Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin.

The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text)

Red berries of Richadella dulcifica, which is a native shrub of tropical West Africa, have a property in modifying sour taste into sweet taste; e.g. lemons elicit a sweet taste after chewing pulps of the berries. Because of this unusual property, the berry has been called "miracle fruit." Recently the active principle of miracle fruit, which is called miraculin, was completely purified (l), and its amino acid sequence was determined (2). Miraculin was a glycoprotein with 191 amino acid residues, and its molecular weight calculated on the basis   (2). It was confirmed that each glycopeptide is highly pure and does not contain other glycopeptides (2). Preparation of Oligosaccharides by Glycopeptidase Digestion-N-Oligosaccharide glycopeptidase (almond) has broad substrate specificity with respect to the carbohydrate moiety; it has been shown that sialylated complex-type oligosaccharides as well as high mannose type and hybrid-type oligosaccharides can be released from a variety of glycoproteins. Any complexities arising from selective release of particular sugars by the glycopeptidase digestion have not so far been encountered (3, 5, and 6). The neutral sugar content of the obtained oligosaccharide fraction and those of the original protein were determined with the orcinol-H&SO4 reagent (14). It was confirmed on the basis of the results of the neutral sugar content determination that less than about 10% of the total carbo- hydrates of the original protein remained undigested by this procedure. The oligosaccharide fraction of each of two kinds of glycopeptides containing Asn-42 and Asn186, respectively, was also separately released by the glycopeptidase digestion. Fractionation of Oligosaccharides and Comparison of Distribution of Each Oligosaccharide between Two Glycosylation Sites-On reverse-phase HPLC (Fig. l), isocratic elution was performed in this case with 10 mM sodium phosphate buffer, pH 3.8, instead of gradient elution. Pyridylamino derivative of each oligosaccharide prepared from whole protein and two glycopeptides containing Asn-42 and Asn-186, respectively, was separated into more than five peaks. Oligosaccharides A-E shown on Fig. 1 were further examined for the homogeneity by use of an amide absorption Amide-80 column (Fig. 2). The elution positions of oligosaccharides on the Amide-80 column reflect primarily the molecular sizes of oligosaccharides.

It was revealed that oligosaccharides
A-E were all homogeneous not only on an ODS-silica column but also in terms of molecular size. Fig. 1, moreover, clearly shows that a signifcant degree of variety exist between two glycosylation sites, Asn-42 and Asn-186. Although several minor peaks are present in addition to the lettered components on Fig. 1, they were obtained in an amount too small for further structural analyses.

'H NMR Analyses of Oligosaccharides A-E-Chemical shift values for the anomeric protons and the methyl protons of oligosaccharides
A-E along with four reference compounds are compiled in Table I. Compounds a, c, and f from lactase, and that from bromelain are xylose-containing N-acetyllactosamine-type oligosaccharides of plant origin glycoprotein (3). 'H NMR spectral data for the H-l and methyl groups clearly indicate that the chemical shifts of oligosaccharides C, A, and E are all in good agreement with those obtained from bromelain, lactase a, and lactase f, respectively. The structures of oligosaccharides B and D, however, have not yet been proved to exist. Comparisons of the chemical shift data separately. They were subjected to a size fractionation on an amide-silica column. The values of elution positions (expressed as glucose unit numbers) of oligosaccharides on ODSand amide-silica columns were plotted on the two-dimensional sugar map prepared using 113 different oligosaccharide standards (6) (Fig. 3). The elution positions of oligosaccharides A, C, and E derived from miraculin coincide within the experimental error with those of reference compounds lactase a, bromelain, and lactase f, respectively. for B and D with those of reference compounds lactase c and lactase f clearly indicate that the substitutions of outer Nacetylglucosamine residue by galactose residue through p1-t 4 linkage and fucose residue through al-6 linkage cause the anomeric proton signals to shift the downfield, from 4.511 (lactase c) to 4.533 ppm (lactase f) for GlcNAc-5, from 4.546 (lactase c) to 4.576 ppm (lactase f) for GlcNAc-5'.
Oligosaccharides B and D each have 1 outer GlcNAc residue linking to galactose and Fuc-2 residues. The H-l resonance observed at 4.535 in oligosaccharide B is characteristic for GlcNAc-5. The chemical shift of an anomeric proton at 4.576 ppm in oligosaccharide D is characteristic for GlcNAc-5'. These results indicate that in oligosaccharides B and D, the outer GlcNAc residue links to Man-4 and Man-4', respectively.

