Chemical structure of neutral sugar chains isolated from human mature milk kappa-casein

The carbohydrate chains linked to human kappa-casein from mature milk were released by alkaline borohydride treatment as reduced oligosaccharides. The neutral oligosaccharides of lower molecular weight were fractionated and purified by gel filtration and preparative thin layer chromatographies. Seven neutral oligosaccharides (a di- (0.5%), two tetra- (30.5%), two penta- (5.4%) and two hexasaccharide alditols (10.9%] were obtained in homogeneity, and followed by methylation analysis with gas-liquid chromatography-mass spectrometry and by anomer analysis with 13C nuclear magnetic resonance. Their chemical structures were identified to be Gal beta 1----3GalNAc-ol (I), Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (II), Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (III), GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (IV), GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (V), Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (VI) and Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (VII). Five oligosaccharide alditols (III-VII) were the novel carbohydrate chains of kappa-casein from mammalian milk.     

Isolation and structural characterization of the smaller-size oligosaccharides from desialylated human kappa-casein. Establishment of a novel type of core for a mucin-type carbohydrate chain

Alkaline borohydride reductive cleavage (beta-elimination) of desialylated human kappa-caseinoglycopeptide resulted in the release of a series of oligosaccharides. The smaller-size compounds among them were purified to virtual homogeneity by gel filtration followed by high-performance liquid chromatography. The structures of 9 oligosaccharides were determined by 1H-NMR spectroscopy in conjunction with sugar analysis. The tetrasaccharide Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol and various partial structures thereof were characterized. Notably, the disaccharide GlcNAc beta(1----6)GalNAc-ol and the trisaccharide Gal beta(1----4)GlcNAc beta(1----6)GalNAc-ol were identified; they represent a novel type of core structure for mucin-type carbohydrate chains, namely a peptide-linked GalNAc that is mono-substituted at C-6. In addition, some oligosaccharides ending in GlcNAc-ol could be characterized. Their possible origin is discussed.     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity

New neutral oligosaccharides from cow colostrum kappa-casein were identified and characterized by 500-MHz 1H-NMR spectroscopy. Their structures are Gal beta(1----3)GalNAc-ol, Gal beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Fuc alpha(1----3)[Gal beta(1----4)]GlcNAc beta(1----6)]GalNAc-ol. The tetrasaccharide and the cow colostrum kappa-caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow kappa-casein as a function of time after calving is studied.     

The structure of the O-glycosidic oligosaccharide chains of the major Zajdela hepatoma ascites-cell-membrane glycoprotein

Glycoprotein MII2, the major cell surface glycoprotein (molecular mass 110 kDa) of Zajdela hepatoma ascites cells, contains about 25 O-glycosidic oligosaccharide chains per molecule. They were released as oligosaccharide-alditols by alkaline borohydride treatment of MII2, and purified by gel filtration on Bio-Gel P-6 followed by high-voltage paper electrophoresis. Four oligosaccharide-alditol fractions (A-D) were obtained in relative yields of 8:6:3:3. The structure of the components of fractions A-C was determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis, to be as follows. (A) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B1) NeuAc alpha(2----3)Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B2) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (C) NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol. On the basis of sugar composition and characteristics on Bio-Gel P-6 filtration, paper electrophoresis and thin-layer chromatography, the structure of the carbohydrate component of fraction D is proposed to be as follows. (D) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol     

Structural analysis of O-glycosidic type of sialyloligosaccharide-alditols derived from urinary glycopeptides of a sialidosis patient

Sialidosis urine was fractionated by gel filtration on Bio-Gel P-6. All pooled fractions containing carbohydrates showed the presence of small amounts of GalNAc in non-reducing position, besides free N-acetyllactosamine type of oligosaccharides as major constituents. The fractions were subjected to reductive alkaline borohydride degradation, after which the major part of GalNAc was recovered as N-acetyl-D-galactosaminitol (GalNAc-ol). The GalNAc-ol-containing material was separated from the N-glycosidic oligosaccharides by a second gel-filtration step on AcA 202. Subsequently, the O-glycosidic sialyloligosaccharide-alditols were subfractionated by anion-exchange chromatography on Mono Q. Structural analysis by 500-MHz 1H-NMR spectroscopy revealed two major components in all fractions, namely: NeuAc alpha 2-3Gal beta 1-3GalNAc-ol and NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc-ol. Furthermore, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6]GalNAc-ol was found as a minor component in some of the fractions. The presence of these carbohydrate chains in Bio-Gel fractions differing in molecular mass suggested that they are derived from glycopeptides which are heterogeneous in their peptide part.     

