Isolation and characterization of an N-acetylgalactosamine specific lectin from Salvia sclarea seeds

Crude extracts from Salvia sclarea seeds were known to contain a lectin which specifically agglutinates Tn erythrocytes (Bird, G. W. G., and Wingham, G. (1974) Vox Sang. 26, 163-166). We have purified the lectin to homogeneity by ion-exchange chromatography and affinity chromatography. The agglutinin was found to be a glycoprotein of Mr = 50,000, composed of two identical subunits of Mr = 35,000 linked together by disulfide bonds. The purified lectin agglutinates specifically Tn erythrocytes and, at higher concentrations, also Cad erythrocytes. Native A, B, or O red blood cells are not agglutinated by the lectin and, even after treatment with sialidase or papain, these cells are not recognized. Tn red cells present 1.45 X 10(6) accessible sites to the lectin which binds to these erythrocytes with an association constant of 1.8 X 10(6) M-1. On Cad red cells, 1.73 X 10(6) sites are accessible to the lectin which binds with an association constant of 1.0 X 10(6) M-1. The carbohydrate specificity of the S. sclarea lectin has been determined in detail, using well defined monosaccharide, oligosaccharide, and glycopeptide structures. The lectin was found to be specific for terminal N-acetylgalactosamine (GalNAc) residues. It binds preferentially alpha GalNAc determinants either linked to Ser or Thr (as in Tn structures) or linked in 1-3 to a beta GalNAc or to an unsubstituted beta Gal. Although more weakly, the lectin binds beta GalNAc residues linked in 1-4 to a beta Gal (as in Cad structures). It does not recognize beta GalNAc determinants linked in 1-3 to a Gal (as in globoside) or the alpha GalNAc residues of blood group A structures.     

Structure of the oligosaccharides of three glycopeptides from calf thymocyte plasma membranes.

The carbohydrate composition and oligosaccharide structure of three glycopeptides isolated from delipidated calf thymocyte plasma membranes following Pronase digestion have been determined. Five major glycopeptide fractions were separated using Bio-Gel P-6 gel filtration and diethylaminoethylcellulose chromatography. The structure of the oligosaccharide chains of three of these glycopeptides was determined by a combination of sequential degradation with glycosidases and methylation analysis. These oligosaccharide structures consist of complex, highly branched N-linked chains containing at their nonreducing termini the unusual sequence Gal(beta1 leads to 3)Gal(beta1 leads to 4)GlcNAc leads to as well as the more usual sequence SA(alpha2 leads to 3)Gal(beta1 leads to 4)GlcNAc leads to. In addition, one glycopeptide also contains short O-linked chains with the structure Gal(beta leads to 3)GalNAc leads to Ser(Thr) which have receptor activity for the lectin from the mushroom Agaricus bisporus.     

Structural studies of the asparagine-linked sugar chains of two immunoglobulin M's purified from a patient with Waldenstr?m's macroglobulinemia

The structures of the sugar chains present in two human monoclonal IgM molecules purified from the serum of a patient with Waldenstr?m's macroglobulinemia have been determined. The asparagine-linked sugar chains were liberated as oligosaccharides by hydrazinolysis and labeled by reduction with NaB3H4 after N-acetylation. Their structures were studied by serial lectin column chromatography and sequential exoglycosidase digestion in combination with methylation analysis. These two IgM's were shown to contain almost the same sugar chains. The sugar chains were a mixture of a series of high-mannose-type and biantennary complex-type oligosaccharides. The complex-type oligosaccharides contain Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc as their core and GlcNAc beta 1----, Gal beta 1----4GlcNAc beta 1---- and Neu5Ac alpha 2----6Gal beta 1----4GlcNAc beta 1---- groups in their outer chain moieties.     

