Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin

Laminin, a high molecular weight (1,000,000) glycoprotein component of basement membranes, was isolated from the EHS murine tumor as a noncovalent complex with entactin by lectin affinity chromatography using the alpha-D-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Compositional analysis showed that the affinity-purified laminin contained 25-30% carbohydrate by weight. Methylation analysis revealed that the oligosaccharides of laminin contained bi- and triantennary chains, the blood group I structure, and repeating sequences of 3Gal beta 1,4GlcNAc beta 1 units. Free oligosaccharides were derived from the asparagine-linked glycans of affinity-purified laminin by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. When fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose, 80% of the oligosaccharides passed through the column unretarded and a single peak corresponding to 20% of the oligosaccharides was adsorbed and specifically eluted with a linear gradient of 0-30 mM methyl alpha-D-glucopyranoside. Further fractionation of the Con A reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing alpha-D-galactopyranosyl unit. The Con A reactive oligosaccharides were subjected to sequential digestion with endo- and exoglycosidases, and the reaction products were analyzed by gel filtration chromatography on a column of Bio-Gel P4. We thereby obtained evidence for a variety of structures not previously reported to exist on murine laminin including hybrid biantennary complex and biantennary complex structures containing poly(lactosaminyl) repeating units. The poly(lactosaminyl) units occur either on one or on both branches of the biantennary chains, as well as in more highly branched blood group I poly(lactosamine) structures. All sialic acid is present as N-acetylneuraminic acid linked alpha 2,3 to galactose.     

Structures of the asparagine-linked sugar chains of laminin

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.     

Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin.

The carbohydrate binding specificity of Mr = 30,000 lectin (CBP30) from baby hamster kidney (BHK) cells has been studied by inhibition of binding of the radiolabeled lectin to asialofetuin-Sepharose using model oligosaccharides and glycopeptides. CBP30 binds type I or II Gal beta(1----3(4))GlcNAc chains but not Gal(beta 1----3)GalNAc. The inhibitory potency of straight chain polylactosamine structures or complex-type branched glycans is increased in proportion to the number of Gal(beta 1----3(4)) units present. Fucosylation or sialylation of terminal galactose residues or further substitution by (alpha 1----3)-linked galactose or N-acetylgalactosamine does not affect binding whereas substitution of the penultimate N-acetylglucosamine residue drastically reduces binding. Thus, blood group A, H type I or H type II structures, shows high affinity whereas Lex, Lea, and Leb structures bind poorly. CBP30 binds to murine Engelbreth-Holm-Swarm (EHS) tumor laminin and human amniotic fluid fibronectin but not human plasma fibronectin. Binding involves polylactosamine glycans as well as tri- and tetraantennary complex-type glycans present in EHS laminin and amniotic fluid fibronectin but absent in plasma fibronectin. Proteolytic fragments of EHS laminin (E1X/Nd, P1, E8, and E3) bind CBP30, but only fragment E8 supports attachment and spreading of BHK cells. BHK cell adhesion to EHS laminin or fragment E8 was not disturbed by CBP30-specific antibodies, but at relatively high concentrations (45 micrograms/ml) CBP30 inhibited spreading and partially attachment of cells on laminin.     

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.     

Structure of the carbohydrate units of human amniotic fluid fibronectin

Human amniotic fluid fibronectin was found to contain three types of carbohydrates: complex-type N-glycosidic glycans, lactosaminoglycans, and O-glycosidic glycans. The structures of the complex-type glycans were established by carbohydrate and methylation analysis, Smith degradation, sequential exoglycosidase treatments, lectin chromatography, and DEAE-Sephadex chromatography. Lactosaminoglycans were analyzed by fast atom bombardment mass spectrometry, and the O-glycosidically-linked oligosaccharides by gas-liquid chromatography-mass spectrometry and high-pressure liquid chromatography. The results show that amniotic fluid fibronectin contains 2 mol of biantennary and 2-3 mol of triantennary, complex-type N-glycosidic glycans. Unlike the N-glycosidic glycans of human adult plasma fibronectin, which contain only traces of fucose and are completely sialylated, the glycans from amniotic fluid fibronectin are fucosylated and only partially sialylated. The complex-type N-glycosidic glycans present in amniotic fluid fibronectin also include a fractional amount (0.1 mol) of glycans with a polylactosaminyl structure. In addition, 4 mol of O-glycosidic oligosaccharides, which have not previously been described in fibronectins, were found in amniotic fluid fibronectin. The major oligosaccharides in this fraction have the structures Gal beta 1----3GalNAcol, NeuNAc alpha 2----3Gal beta 1----3GalNAcol and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcol. O-glycosidically linked oligosaccharides were also detected in human adult plasma fibronectin but in smaller amounts than in amniotic fluid fibronectin. These results show that amniotic fluid fibronectin differs from plasma fibronectin with regard to the number of glycans attached to the polypeptide and that the glycans present in these two fibronectins differ in structure.     