Further characterization of allergenically active oligosaccharitols isolated from a sea squirt H-antigen.

Complete primary structures of five allergenically active oligosaccharitols (HPG-beta 2-N5a, -N6, -N7a, -N7b, and -N9) derived from a sea squirt H-antigen were studied. Structural characterization was carried out by a new method in which products of limited periodate oxidation, followed by derivatization with p-aminobenzoic acid ethyl ester, were analyzed by a combination of HPLC, fast atom-bombardment mass spectrometry, sequential glycosidase digestion, methylation analysis, and 500-MHz 1H NMR. Established structures of GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3) GlcNAc beta 1-3GalNAc-ol, GalNAc beta 1-4GlcNAc beta 1-3 (GalNAc beta 1-4GlcNAc beta 1-6) GalNAc-ol, GalNAc beta 1-4GlcNAc beta 1-3[GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-6] GalNAc-ol, GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-3[GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-6] GalNAc-ol, and GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3)GlcNAc beta 1-3 [GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3)GlcNAc beta 1-6]GalNAc-ol are represented by HPG-beta 2-N5a, -N6, -N7a, -N7b, and -N9, respectively. These structures have not been encountered previously. Oligosaccharide units GalNAc alpha 1-2Fuc alpha 1-, GalNAc beta 1-4GlcNAc beta 1-, and Fuc alpha 1-3GlcNAc beta 1- are considered to be the allergenically specific epitopes. Partial assignments of 500-MHz 1H NMR spectra of these novel O-linked oligosaccharitols were attempted.     

Presence of an O-glycosidically linked hexasaccharide in fetuin

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.     

Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy

The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.     

GalNAc beta 1----3 terminated glycosphingolipids of human erythrocytes

Nonacid glycosphingolipids with 4 to 10 sugar residues isolated from pooled erythrocytes of blood group O donors have been efficiently separated as peracetylated derivatives on silicic acid. This procedure enabled a quantitative estimate of individual compounds and also revealed several GalNAc beta 1----3 terminated structures. The structural characterization of these glycolipids with 1H-NMR spectroscopy, direct inlet mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry identified the compounds as GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl sphingosine and GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl phytosphingosine, GalNAc beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 ceramide, and GalNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.     

Sulfated N-linked carbohydrate chains in porcine thyroglobulin

N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3[(SO3Na----3)Gal beta 1----4GlcNAc beta1----2-Mana alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc-ol and NeuAc alpha 2----6Gal beta 1----4(SO3Na----)0-1 GlcNAc beta 1----2-Man alpha 1----3[NeuAc alpha 2----6Gal beta 1----4(SO3Na----6)1-0GlcNAc beta 1----2Man alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc- ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity.     

Structures of mucin-type sugar chains of the galactosyltransferase purified from human milk. Occurrence of the ABO and Lewis blood group determinants

The mucin-type sugar chains of human milk galactosyltransferase samples purified from two donors with different blood types were released by alkaline borohydride treatment and quantitatively labeled by N-[3H]acetylation. The radioactive oligosaccharides thus obtained were fractionated by high performance liquid chromatography and immobilized lectin chromatography, and their structures were studied by sequential digestion with endo- or exoglycosidases, methylation analysis, and periodate oxidation. It was revealed that the structures of the mucin-type sugar chains of galactosyltransferase are extremely various, and many blood group determinants are expressed on more than 13 different backbone sugar chains. The characteristic features of the sugar chains could be summarized as follows. 1) The sugar chains of both samples are composed of core 1, Gal beta 1----3GalNAc, and core 2, GlcNAc beta 1----6(Gal beta 1----3)GalNAc. 2) One or two N-acetyllactosamine repeating units extend from the core through GlcNAc beta 1----6Gal and GlcNAc beta 1----3 Gal linkages. 3) Blood group determinants are expressed in accord with the blood types of the donors: sample 1 from a donor of blood type O, Lea+b- contains oligosaccharides with Lea and X determinants, and sample 2 from a donor of B, Lea-b- contains those with H, X, Y, and B determinants.     