Oligosaccharides
B and D, however, do not correspond to any oligosaccharide on the present map, indicating that they are new or unusual oligosaccharides.
Oligosaccharides A-E and reference compounds prepared from sycamore cell lactase and stem bromelain were always processed at the same time and in the same manner and their HPLC profiles were compared. Jack bean /3-galactosidase cannot release galactose residue from lactase f under usual conditions (5 milliunits of enzyme/500 pmol of substrate) because of the existence of fucose al-& residue attached to the neighbor GlcNAc residue. Furthermore, any commercial cu-L-fucosidase such as from bovine kidney, Fusarium oxysporum, Corynebacterium, and from Charonia lampas cannot release the fucose al-3 residue attached to the reducing end GlcNAc residue in oligosaccharides of plant origin, under usual conditions (20 milliunits of enzyme/500 pmol of substrate). It was observed that on incubation with cu-L-fucosidase (bovine kidney), each 1 mol of fucose residue is released from oligosaccharides B and D, and 2 mol are released from oligosaccharide E. After sequential digestion of a-L-fucosidase (bovine kidney) and P-galactosidase (jack bean), the elution positions of oligosaccharides B, D, and E were shifted on the map to the positions identical with those of the reference compounds lactase b-2, lactase b-1, and lactase C, respectively (Fig. 3). These results suggest that each 1 mol of fucose and galactose residue is released from both oligosaccharides B and D, and 2 mol of both fucose and galactose residues are released from oligosaccharide E. As shown in Fig. 3, after following /?-hexosaminidase (jack bean) digestion, oligosaccharides B, D, and E were all converted finally to the common structure, which is the same as lactase a, The chemical shift of the H-l proton of Man-4 of oligosaccharide D is identical with that for oligosaccharide A. By contrast, chemical shifts observed for Man-4 of oligosaccharides B and E are identical but are significantly different from those observed for oligosaccharides A and D. These results indicate that 1) in oligosaccharides A and D, Man-4 is at a non-reducing terminal and 2) an N-acetylglucosamine residue is bonded to Man-4 in oligosaccharides B and E. This situation is quite similar for the chemical shift of H-l proton of xylose residue, that is, in oligosaccharides A and D, the chemical shifts of H-l proton of xylose residue are 4. Technique-Each oligosaccharide which was clearly separated on ODS-silica column (Fig. 1) was collected 4GlcNAc structure was found for the first time (3). Since the xylose residue is specific in plant glycoprotein, the mechanism of biosyntheses of Xyl@1+2 and Fucal-3 residues are of great interest. Johnson and Chrispeels (11) and Kimura et al. (12) proposed independently different biosynthetic pathways for plant oligosaccharides. However, oligosaccharides B-E characterized in the present study cannot consistently be placed on either of the pathways. Unless more detailed information about substrate specificities of glycosyl transferases is available, it is virtually impossible to align these oligosaccharides in the order of biosynthesis. The function of the carbohydrate moiety of miraculin has not yet been elucidated. The functional role of the oligosaccharide moiety of the glycoprotein should be studied by using deglycosylated miraculin molecule. The taste modifying function of miraculin is completely damaged even in the mild condition such as a 7-h incubation at 37 "C in a phosphate buffer, pH 6.8 (data not shown). The release of oligosaccharide moiety by enzyme digestion, therefore, is difficult without loss of the miraculin activity. Sasaki et al. (13) reported that in erythropoietin, the peptide structures surrounding the glycosylation sites are important factors influencing the kind of carbohydrate chains formed. In miraculin molecule, threonine and proline residues are predominant and no charged groups are in the vicinity of Asn-42. By contrast, a number of phenylalanine residues and also amino acid residues with acidic and basic charged groups surround the Asn-186 position, It is difficult to draw any definite conclusion from the observed data about the relationship between the peptide structure and carbohydrate biosyntheses.