Precipitation of concanavalin A by a high mannose type glycopeptide

The interactions of a high mannose type glycopeptide with Concanavalin A has been investigated by quantitative precipitation analysis. The equivalence points of the precipitin curves indicate that the glycopeptide is bivalent for lectin binding. These results and others demonstrate that there are two lectin binding sites per molecule of the glycopeptide: one site on the alpha (1-6) arm of the core beta-mannose residue involving a trimannosyl moiety, and another site on the alpha (1-3) arm of the core beta-mannose residue involving an alpha (1-2) mannobiosyl group. The two sites are unequal in their affinities, and bind by different mechanisms. These results are related to the possible structure-function properties of high mannose type of glycopeptides on the surface of cells.     

Purification of acetyllactosamine-specific tomato lectin by erythroglycan-sepharose affinity chromatography

Tomato lectin is specific for oligomers of poly-N-acetyllactosamine containing 3 repeating Gal(beta 1-4)GlcNAc (beta 1-3)-disaccharides. As such it is highly useful for purifying oligosaccharides or glycopeptides with poly-N-acetyllactosamine character. We have found the lectin very useful as an affinity reagent for isolating glycoproteins or glycoprotein domains having poly-N-acetyllactosamine glycosylation. Conventional preparation of tomato lectin by ovomucoid-Sepharose affinity chromatography was found to be unsatisfactory due to instability of column and bleeding of ovomucoid into eluents requiring the necessity for additional purification steps following affinity chromatography. We prepared a column of human erythrocyte band 3 carbohydrate glycopeptide (erythroglycan) attached to Sepharose as an affinity matrix. The purification of tomato lectin to homogeneity in one step on this column matrix is described in this report.     

Isolation and characterization of a family of alpha-D-galactosyl-containing glycopeptides from Ehrlich ascites tumor cells

A family of glycopeptides that contain nonreducing terminal alpha-D-galactosyl residues has been isolated from Pronase digests of delipidated Ehrlich ascites tumor cells. The glycopeptides, which comprise 17.2% of the total plasma membrane hexose, have an average molecular weight of 7500 and are precipitated by Griffonia simplicifolia B4 isolectin, wheat germ agglutinin, and Ricinus communis lectin. Exo- and endoglycosidase digestion, periodate oxidation, permethylation analysis, and lectin reactivity provided evidence for a tentative carbohydrate structure for the glycopeptide mixture. The glycopeptides possess tetraantennary branched structures containing a trimannosyl core N-glycosidically linked via an N,N'-diacetylchitobiosyl unit to an asparagine residue. Each branch contained repeating leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units resulting in a keratan-like structure, terminated with alpha-D-Galp-(1 leads to 3)-[alpha-D-Galp-(1 leads to 6)]-beta-D-Galp-units. The variation in the molecular weight observed for the glycopeptide mixture can be attributed to the variable amounts of leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units found in the branch chains.     

Isolation and characterization of cyanogen bromide fragments and a glycopeptide from the Dolichos biflorus lectin.

The 110000 molecular weight Dolichos biflorus lectin is a glycoprotein composed of four subunits of approximately 27000 molecular weight with one methionine residue per subunit (Carter and Etzler, 1975b). Cyanogen bromide cleavage of the lectin yielded two fragments with approximate molecular weights of 15000 and 12000 as determined by electrophoresis on sodium dodecyl sulfate gels. Only the 15000 molecular weight fragment stained for carbohydrate with the periodic acid-Schiff stain. The two fragments were isolated, and their amino acid compositions were determined. The 15000 molecular weight fragment was identified as the amino terminal segment of the lectin subunits by NH2-terminal amino acid analysis. A glycopeptide with a minimum molecular weight of 1100 was isolated from the lectin by exhaustive Pronase digestion. Complete acid hydrolysis of the glycopeptide yielded aspartic acid, mannose, and N-acetylglucosamine in the ratio of 1:4-5:1-2. Partial acid hydrolysis of the glycopeptide produced a component which had an identical mobility with commercial N-acetylglucosaminylasparagine in high voltage paper electrophoresis. The data indicate that the carbohydrate unit of the lectin is bound to the amino terminal half of the subunits by a glycosylamine linkage between N-acetylglucosamine and asparagine.     

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.     

Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma

The sugar chains of transferrin samples, purified from sera of patients with hepatocellular carcinoma and of healthy individuals, were released quantitatively as radioactive oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Comparative study of their structure by serial lectin column chromatography, by Bio-Gel P-4 column chromatography, and by sequential exoglycosidase digestion revealed that prominently altered glycosylation is commonly found in the hepatoma transferrins, although they all contain two complex-type asparagine-linked sugar chains in one molecule like in the case of normal transferrins. The alteration is quite various, including the increase of highly branched sugar chains, of those with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----groups in their outer chain moieties and of those with a fucosylated trimannosyl core. Many but not all of the hepatoma transferrin samples contained a small amount of a bisected biantennary sugar chain, which was not detected in the normal transferrin samples.     

Products of trypsin digestion of haptoglobin beta (heavy) chain

Trypsin digestion of haptoglobin beta (heavy) chain resulted in five glycopeptides. The glycopeptides were characterized by carbohydrate and sulphydryl groups content; their molecular mass was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. None glycopeptide possessed hemoglobin-binding capacity. glycopeptide I did not form any precipitate with antihaptoglobin serum but was shown to inhibit strongly the reaction of haptoglobin or beta chain with the antiserum. Glycopeptide II showed dominant antigenic determinants in relation to native haptoglobin and to beta chain. Reaction of this glycopeptide with concanavalin A was almost twice higher than the corresponding reaction of haptoglobin. Glycopeptides IV and V were inactive in the reaction with the lectin. Glycopeptide III exhibited relatively the strongest cross-reactivity with the specific antihaptoglobin serum while its inhibitory activity in the immunoreaction was the lowest.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

Cloning and expression of human core 1 beta1,3-galactosyltransferase.

The common core 1 O-glycan structure Galbeta1--> 3GalNAc-R is the precursor for many extended mucin-type O-glycan structures in animal cell surface and secreted glycoproteins. Core 1 is synthesized by the transfer of Gal from UDP-Gal to GalNAcalpha1-R by core 1 beta3-galactosyltransferase (core 1 beta3-Gal-T). Amino acid sequences from purified rat core 1 beta3-Gal-T (Ju, T., Cummings, R. D., and Canfield, W. M. (2002) J. Biol. Chem. 277, 169-177) were used to identify the core 1 beta3-Gal-T sequences in the human expressed sequence tag data bases. A 1794-bp human core 1 beta3-Gal-T cDNA sequence was determined by sequencing the expressed sequence tag and performing 5'-rapid amplification of cDNA ends. The core 1 beta3-Gal-T predicts a 363-amino acid type II transmembrane protein. Expression of both the full-length and epitope-tagged soluble forms of the putative enzyme in human 293T cells generated core 1 beta3-Gal-T activity that transferred galactose from UDP-Gal to GalNAcalpha1-O-phenyl, and a synthetic glycopeptide with Thr-linked GalNAc and the product was shown to have the core 1 structure. Northern analysis demonstrated widespread expression of core 1 beta3-Gal-T in tissues with a predominance in kidney, heart, placenta, and liver. Highly homologous cDNAs were identified and cloned from rat, mouse, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that the enzyme is widely distributed in metazoans. The core 1 beta3-Gal-T sequence has minimal homology with conserved sequences found in previously described beta3-galactosyltransferases, suggesting this enzyme is only distantly related to the known beta3-galactosyltransferase family.     

The oncofetal structure of human fibronectin defined by monoclonal antibody FDC-6. Unique structural requirement for the antigenic specificity provided by a glycosylhexapeptide

