Glycobiology, 2001, Vol. 11, No. 1 31-36
© 2001 Oxford University Press
Carrier protein-modulated presentation and recognition of an N-glycan: observations on the interactions of Man8 glycoform of ribonuclease B with conglutinin
Instituto de Química Física "Rocasolano," C.S.I.C., Serrano 119, 28006 Madrid, Spain, 2Instituto de Estructura de la Materia, C.S.I.C., Serrano 119, 28006 Madrid, Spain, 3Instituto de Química Orgánica General, C.S.I.C., Juan de la Cierva 3, 28006 Madrid, Spain, and 4The Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Campus, Watford Road, Harrow, Middlesex HA1 3UJ, UK
Received on May 30, 2000; revised on August 17, 2000; accepted on September 4, 2000.
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Abstract |
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Conglutinin is a serum lectin of the innate immune system, which binds high mannose N-glycans when these are appropriately presented on proteins. Here we use the conglutinin-ribonuclease B (RNaseB)-recognition system as a model to investigate the structural basis of selective recognition of protein-bound oligosaccharides by this carbohydrate-binding receptor. Conglutinin shows little binding to the isolated RNaseB-Man8 glycoform, and no binding to Man5-6 glycoforms. In contrast, when the protein moiety is reduced and denatured we observe that conglutinin binds strongly to the isolated RNaseB-Man8 glycoform and weakly to the Man5-6 glycoforms. These results are in accord with observations on the binding to the N-glycans in the absence of carrier protein. NMR analyses of native RNaseB-Man8 and -Man5-6 glycoforms reveal that the three-dimensional structure of the protein moiety is essentially identical to that of non-glycosylated RNase (RNaseA). Thus there are no perceptible differences between the RNase protein forms that could account for differential availability of the N-glycan for conglutinin-binding. After reduction and denaturation, the NMR spectrum became typical of a non-structured polypeptide, although the conformational preferences of the N-glycosidic linkage were unchanged, and most importantly, the Man8 oligosaccharide retained the average conformational behavior of the free oligosaccharide irrespective of the carrier protein fold. This conformational freedom is clearly not translated into full availability of the oligosaccharide for the carbohydrate-recognition protein. We propose, therefore, that the differing bioactivity of the N-glycan is a reflection of the existence of different geometries of presentation of the carbohydrate determinant in relation to the protein surface within the glycan:carrier protein ensemble.
Key words: carbohydrate recognition/conformational analysis/conglutinin/oligosaccharide presentation/ribonuclease B
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentsAbbreviationReferences |
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Carbohydrate recognition by proteins is acknowledged to play a role in a considerable number of physiological and pathological processes such as the mechanisms of inflammation and host defense (Drickamer and Taylor, 1993
). The endogenous lectins may distinguish not only between different oligosaccharide structures, but also between different presentations of oligosaccharides in the context of specific carrier proteins (Crocker and Feizi, 1996).
The serum proteins conglutinin and mannan-binding protein are soluble carbohydrate-binding receptors of the innate immune system, and are thought to promote clearance of bacteria and yeasts through their interactions with carbohydrates at the surface of the infectious agents (Holmskov et al., 1994). The two carbohydrate-binding receptors contain homologous, "C-type," carbohydrate-recognition domains (CRDs) and show qualitatively similar specificity toward high mannose N-glycans, but only conglutinin binds to iC3b, a glycoprotein which is a proteolytically pruned form of the major serum complement glycoprotein C3, containing saccharides of this type (Childs et al., 1989; Solís et al., 1994). This interaction is mediated by recognition of the Man8 or Man9 N-glycan at Asn-917, as presented on iC3b but not on the parent glycoprotein C3 nor on the further proteolysed glycoprotein fragment C3c on which the Man8/9 N-glycan is preserved (Solís et al., 1994). A similar phenomenon occurs with the glycoprotein ribonuclease B (RNaseB), which contains at a single glycosylation site, Asn-34, one or other of the high-mannose N-glycans, Man5-9, their relative molar proportions being 57, 31, 4, 7, and 1%, respectively (Fu et al., 1994). On the native glycoprotein, the oligosaccharides are not bound by conglutinin and mannan-binding protein, whereas binding occurs when the protein is reduced and denatured (Solís et al., 1994). RNaseB is a small glycoprotein (124 amino acid residues) and is an ideal model to probe in depth the structural basis of presentation of oligosaccharide for recognition by conglutinin.
