T., and Yamaguchi, H. (2002). N-glycans stabilize human erythropoietin through hydrophobic interactions with the hydrophobic protein surface: Studies by surface plasmon resonance analysis. J. Biochem. 131, 511–515. van Kooyk, Y., and Rabinovich, G. A. (2008). Protein-glycan interactions in the control of innate and adaptive immune responses. Nat. Immunol. 9, 593–601. Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., and Mee, E. (2009). Essentials of Glycobiology Cold Spring Harbor Press, Cold Spring Harbor, New York. Vliegenthart, J. F. G., Dorland, L., and Van Halbeek, H. (1983). 1H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Adv. Carbohydr. Chem. Biochem. 41, 209–374. Wang, L. X. (2008). Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation. Carbohydr. Res. 343, 1509–1522.

388

Adam W. Barb et al.

Weller, C. T., Lustbader, J., Seshadri, K., Brown, J. M., Chadwick, C. A., Kolthoff, C. E., Ramnarain, S., Pollak, S., Canfield, R., and Homans, S. W. (1996). Structural and conformational analysis of glycan moieties in situ on isotopically C-13, N-15-enriched recombinant human chorionic gonadotropin. Biochemistry 35, 8815–8823. Whelan, S. A., and Hart, G. W. (2006). Identification of O-GlcNAc sites on proteins. In ‘‘Methods in Enzymology,’’ (M. Fukuda, ed.)., Vol. 415, pp.113–133. Wyss, D. F., Choi, J. S., Li, J., Knoppers, M. H., Willis, K. J., Arulanandam, A. R. N., Smolyar, A., Reinherz, E. L., and Wagner, G. (1995). Conformation and function of the N-linked glycan in the adhesion domain of human CD2. Science 269, 1273–1278. Yamaguchi, Y., Kato, K., Shindo, M., Aoki, S., Furusho, K., Koga, K., Takahashi, N., Arata, Y., and Shimada, I. (1998). Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective C-13 labeling of the glycans. J. Biomol. NMR 12, 385–394. Yamaguchi, Y., Nishimura, M., Nagano, M., Yagi, H., Sasakawa, H., Uchida, K., Shitara, K., and Kato, K. (2006). Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy. Biochim. Biophys. Acta Gen. Subj. 1760, 693–700. Yamamoto, N., Tanabe, Y., Okamoto, R., Dawson, P. E., and Kajihara, Y. (2008). Chemical synthesis of a glycoprotein having an intact human complex-type sialyloligosaccharide under the Boc and Fmoc synthetic strategies. J. Am. Chem. Soc. 130, 501–510.