Previously, monoclonal antibody FDC-6 was established, which defines a structure specific for fibronectins isolated from fetal and malignant cells and tissues. The presence of the FDC-6-defined structure at type III connecting segment (III CS) is characteristic of oncofetal fibronectin (onf-FN), and its absence is characteristic of normal fibronectin (nor-FN) (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). Hepatoma fibronectin was sequentially digested by various proteases, followed by subsequent chromatography on an FDC-6 affinity column and reverse-phase columns at each step of digestion. A single strongly active glycosylhexapeptide (glycopeptide 1) and an inactive glycosylpentapeptide (glycopeptide 3) were isolated from glycopeptide A containing 35 amino acid residues. The minimum essential structure required for the FDC-6 activity was found to be a hexapeptide sequence Val-Thr-His-Pro-Gly-Tyr having NeuAc alpha 2----3Gal beta 1----3GalNAc or its core (Gal beta 1----3GalNAc or GalNAc) linked at threonine. Various synthetic peptides including the Val-Thr-His-Pro-Gly-Tyr sequence and a glycopeptide having the Val-Thr-His-Pro-Gly pentapeptide with the same glycosylation at threonine were all inactive. Elimination of sialic acid slightly increased the activity, and subsequent elimination of galactose did not alter the activity; however, removal of the Gal beta 1----3GalNAc residue by endo-alpha-N-acetylgalactosaminidase from desialylated glycopeptide A resulted in total inactivation of the reactivity with FDC-6 antibody. Thus, a single glycosylation at a defined threonine residue of the III CS region may induce conformational changes in the peptide to form the specific oncofetal epitope recognized by FDC-6 antibody. This finding opens the possibility that a number of other oncofetal epitopes consist of a peptide and a common O-linked carbohydrate and that the combination produces a conformation specific to cancer or to a stage of development.     

Structures of asparagine-linked oligosaccharides of human placental fibronectin

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.     

Structural study of the carbohydrate moieties of two human immunoglobulin subclasses (IgG2 and IgG4)

Asparagine-linked sugar chains were quantitatively released as oligosaccharides from human IgG2 and IgG4 myeloma proteins by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Each oligosaccharide was isolated by serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion and methylation analysis, revealed that all of them were of the bi-antennary complex-type containing Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc as core structures, and GlcNAc beta 1-, Gal beta 1-4GlcNAc beta 1- and Sia alpha 2-6Gal beta 1- in their outer chain moieties. However, the molar ratio of each oligosaccharide was different in each IgG sample, indicating that clonal variation is included in the sugar chain moieties of IgG molecules. One of the IgG2 contained four asparagine-linked sugar chains in one molecule, two on the Fc fragment and the remainder on the Fab fragment. The sugar chains in the Fc fragment contained much less galactose as compared with the Fab fragment.     

Specificity of the sialic acid-binding lectin from the snail Cepaea hortensis.

The specificity of the sialic acid-binding lectin from the snail Cepaea hortensis, purified by affinity chromatography on fetuin-Sepharose, was studied by hemagglutination inhibition assay applying 32 sialic acid derivatives and 14 glycoproteins. 2-alpha-Methyl-9-O-acetyl-NeuAc was the most potent inhibitor, followed closely by 2-alpha-methyl-NeuAc and 2-alpha-benzyl-NeuAc. An axially orientated carboxyl group is a prerequisite for maximal lectin-sugar binding. Neither size nor polarity of the alpha-anomeric substituent significantly influenced inhibition potency. An intact sialic acid N-acetyl group is essential for optimal lectin-sugar interaction. The trihydroxypropyl side chain also is of great importance. However, a bulky hydrophobic substituent at the side chain like a 9-O-tosyl residue did not decrease binding to the lectin. The lectin did not distinguish between NeuAc alpha 2----3Gal beta 1----4Glc and NeuAc alpha 2----6Gal beta 1----4Glc. Among other sugars tested, only N-acetylglucosamine showed inhibition, although 50-fold less. The most potent glycoprotein inhibitors were those carrying O-chains only or preferentially, as ovine submaxillary mucin, bovine submaxillary mucin, and glycophorin A. Tamm-Horsfall protein was an exception being a strong inhibitor, although carrying only N-chains. Asialoglycoproteins were inactive. Glycoproteins containing the NeuAc alpha 2----3Gal sequence inhibited the lectin as well as those with NeuAc alpha 2----6GalNAc. From the results a model of the lectin's binding site for sialic acid is suggested.     