The three-dimensional structure of the non-glycosylated RNase form (RNaseA) and of RNaseB has been investigated by x-ray diffraction and NMR. No significant differences have been observed (Williams et al., 1987; Joao et al., 1992) indicating that the N-glycan has no effect on the average conformation of the protein moiety. Moreover, 13C NMR spectroscopy of RNaseB-Man5-6 indicated that there are no interactions between the oligosaccharide and the protein (Berman et al., 1981). Glycosylation does alter, however, the stability and unfolding kinetics of ribonuclease (Joao et al., 1992; Arnold and Ulbrich-Hofmann, 1997), and, importantly, recognition of the oligosaccharide by several lectins and enzymes is crucially influenced by the folded/unfolded state of the protein (Williams and Lennarz, 1984; Solís et al., 1994).
Here we have compared the binding of conglutinin to RNaseB-Man8, with the Man5-6 form, when the protein is in the native and in the reduced and denatured state. The N-glycan on the Man8 form is one of the preferred ligands for conglutinin. We show that conglutinin binds strongly to the Man8 oligosaccharide on reduced and denatured RNaseB but not on the native glycoprotein. Pursuing the molecular basis of this different oligosaccharide bioactivity, we have isolated in relatively large scale (milligram amounts) the RNase-Man8 glycoform, for which conformational information is not available so far, and have examined, by a combination of NMR and molecular dynamics calculations, the conformation of the Man8 oligosaccharide on the native and on the reduced and denatured RNaseB.
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Results and discussion |
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Conglutinin binds to the Man8 oligosaccharide on reduced and denatured RNaseB
Previous binding studies (Solís et al., 1994
) with high mannose-type neoglycolipids derived from individual Man5-9 N-glycans showed binding of conglutinin predominantly to the Man7, Man8, and Man9 species, which are minor components of total RNaseB oligosaccharides, rather than to the major components, Man5-6. To dissect the influence of carrier protein on recognition of RNaseB oligosaccharides by conglutinin, we performed microwell and nitrocellulose overlay assays (Figure 1), using equimolar amounts of the isolated RNase-Man8 and RNase-Man5-6 glycoforms before and after reduction and denaturation. With the non-reduced glycoproteins, weak binding was detected only to RNaseB-Man8 after electrophoresis in SDS, and electrotransfer (Figure 1a, panel B). After reduction and denaturation of the proteins, however, strong binding was observed to Man8 immobilized either on nitrocellulose (Figure 1a, panel C) or on microwells (Figure 1b) and weak binding to Man5-6. This is in accord with the reactivities observed with the oligosaccharides in the absence of the carrier protein (Solís et al., 1994). Thus, the protein moiety exerts a qualitatively similar influence on the presentation of the Man8 and Man5-6 oligosaccharides for recognition by conglutinin.
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Inhibition experiments of the binding of 125I-conglutinin to reduced and denatured RNase-Man8 using liquid-phase native RNaseB and reduced heat-denatured RNaseB as inhibitors (Table I) were in accord with the results of the binding patterns observed to the glycoproteins immobilized on plastic microwells. In the presence of dithiothreitol, RNaseB retained the three-dimensional structure of the native protein, as evidenced by NMR spectroscopy analysis (see below), and accordingly, the inhibitory activity of the glycoprotein toward conglutinin binding at the levels tested (10 mg/ml) was only 9% as compared with
70% inhibition exerted by reduced heat-denatured RNaseB. Due to the multivalent binding ability of conglutinin, any kind of self-aggregation of the reduced heat-denatured glycoprotein could potentially result in multivalent recognition with a concomitant increase in the binding avidity. However, no evidence for such an aggregation has been observed. On gel filtration chromatography, both native and reduced heat-denatured RNaseB eluted as a major peak (about 97% of the protein) with an apparent molecular mass of 15 kDa corresponding to the monomeric glycoprotein (results not shown).