Localization and structure of the asparagine-linked oligosaccharides of type IV collagen from glomerular basement membrane and lens capsule.

Analysis of the Sephacryl S-200 fractionated type IV collagen domains from bovine and human glomerular basement membranes (GBM) and calf anterior lens capsule (ALC) indicated that Asn-linked oligosaccharides are primarily or exclusively localized in the 7 S region, whereas the hydroxylysine-linked Glc alpha 1----2Gal disaccharides (Glc-Gal-Hyl) are present in all the major segments of the molecule (7 S, NC1, and helical domain); no Ser/Thr-linked saccharide were detected. The Asn-linked carbohydrate units observed in the 7 S domain (Mr approximately 300,000) occurred in a number equal to the 12 polypeptide chains constituting this cross-linked region, and this was consistent with lectin blots of the reduced electrophoretically resolved 7 S components. Fractionation of the N-glycanase and endo-beta-N-acetylglucosaminidase-released oligosaccharides by concanavalin A affinity and high performance liquid chromatography indicated that the Asn-linked carbohydrate occurred predominantly in the form of complex tri- and biantennary units, although submolar amounts of polymannose variants (Man5-7GlcNAc2) were also present in calf ALC and bovine GBM. Structural studies of the complex N-linked oligosaccharides employing hydrazine/nitrous acid fragmentation and glycosidase digestions indicated a pattern in which there was complete fucosylation of the innermost GlcNAc residue of the Man3GlcNAc2 core but only sparse substitution with capping groups of the nonrepeating N-acetyllactosamine branches. Whether tri- or biantennary, the oligosaccharides from bovine GBM contained only one capping residue, in the form of either NeuAc or alpha-D-Gal, whereas those from ALC had only a single alpha-D-Gal and no NeuAc; human GBM oligosaccharides were devoid of both NeuAc and alpha-D-Gal. The absence of terminal alpha-D-Gal in the human 7 S domain was reflected in its lack of reactivity with Bandeiraea simplicifolia I and from its failure to yield Gal alpha 1----3Gal beta 1----4 [3H]anhydromannitol after hydrazine/nitrous acid/NaB3H4 treatment. Application of the latter procedure to the collagen domains yielded, in addition to fragments from the N-linked oligosaccharides, a disaccharide (Glc alpha 1----2[3H]galactitol) derived from the Glc-Gal-Hyl units. The localization of Asn-linked carbohydrate units in the evolutionarily conserved 7S domain of type IV collagens suggests that these oligosaccharides may play a role in the assembly of the collagen network of basement membranes.     

Developmental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane.

The chemical structure of Band 3 glycopeptide prepared from erythrocytes of normal adult (blood group OI), umbilical cord vessels (Oi), and an i adult variant who fails to develop I antigen (Oi), has been compared. Band 3 glycopeptide of cord erythrocytes gave, on permethylation analysis, predominantly 2,4,6-tri-O-methylgalactose and 3,6-di-O-methyl-2-N-methylacetamido-2-deoxyglucose, whereas the same glycopeptide of normal adult erythrocytes gave much higher amounts of 2,3,4,6-tetra-O-methylgalactose and 2,4-di-O-methylgalactose as compared with that of cord erythrocytes. Band 3 glycopeptide from i adult showed the same methylation pattern as cord-Band 3 glycopeptide. In accordance with these results, Band 3 glycopeptide of cord and i adult erythrocytes were hydrolyzed to mostly small oligosaccharides by endo-beta-galactosidase from Escherichia freundii, whereas that of normal adult produced a number of oligosaccharides with various sizes which was caused by branched structures. Based on these results and structures of released oligosaccharides, the major developmental change of carbohydrate structure in the erythrocyte membrane is the conversion of linear repeating Galbeta1 leads to 4GlcNAcbeta1 leads to 3Gal to a branched Galbeta 1 leads to 4GlcNAcbeta 1 leads to 3 (R leads to 6) Gal structure. i individual may result from the lack of the branching enzyme.     

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.     

Location and characterization of the three carbohydrate prosthetic groups of human protein HC.