The beta 1----2-D-xylose and alpha 1----3-L-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Isolation, characterisation and comparison with other legume lectins

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 1H-NMR showed that the major component was the fucose-containing heptasaccharide Man alpha 3(Man alpha 6)(Xyl beta 2)Man beta 4GlcNAc beta 4(Fuc alpha 3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components. The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Man alpha 3(Man alpha 6)Man beta 4GlcNAc beta 4GlcNAc] by a D-xylose residue linked beta 1----2 to the beta-mannosyl residue and an L-fucose residue linked alpha 1----3 to the reducing terminal N-acetylglucosamine residue. The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.     

Lectin from rice

N-Acetyl-D-glucosamine-binding lectin was isolated and purified from rice by ammonium sulphate fractionation and affinity chromatography using N-acetyl-D-glucosamine linked Sepharose 6B column. It gave a single hand on Polyacrylamide disc gel. It was identified as a glycoprotein. The purified lectin dissociated into two components on Sephadex G-100 column chromatography,-a higher molecular weight fraction not containing any carbohydrate and a lower molecular weight glycoprotein fraction. The apparent molecular weights of these fractions were 85,000 and 14,500. The lectin agglutinated erythrocytes of human A,B,O groups and of several other mammals and its activity was inhibited only by N-acetyl-D-glucosamine. The glycopeptide isolated by pronase digestion of the lectin was homogeneous and did not possess agglutinating activity. It contained about 10% carbohydrate of which xylose, arabinose and glucose were the major components.     

The carbohydrate units of asialo-ovomucoid: their heterogeneity and enzymic degradation

An ovomucoid variant free from sialic acid has been prepared in a pure state by ion-exchange chromatography on DEAE-cellulose. The purified glycoprotein contained 10-11 residues of mannose, 2-3 residues of galactose, and 21 residues of 2-acetamido-2-deoxyglucose. Glycopeptides have been prepared by exhaustive digestion with Pronase followed by ion-exchange chromatography on Dowex 50 (X2) resin. Three fractions were obtained, all with similar contents of mannose and hexosamine but with various contents of galactose. The sugar-aspartic acid ratios indicated that all of the fractions were heterogeneous, the major fraction having mannose-galactose-hexosamine-aspartic acid ratios of 2.6:0.5:5.8:1.0. Cleavage of asialo-ovomucoid with cyanogen bromide and proteolytic digestion of the isolated fragments gave two heterogeneous glycopeptide fractions of similar composition. Both asialo-ovomucoid and the principal glycopeptide fraction were degraded with beta-D-galactosidase, alpha-D-mannosidase, and beta-N-acetylglucosaminidase singly and in sequence. Removal of much of the carbohydrate from asialo-ovomucoid had no appreciable effect on its anti-tryptic activity. By sequential degradation of the glycopeptide, a pentasaccharide core alpha-D-Man-[alpha-D-Man]-beta-D-Man-beta-D-GlcNAc-beta-D-GlcNAc-Asn was obtained. Other structural features revealed by enzymic degradation are discussed.     

Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra)

A novel lectin was purified from the coelomic fluid of the sea cucumber Holothuria scabra (HSL), subjected to bacterial challenge. HSL is a monomeric glycoprotein of molecular mass 182 kDa. The lectin is highly thermostable as it retains full activity for 1 h at 80 degrees C. Further, the hemagglutination activity of HSL is unaffected by pH in the range 2-11. Unlike other lectins purified from marine invertebrates, the hemagglutination activity of HSL does not require any divalent metal ions. The affinity profile of HSL was studied by a combination of hemagglutination inhibition and fluorescence spectroscopy. HSL binds to desialylated glycoproteins, MealphaGal, T-antigen and T (alpha-ser)-antigen with a distinction between beta1-4 and beta1-3 linkages. Mealpha-T-antigen was a potent ligand having highest affinity (Ka 8.32 x 10(7)M(-1)). Monosaccharide binding is enthalphically driven while disaccharide binding involves both entropic and enthalpic contributions.