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The binding of conglutinin to reduced and denatured RNase-Man8 was inhibited by mannose and N-acetylglucosamine (Table I). Comparable levels of inhibition were observed when these monosaccharides were used as inhibitors of conglutinin binding to immobilized invertase, a highly glycosylated protein which is generally assumed to present only carbohydrate ligands to the lectin. Furthermore, no significant differences in conglutinin binding to either reduced heat-denatured RNase-Man8 or invertase were observed under various conditions typically promoting (high ammonium sulfate concentrations) or weakening (low salt concentrations, water-miscible alcohols or detergents) hydrophobic interactions (Table I). Thus, the binding of conglutinin to reduced and denatured RNase-Man8 seems to be exclusively carbohydrate-mediated and no additional hydrophobic protein–protein interactions appear to be involved.
The Man8 oligosaccharide exhibits a similar average conformation on native RNaseB and on the reduced and denatured protein
There were no significant differences in the NOE patterns of the protein cross peaks of the isolated RNase-Man8 (Figure 2a) and -Man5-6 (not shown) glycoforms relative to RNaseA. Therefore, the protein moiety of RNase-Man8 exhibits a three-dimensional structure essentially identical to that previously found for the non-glycosylated protein (Santoro et al., 1993). A similar conclusion can be drawn from these and previous studies (Joao et al., 1992) for the major RNase-Man5-6 glycoform. Thus, no differences are apparent in the protein moiety of the individual glycoforms that could be related to the different processing of the oligosaccharide in these glycoforms. In the presence of dithiothreitol, no significant changes in the chemical shift dispersion characteristic of the native protein were observed. However, after incubation at 65°C, the NMR spectra of the resulting reduced heat-denatured RNaseB-Man8 (Figure 2b) were characterized by limited dispersion of chemical shifts, coupling constants and smaller NOEs, close to random coil values, which is indicative of a great motion as expected for a non-structured polypeptide.
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By TOCSY and NOESY NMR experiments all the spin systems of the sugar residues could be assigned and a number of sequential and remote NOE contacts between different monosaccharides deduced (Figure 3). For both the native (Figure 2c) and the reduced heat-denatured (Figure 2d) RNaseB-Man8 glycoprotein, the protein-bound oligosaccharide exhibited 1H chemical shifts and NOE data very similar to the free (-D2)Man8 oligosaccharide glycomer investigated previously (Wooten et al., 1990
; Fu et al., 1994). Possible oligosaccharide conformations compatible with the NMR data were explored by molecular modeling. Full NMR data and details of the MD simulations have been described elsewhere (González et al., forthcoming). The best adjustment between modeled and experimental data was found for the simulation of the gtgt conformation of the two 
1
6 linkages. In particular, the observed long range NOE between ManA-H1 and GlcNAc2-methyl group indicates that the gt conformation of the Man4'(16)Man3 linkage, where the Man4' arm is folded back toward the trisaccharide core, is significantly populated. Thus, both on the native and on the reduced heat-denatured protein, the RNaseB-Man8 oligosaccharide exhibits a conformational behavior similar to that described for the free oligosaccharide (Wooten et al., 1990), with the Man4'-ManA arm folding in and out over the rest of the glycan chain, thus approaching and moving far away from the polypeptide backbone.