Three different carbohydrate prosthetic groups associated to three chymotryptic peptides, Q1, Q2 and Q3, were isolated from the reduced and carboxymethylated human protein HC. The first oligosaccharide forms an O-glycosidic linkage with a threonine residue at position 5 in the polypeptide chain of protein HC. The second and third carbohydrate prosthetic groups form N-linkages with asparagine residues at positions 17 and 96. Oligosaccharides present in Q1 contain 1 residue of NANA, 2 of GalNAc and 1 of Gal corresponding to the following structure: -O-GalNAc-GalNAc-Gal-NANA. Q2 contains 3 NANA, 9 GlcNAc, 2 Gal and 3 Man, and Q3 contains 2 NANA, 5 GlcNAc, 1 Gal and 2 Man. The sugar compositions of Q2 and Q3 oligosaccharides are compatible with that of the complex kind. The amount of oligosaccharides present in Q1, Q2 and Q3 corresponded respectively to 3.0%, 12.2% and 7.3% of the weight of protein HC. No difference was found between the carbohydrate composition of urinary and plasma protein HC.     

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.     

Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion

A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW ∼ 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galβ1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc) as the common core structure, and most of them contained Fucα1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galβ1-4GlcNAcβ1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.     

Structural characterization of N-linked oligosaccharides of laminin from rat kidney: changes during diabetes and modulation by dietary fiber and butyric acid

Carbohydrates of laminin, a family of large multidomain glycoproteins, have been implicated in various cellular activities including maintaining the protein structure, its function and also basement membrane integrity. During the course of our investigation, we observed that purified laminin from kidneys of control, diabetic, and dietary fiber- and butyric acid-treated diabetic rats showed differences in binding to extracellular matrix components. This prompted us to determine whether there are structural changes in laminin oligosaccharides. In this study, we have characterized a few major N-linked oligosaccharides isolated from purified laminin in various experimental groups, viz. normal, diabetic and diabetic rats fed with dietary fiber and butyric acid. Sugar composition, as identified by GLC, revealed the presence of mannose, galactose and N-acetylglucosamine. In order to study fine structures of the oligosaccharides, N-linked oligosaccharides of laminin were released by Peptide-N-glycosidase F digestion, end-labeled with 2-anthranilic acid and fractionated by lectin affinity chromatography. Furthermore, structural elucidation carried out by MALDI-TOF MS/MS analysis showed variations in the oligosaccharide sequence of laminin during diabetes which were altered by the feeding of dietary fiber and butyric acid.     

Characterization of oligosaccharides in milk of a mink, Mustela vison

Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.     

Chemical characterization of milk oligosaccharides of the koala (Phascolarctos cinereus)

Previous structural characterizations of marsupial milk oligosaccharides had been performed in only two macropod species, the tammar wallaby and the red kangaroo. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of the koala milk carbohydrate fraction were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of milk of the koala, a non-macropod marsupial, and characterized by ¹H-nuclear magnetic resonance spectroscopy. The structures of the neutral saccharides were found to be Gal(β1-4)Glc (lactose), Gal(β1-3)Gal(β1-4)Glc (3′-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3′,3″-digalactosyllactose), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I) and Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl lacto-N-novopentaose I), while those of the acidic saccharides were Neu5Ac(α2-3)Gal(β1-4)Glc (3′-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Gal (sialyl 3′-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), and Neu5Ac(α2-3)Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl sialyl lacto-N-novopentaose a). The neutral oligosaccharides, other than fucosyl lacto-N-novopentaose I, a novel hexasaccharide, had been found in milk of the tammar wallaby, a macropod marsupial, while the acidic oligosaccharides, other than fucosyl sialyl lacto-N-novopentaose a had been identified in milk carbohydrate of the red kangaroo. The presence of fucosyl oligosaccharides is a significant feature of koala milk, in which it differs from milk of the tammar wallaby and the red kangaroo.     

The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1

Two size classes of O-glycosidically linked oligosaccharides were liberated from glycoprotein E1 of mouse hepatitis virus (MHV) A59 by reductive beta-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Ac alpha 2----3 Gal beta 1----3 GalNAc) comprised 35% of the total carbohydrate side chains, while the remaining 65% of the oligosaccharides of E1 had the branched structure B: Neu5Ac alpha 2----3 Gal beta 1----3 (Neu5Ac alpha 2----6) GalNAc. Both oligosaccharides were linked to the E1 polypeptide via N-acetylgalactosamine, and 20% of the sialic acids present in E1 glycopeptides were found to consist of N-acetyl-9-mono-O-acetylneuraminic acid. The reported structures of the O-linked glycans are discussed in the context of the amino acid sequence of E1, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential O-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical O-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.     