Isolation of a novel O-linked, sulfated polysaccharide of high molecular weight from an ovarian cyst glycoprotein having blood group "A" activity

Treatment of a blood group A-active ovarian cyst mucin glycoprotein with alkaline borohydride under conditions expected to cleave O-glycosidic linkages between carbohydrate and peptide releases a sulfated polysaccharide of average molecular weight 20,000. Its peptide and mannose content is less than 1%, and carbohydrate analysis gives Fuc/GalNAc/Gal/GlcNAc in the ratio of 1:1:2.2:2.2. Galactosaminitol is recovered at the level of one residue per 112-residue average polysaccharide chain. The 13C- and 1H-NMR spectra show that the polysaccharide has side chains whose non-reducing terminals have the blood group A structure on a type 1 chain: (Formula: see text). Methylation analysis confirms the presence of these blood group A type 1 sidechains as well as 4-substituted GlcNAc, 3-substituted galactose and 3,6-substituted galactose branch points. Periodate oxidation removes all the fucose and GalNAc from the non-reducing terminal but leaves intact the backbone composed of beta-linked Gal and GlcNAc, as would be expected for a polylactosamine. Although the native polysaccharide is resistant to endo-beta-galactosidase digestion, the product of periodate degradation is partially digested, giving a 30% yield of a trisaccharide shown by 1H-NMR spectroscopy to be: Gal(beta 1----3)GlcNAc(beta 1----3)Gal We conclude that this is a high molecular weight sulfated polysaccharide which is related to the asparagine-linked polylactosamine chains of cell surface glycoproteins which have been implicated in cell differentiation. However, the blood group A polysaccharide from the ovarian cyst mucin is unique in several respects. It is linked to the protein by an O-glycosidic bond rather than the N-asparagine linkage of the previously known polylactosamines which have a trimannosyl core, and its blood group A side chains are on a type 1 core rather than type 2 which is found on other polylactosamines.     

Carbohydrate determinants associated with carcinoembryonic antigen (CEA)

The reactivities of eight purified preparations of carcinoembryonic antigen with monoclonal antibodies directed to tumor-associated carbohydrate determinants have been studied. All eight preparations showed strong reactivities with AH6, which defines Y structure (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3] GlcNAc beta 1----R), whereas only a few preparations showed reactivity with FH4-defining dimeric X determinants, (Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4 [Fuc alpha 1----3]GlcNA beta 1----3Gal beta 1----R). No other antibodies tested showed any reactivity with these preparations. These carbohydrate markers associated with carcinoembryonic antigen will be useful to enhance the diagnostic value of the antigen.     

The structures of the carbohydrate moieties of bovine blood coagulation factor X

Bovine blood coagulation factor X contains both asparagine-linked and threonine-linked oligosaccharides. The asparagine-linked chain is a mixture of a tridecasaccharide NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and a dodecasaccharide NeuAc alpha 2 leads to 6 Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and their partial desialylation products. The threonine-linked chain is a mixture of NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc, NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuGly alpha 2 leads to 6)GalNAc, NeuGly alpha 2 leads to 3Gal beta 1 leads to 3 (NeuAc alpha 2 leads to 6)GalNAc, and NeuGly alpha 2 leads to 3Gal beta 1 leads to 3(NeuGly alpha 2 leads to 6)GalNAc, and their partial desialized forms. The carbohydrate moieties of the factor X subgroups, factors X1 and X2, are identical.     

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.     

Structure elucidation of sulphated oligosaccharides from recombinant human tissue plasminogen activator expressed in mouse epithelial cells.

Sulphated N-linked carbohydrate chains isolated from recombinant human tissue plasminogen activator expressed in mouse epithelial (C127) cells were analysed as oligosaccharide alditols by methylation analysis, liquid secondary ion mass spectrometry, and one- and two-dimensional 1H-NMR spectroscopy. The results demonstrate that the major component has the following novel structure: NeuAc-alpha 2-6Gal beta 1-4GlcNAc beta 1-2[NeuAc alpha 2-3Gal beta 1- 4GlcNAc beta 1-4]-Man alpha 1-3[NeuAc alpha 2-3(SO4-6)Gal beta 1- 4-GlcNAc beta 1-2Man alpha 1-6]-Man beta 1-4GlcNAc beta 1- 4[Fuc alpha 1-6]GlcNAc-o1.     

Carbohydrate moieties of peanut agglutinin receptors isolated from human gastric cancer cells

Receptors for peanut agglutinin (PNA) were isolated from Kato III human gastric cancer cells by affinity chromatography on PNA agarose, and were labeled by the galactose oxidase-NaB3H4 method. Alkaline NaBH4 treatment of the labeled receptors released two small oligosaccharide alcohols, which were identified as Gal beta 1----3GalNAc-ol and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc-ol. Higher oligosaccharides and glycopeptides of both N- and O-linked type were also detected, but they did not appear to bear PNA binding sites. The presence of oligo-N-acetyllactosamine units in the N-linked type sugars was indicated by endo-beta-galactosidase digestion.     

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.