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NOEs to the protein were only detected for GlcNAcl, and all of them were to the Asn34 protons, as reported similarly for the N-glycan at Asn52 of the free
subunit of human chorionic gonadotropin (De Beer et al., 1996), showing that GlcNAc1 is solvent exposed. NOE data for the N-glycosidic linkage in native RNaseB-Man8 (Figure 3, inset) indicate that the GlcNAc1-C1H/Asn34-N
H and Asn34-NH/C
O bonds are in the trans conformation. The GlcNAc1-NAcH conformation is also trans. The same conclusions were drawn for the reduced heat-denatured glycoprotein. Only the weak NOE from Asn34-NH to GlcNAc1-amide NH assigned in native RNaseB-Man8 was not detected in the spectra of the reduced heat-denatured protein, possibly a reflection of the high degree of disorder exhibited by the protein moiety. Overall, the three-dimensional architecture of the native RNaseB-Man8 glycoprotein is well defined. A model generated from the protein and oligosaccharide NMR structures is shown in Figure 4. The intrinsic conformational properties of the oligosaccharide are preserved on reduced heat-denatured RNaseB-Man8. However, the disorder exhibited by the protein moiety implies that there is a repertoire of oligosaccharide orientations with respect to the protein surface. Thus, the oligosaccharide–protein ensemble contains several very different geometries (Figure 4, inset).
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On the structural basis of selective recognition by conglutinin of the RNaseB oligosaccharide
Conglutinin recognizes predominantly the terminal
(12)-linked mannose units (Young and Leon, 1987) which are fully exposed on the native RNaseB-Man8. Yet, the affinity of conglutinin for the N-glycan on the native glycoprotein is not high enough for efficient binding. Mannan-binding protein behaves similarly. Crystallographic studies on recombinant mannan-binding protein CRD in complex with an asparaginyl Man6 oligosaccharide (Weis et al., 1992) have shown that, in addition to the direct interaction with the terminal mannose, there are water-mediated interactions of the mannan-binding protein with internal sugars of the oligosaccharide which may play a significant role in binding. When the oligosaccharide is on a carrier protein, the ability to form these water-mediated lectin–glycan chain interactions may be crucially dependent on the precise geometry of the oligosaccharide–carrier protein ensemble. Thus, the observed differences in binding to native and to reduced heat-denatured RNaseB could arise from differences in the oligosaccharide presentations, that is, oligosaccharide orientations relative to the carrier protein surface that are available to the carbohydrate-binding proteins.
The extent to which oligosaccharide presentation determines recognition by different carbohydrate-binding proteins will depend on the specific primary and secondary requirements of each recognition protein. For example, binding of calnexin and trimming by glucosidase II of monoglucosylated RNase Man7-Man9 glycoforms in the early stages of glycan processing have been reported to be independent of the conformation of the glycoprotein (Zapun et al., 1997). In contrast, the processing enzymes in preparations of Golgi membranes from bovine pancreas can process into complex type chains the high-mannose chains on the reduced and alkylated RNaseB, but not on the native protein (Williams and Lennarz, 1984).
It has been proposed that recognition of specific protein-constrained oligosaccharide conformations may play a role in the control of N-linked oligosaccharide biosynthesis and carbohydrate-mediated recognition processes (Carver, 1993). The present study suggests that different carrier protein-related presentations of a conformationally free oligosaccharide may also modulate recognition.
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Materials and methods |
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RNaseB glycoforms
Fractionation of RNaseB glycoforms has been described in detail elsewhere (González et al., forthcoming). In brief, RNaseB-Man8 and a mixture of RNaseB-Man5-6 were isolated by gradient-elution affinity chromatography of RNaseB (Sigma) on concanavalin A–Sepharose and the composition of the fractions was determined by capillary electrophoresis and electrospray ionization mass spectrometry. Reduced and alkylated RNase-Man5-6 and RNase-Man8 were prepared as described previously (Solís et al., 1994
). Reduced and heat-denatured glycoproteins were prepared by incubation with 10–15 mM dithiothreitol at 65°C for 10 min. The aggregation state of reduced heat-denatured RNaseB was checked by gel filtration chromatography on a Superose 12 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated with 10 mM Tris/HCl, pH 7.8, 0.15 M NaCl, 15 mM DTT. The flow rate was 0.5 ml/min and the elution was monitored at 280 nm. Native RNaseB as well as standard proteins for column calibration were chromatographed under similar conditions.