Purification and structures of oligosaccharide chains in swine trachea and Cowper's gland mucin glycoproteins

Mucin glycoproteins were purified from extracts of swine trachea mucosa and Cowper's gland. The gelatinous extracts were solubilized by reduction and carboxymethylation and then purified by chromatography on Sepharose CL-6B and DEAE-Sepharose. The structure of some of the carbohydrate units in these glycoproteins were determined and compared. Alkaline borohydride treatment indicated that more than 85% of the carbohydrate chains in these glycoproteins were linked to serine or threonine residues in the polypeptide chain through O-glycosidic bonds with N-acetylgalactosamine. Reduced oligosaccharides released by treatment with alkaline borohydride were isolated by gel filtration on Bio-Gel P-6 and chromatography on DEAE-cellulose and paper. The structures of the oligosaccharides were established by methylation analysis, gas chromatography, and sequential hydrolysis with specific exoglycosidases. The major oligosaccharides in Cowper's gland mucin glycoproteins were sialylated short chains: NeuAc alpha 2,6GalNAcol and NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAcol. In marked contrast, branched chains containing a Gal beta 1,3(GlcNAc beta 1,6)GalNAc core unit were the major components of trachea mucin glycoprotein. Ten of these chains had the following structures: (Formula: see text).     

Chemical characterization of the lectin from the freshwater prawn Macrobrachium rosenbergii (De Man) by MALDI-TOF

The serum of the freshwater prawn contains a sialic acid specific lectin (MrL) that agglutinates erythrocytes from rat and rabbit, as well as some Gram negative and positive bacterial strains. In this work, we performed the chemical characterization of the MrL purified by affinity chromatography on stroma from rat erythrocytes and by ion exchange chromatography. In its active form, MRL is a dimeric glycoprotein with 9.5 kDa per subunit. The amino acid sequence of the lectin was deduced from peptides obtained after trypsin treatment by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis (MALDI-TOF). The predicted amino acid sequence of the lectin showed 54% homology with the hyperglycemic hormone from Macrobrachium rosenbergii. It also showed homology with the variable region of the human immunoglobulin kappa (22%) and lambda (27%) light chains. The lectin is a glycoprotein with 11% (w/w) carbohydrate content and is constituted by Gal, Man, GlcNAc, GalNAc and NeuAc in a molar ratio of 4:3:2:1:0.6. The primary structure of the carbohydrate chains of the lectin from the freshwater prawn was determined by affinity chromatography of MrL-glycopeptides on Con A and LCA lectin columns, which indicated that the main carbohydrate chains conforming the lectin are N-glycosidically linked. Man3 GlcNAc2.1 oligosaccharides were the most abundant structures with 57%) followed by Gal1.3 Man3 GlcNAc2.8 with 24%. Our results suggest that the freshwater prawn possess a lectin in the hemolymph plasma, related to those from the immunoglobulin superfamily.     

Structural characterization of the oligosaccharides of a human monoclonal anti-lipopolysaccharide immunoglobulin M

The oligosaccharide side chains of a human anti-lipopolysaccharide IgM produced by a human-human-mouse heterohybridoma were analyzed at each of its five conserved N-glycosylation sites. This antibody also has a potential sixth N-glycosylation site in the variable region of its heavy chain which is not glycosylated. The oligosaccharides were released by digestion with various endo- and exoglycosidases and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and fluorophore-assisted carbohydrate electrophoresis. The antibody has various complex- and hybrid-type oligosaccharide structures at Asn 171, various sialylated complex-type oligosaccharides at Asn 332 and 395, and high-mannose-type oligosaccharides at Asn 402 and 563. Of note is the presence in this human IgM of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 98:2 as determined using anion- exchange chromatography. Furthermore, we observed oligosaccharide structures containing Gal alpha (1,3)Gal that have not been reported as components of human glycoproteins.      

Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.     

The structure of the oligosaccharides of alpha3beta1 integrin from human ureter epithelium (HCV29) cell line

There is a growing line of evidence that glycosylation of alpha and beta subunits is important for the function of integrins. Integrin alpha3beta1, from human ureter epithelium cell-line HCV29, was isolated by affinity chromatography on laminin GD6 peptide. Characterization of its carbohydrate moieties was carried out using sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by Western blotting on Immobilon P and on-blot deglycosylation with peptide N-glycosidase-F. Profiles of N-glycans for each subunit were obtained by matrix-assisted laser desorption/ionization mass spectrometry. Our findings demonstrated, in both subunits of integrin alpha3beta1, the presence of complex type oligosaccharides with a wide heterogeneity. Bi- tri- and tetraantennary structures were the most common, while high-mannose type structures were minor. Also the presence of short poly-N-acetyllactosamine entities was shown. These results show that while the predominant oligosaccharides of both subunits are identical, some slight differences between them do exist.