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The IgE of sera from patients with a history of allergy to oranges (Citrus sinensis) binds a number of proteins in orange extract, including Cit s 1, a germin-like protein. In the present study, we have analyzed its immunological cross-reactivity and its molecular nature. Sera from many of the patients examined recognize a range of glycoproteins and neoglycoconjugates containing beta1,2-xylose and core alpha1,3-fucose on their N-glycans. These reagents also inhibited the interaction of Cit s 1 with patients' sera, thus underlining the critical role of glycosylation in the recognition of this protein by patients' IgE and extending previous data showing that deglycosylated Cit s 1 does not possess IgE epitopes. In parallel, we examined the peptide sequence and glycan structure of Cit s 1, using mass spectrometric techniques. Indeed, we achieved complete sequence coverage of the mature protein compared with the translation of an expressed sequence tag cDNA clone and demonstrated that the single N-glycosylation site of this protein carries oligosaccharides with xylose and fucose residues. Owing to the presumed requirement for multivalency for in vivo allergenicity, our molecular data showing that Cit s 1 is monovalent as regards glycosylation and that the single N-glycan is the target of the IgE response to this protein explain the immunological cross-reactive properties of Cit s 1 as well as its equivocal nature as a clinically relevant allergen.

Fast atom bombardment mass spectrometry (FABMS) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) were applied to the investigation of the anomeric isomerism of synthetic trisaccharides consisting of xylose, galactose and sulfated fucose {Xyl1-->3Gal alpha 1-->3(4-OSO3Na)Fuc} and {Xyl1-->3Gal alpha 1-->4(3-OSO3Na)Fuc} and the linkage position of the sulfate group. It was possible to differentiate between various glycosidic linkages in several synthetic trisaccharides. The position of a sulfate group in synthetic methyl O-sulfo-alpha-L-fucopyranoside isomers was elucidated from the fragmentation patterns. Comparing the data from the synthetic sulfated trisaccharides with the spectra from the natural compound derived from glycan chains of the acrosome reaction-inducing substance (ARIS) from starfish, the anomeric structure and the position of the sulfate group in the natural sample were determined without ambiguity as Xyl beta 1-->3Gal alpha 1-->3(4-OSO3-)Fuc, in agreement with the result from an independent study based on nuclear magnetic resonance.

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz 1H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man3Xyl1GlcNac2, Man3Fuc1Xyl1GlcNac2; 3.4%).

Cell walls of Chlamydomonas gymnogama, shed during sexual mating, were collected and analyzed. Ultrastructural examination indicates that the walls are free of cytoplasmic contamination and that they exhibit a regular lamellate structure. The walls are composed of glycoprotein rich in hydroxyproline. The hydroxyproline is linked glycosidically to a mixture of heterooligosaccharides composed of arabinose and galactose. Altogether, the glycoprotein complex accounts for at least 32% of the wall. The amino acid composition of the walls is extraordinarily similar in widely different plant species. The implications of these similarities as well as the widespread occurrence of these glycoproteins are discussed.

Hemocyanin from the freshwater snail Lymnaea stagnalis is a high-molecular-mass copper-containing oxygen-transport protein, which occurs freely dissolved in the hemolymph. It is a glycoprotein containing fucose, xylose, 3-O-methylmannose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues as sugar constituents. The N-glycosidic carbohydrate chains of this glycoprotein were released by hydrazinolysis of a pronase digest and subsequently fractionated as oligosaccharide-alditols on Bio-Gel P-4 followed by Lichrosorb-NH2. Investigation with 500-MHz 1H-NMR spectroscopy, in conjunction with sugar and methylation analysis revealed the lowest-molecular-mass glycan chain to have the structure: (Formula: see text).

A glycoprotein unique to the cytoplasm of the unsporulated oocyst of Eimeria tenella has been purified and partially characterized. The protein has a molecular weight of 30,000, of which approximately 40% is carbohydrate. The carbohydrate portion of the molecule consists of glucose, galactose, mannose, xylose, glucosamine, and galactosamine, with no detectable sialic acid. The protein portion contains approximately 141 residues, being rich in hydrophilic amino acids with very few aromatic amino acids and no cystine. The protein comprises 14% of the total soluble protein of the unsporulated oocyst but has not been identified in the cytoplasm of any other developmental stage of the organism. Using polyacrylamide gel electrophoresis and a radioimmunoassay specific for the protein, it has been shown to disappear from the cytoplasm between the 15th and 20th hour of the 20-hour sporulation process. Subsequent immunofluorescence experiments have shown a reactive material as a component of the sporozoite membrane. These results indicate that the glycoprotein is a structural protein of the sporozoite membrane, apparently synthesized by the unsporulated oocyst and incorporated into the sporozoite membrane as one of the last steps involved in the sporulation process.

1. The isolation of two proteins from the seeds of kidney bean is described. 2. The individual steps in the purification procedure included: extraction of the seeds at pH9.0, dialysis, first against pH9.0 and then against pH5.0 buffers, high-voltage electrophoresis of the proteins soluble at pH5.0 and chromatography on Sephadex G-200, Sephadex G-75 and DEAE-Sephadex columns. 3. Of the two proteins isolated, the first and larger component was a glycoprotein and its carbohydrate part was mainly composed of d-mannose and d-glucosamine together with smaller amounts of arabinose, xylose and fucose. 4. The second protein component isolated was a trypsin inhibitor and was almost entirely devoid of sugars but contained a firmly bound pinkish-blue pigment. 5. The amino acid composition of the two proteins was determined. 6. The glycoprotein contained very little if any cyst(e)ine but was relatively rich in aromatic amino acids, whereas the trypsin inhibitor had an unusually high cystine content (nearly 15%) but was relatively poor in valine and in aromatic amino acids.

Dopamine-beta-hydroxylase (EC 1.14.2.1) has been isolated as a pure protein from bovine-adrenal glands. Although the molecular weight of the native protein is well established (290,000), sodium dodecyl sulfate-gel electrophoresis under dissociating conditions gave a single band with a molecular weight of about 75,000. The enzyme contains about 4% carbohydrate, which consists of residues of mannose, glucosamine, galactose, glucose, fucose, and sialic acid. The pure enzyme also contains about 4 atoms of copper per molecule. It is concluded that dopamine-beta-hydroxylase is a tetrameric glycoprotein.

(1) Polysaccharides were isolated from bovine liver capsule by extraction with 2m-potassium chloride followed by precipitation from 0.8m-potassium chloride with cetylpyridinium chloride. Chondroitin sulphate was eliminated by digestion with hyaluronidase. The yield of heparin was approx. 40% of that obtained after extraction of the papain-digested tissue. (2) The macromolecular properties of the hyaluronidase-digested polysaccharide were studied by gel chromatography on Sephadex G-200 of the intact, as well as of the alkali-degraded, material. The results suggested the presence of single heparin chains in addition to a dermatan sulphate proteoglycan. (3) A purified heparin preparation was analysed for amino acids and neutral sugars. Xylose, galactose and serine were found in amounts corresponding to 0.1, 0.2, and 0.4 residue/polysaccharide chain (mol.wt. 7400), respectively. It is suggested that the isolated material had been degraded by a polysaccharidase with endo-enzyme properties.