Conglutinin binding assays
Conglutinin isolated from heat-inactivated bovine serum (Lachmann, 1962) was radioiodinated using IODO-GEN (Pierce Eurochemie) following the manufacturer’s recommendations.
Nitrocellulose binding assays with 125I-labelled conglutinin were carried out essentially as described (Solís et al., 1994), except that the buffer used was 10 mM Tris/HCl, pH 7.8, containing 0.15 M NaCl. Prior to overlay with labeled lectin, the amount of nonreduced and reduced glycoproteins electrotransferred onto nitrocellulose was checked by densitometric quantitation of the protein bands after Ponceau S staining, using an Imaging densitometer GS-670 from Bio-Rad (Hercules, CA). Equivalent amounts (4.8 ± 0.2 µg) of the two protein forms were detected.
Microwell binding assays were carried out also as described (Solís et al., 1994). Unless otherwise specified, the buffer used was 10 mM Tris/HCl, pH 7.8, 0.15 M NaCl. The adsorption efficiency of the native glycoproteins compared to the reduced and denatured proteins was evaluated by kinetic silver staining (Root and Wang, 1993) of wells coated in parallel with 100 µg/ml solutions of the proteins. After addition of the silver stain reagent to the wells, time courses for silver staining were followed by measuring the absorbance at 405 nm at 5 min intervals, using a Bio-Rad 3550 microplate reader. Both the lag time and the rate of stain development for wells coated with native and with reduced and denatured glycoproteins were similar, indicating that comparable amounts of the proteins were adsorbed onto the wells.
NMR experiments
Native glycoproteins were dissolved at 1.5 mM in 0.5 ml H2O:D2O (9:1 by vol.) or D2O, adjusted to pH 4.0. Reduced heat-denatured glycoproteins were prepared as described above using 10 mM deuterated dithiothreitol. Data were collected at 35°C, using sodium 3-trimethylsilyl(2,2,3,3–42H4) propionate as internal reference.
NMR experiments were performed on a Bruker AMX-600 spectrometer. Water suppression was achieved by including the WATERGATE module (Piotto et al., 1992) in the original pulse sequence. Conventional 1D and 2D pulse sequences and phase-cycling procedures were used.
Molecular dynamics simulations
The AMBER-Homans (Homans, 1990) and Sybyl (Imberty et al., 1991) programs were used for the calculations. Starting oligosaccharide conformations were generated using gg and gt conformations (Bock and Duus, 1994). For both force fields, four independent unrestrained and four independent restrained runs (using NMR-derived interproton distances) were carried out.
A three-dimensional model of RNase-Man8 was generated using the MD-derived structure for the gtgt conformer of the Man8 oligosaccharide and the previously deduced NMR structure of RNaseA (PDB code 2AAS). The MD-derived oligosaccharide structure was attached to the Asn-34 side chain, and submitted to a short (20 ps) restrained MD period, by including the distances estimated from the observed NOEs between the protons at GlcNAc1 with those at Asn-34. The polypeptide was kept rigid during the MD. As an approach to generate a qualitative structure of the reduced and denatured glycoprotein, the disulfide bridges in the RNaseB-Man8 model were manually removed and the resulting structure was minimized using AMBER and then subjected to a high temperature (1000 K) MD run (50 ps). Several snapshots from this simulation were taken.
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Acknowledgments |
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We thank R. W. Loveless for help with the conglutinin binding studies and H. Kogelberg for helpful discussions. This work was supported by the Dirección General de Investigación Científica y Técnica (PB93-0189 and PB96-0833), a Programme Grant (GP601454) from the Medical Research Council, and by travel grants (HB93-114 and HB96-0106) from the Acciones Integradas Hispano-Británicas programme.
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Abbreviation |
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RNaseB, ribonuclease B.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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