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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Key words: carbohydrate recognition/conformational analysis/conglutinin/oligosaccharide presentation/ribonuclease B
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Introduction |
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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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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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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 oligosaccharideprotein ensemble contains several very different geometries (Figure 4, inset).
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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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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 manufacturers 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(2,2,3,342H4) 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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Abbreviation |
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Footnotes |
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References |
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