Synthetic glycopeptides for the development of tumour-selective vaccines.

Based on structural information reported for the tumour-associated epithelial mucin MUC1, glycopeptides have been synthesized which contain tumour-associated saccharide antigens. such as the Thomsen-Friedenreich (T), TN or sialyl TN antigen. in combination with peptide sequences of the tandem repeat region of MUC1. Solid-phase syntheses have been carried out using N-Fmoc protected O-glycosyl serine and threonine building blocks and an allylic anchor which is stable to basic and acidic conditions, but can be cleaved under neutral conditions in a palladium(0)-catalysed allyl transfer reaction. In addition. a (2-3)sialyl T antigen threonine building block was prepared by a chemoenzymatic strategy and used in the synthesis of an N-terminal glycopeptide antigen of leukosialin (CD43). The proliferation of cytotoxic T cells could be induced using a construct consisting of a MUC1-glycopeptide antigen and a T cell epitope.     

A structural basis for four distinct elution profiles on concanavalin A--Sepharose affinity chromatography of glycopeptides.

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanvalin A (Con A)--Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains: (abstract:see text). Such glycopeptides have only a single mannose residue capable of interacting with Con A--Sepharose; an interacting mannose residue is either an alpha-linked nonreducing terminal residue or an alpha-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl alpha-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures: (abstract: see text) where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A--Sepharose and elution as a sharp peak with 0.1 M methyl alpha-D-glucopyranoside; glycopeptides giving this pattern had the structures: (abstract: see text) where R2 is either H, glcNAc, Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the mimimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl alpha-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures: (abstract: see text) where R3 is either GlcNAc,Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc.Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A--Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.     

1-Deoxymannojirimycin inhibits capillary tube formation in vitro. Analysis of N-linked oligosaccharides in bovine capillary endothelial cells.

Capillary endothelial cells can be induced to form capillary-like structures in vitro by plating on fibronectin-coated dishes (Ingber, D. E., and Folkman, J. (1989) J. Cell Biol. 109, 317-330), thereby mimicking angiogenesis. To assess the role of glycoproteins bearing asparagine-linked oligosaccharides in this process, we tested the effect of oligosaccharide processing inhibitors on the formation of capillary tubes. Deoxymannojirimycin, a compound that prevents synthesis of hybrid and complex-type oligosaccharides, inhibited the formation of capillary tubes. In contrast, swainsonine, an inhibitor that blocks synthesis of complex- but not hybrid-type oligosaccharides, did not inhibit tube formation. Lectin affinity chromatography of 2-[3H] mannose-labeled glycopeptides from endothelial cells induced to form tubes did not reveal a striking difference in the spectrum of oligosaccharides compared to uninduced cells. Since endothelial cells formed tubes normally in the presence of swainsonine, we analyzed glycopeptides from swainsonine-treated induced and uninduced cells. Cells induced to form tubes were enriched in monosialylated hybrid-type oligosaccharides sensitive to alpha-fucosidase, beta-galactosidase, and beta-N-acetylhexosaminidase, suggestive of sialyl Lewis-X determinants. We used an enzyme-linked immunoassay to measure sialyl Lewis-X epitopes on capillary endothelial cells and found that both induced and uninduced cells expressed sialyl Lewis-X epitopes. Deoxymannojirimycin and, to a lesser extent, swainsonine reduced the level of sialyl Lewis-X epitopes in cells induced to form capillary tubes, but neither compound affected the level of epitopes in cell monolayers. We conclude that synthesis of at least hybrid-type oligosaccharides is required for capillary tube formation in vitro and that an increase in monosialylated, fucosylated glycans on asparagine-linked oligosaccharides occurs during this process.     

Synthesis of tumor-associated glycopeptide antigens

Carbohydrates and peptides linked together in glycoproteins constitute important components of the molecular communication between cells in multicellular organisms. Cell morphogenesis and tumorigenesis are accompanied by changes in the glycoprotein profiles of the outer cell membranes. Glycopeptide fragments of glycoproteins that have altered structures in tumor cells are of interest as tumor-associated antigens for the distinction between normal cells and tumor cells. In contrast to glycoproteins isolated from biological sources, synthetic glycopeptides are obtained in pure form and exactly specified structures. The methods developed for the synthesis of glycopeptides with tumor-associated antigen structure are outlined in this article by means of a series of typical examples. Beginning with O-glycopeptides of the relatively simple alpha-O-galactosamine-serine/threonine (T(N)-antigen) type, glycopeptide antigens of increasing complexity are described. The review includes syntheses of the saccharide components, the glycosylation reactions to furnish the O-glycosyl amino acid building blocks, their selective C- and N-terminal deprotection and the use of these building blocks for glycopeptide syntheses both in solution and on the solid support. Particular attention is given to glycopeptides containing sialic acid residues, whose syntheses are demanding since reversible protection of the sialic carboxylic group is required. Synthetic methods for the construction of N-glycopeptides carrying the important cell adhesion ligands sialyl Lewis x and sialyl Lewis a antigen are also described. Strategies for the construction of glycopeptides of this type require methods compatible with the presence of the sialic acid residue, as well as with the acid-sensitivity of the fucoside bonds.     

Poly-N-acetyllactosaminyl O-glycans attached to leukosialin. The presence of sialyl Le(x) structures in O-glycans.

Poly-N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids. Attempts were made in HL-60 and K562 cells to determine the amount of poly-N-acetyllactosaminyl O-glycans in the major sialoglycoprotein, leukosialin. Leukosialin was immunoprecipitated from [3H]glucosamine-labeled HL-60 and K562 cells. Glycopeptides were prepared by Pronase digestion, and O-glycan-containing glycopeptides were isolated by affinity chromatography using Jacalin-agarose. The glycopeptides bound to Jacalin-agarose and those unbound were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Sequential glycosidase digestion of the O-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following conclusions. 1) Leukosialin from HL-60 cells contains about 1-2 poly-N-acetyllactosaminyl O-glycan chains/molecule. 2) About 50% of these poly-N-acetyllactosaminyl O-glycans contain sialyl Le(x) termini, NeuNAc alpha 2-->3Gal beta 1-->4 (Fuc alpha 1-->3)GlcNAc beta 1-->R. The amount of sialyl Le(x) structure in leukosialin is roughly equivalent to that on cell surfaces of HL-60 cells. 3) Leukosialin from K562 cells, on the other hand, contains no detectable amount of poly-N-acetyllactosaminyl O-glycans. 4) The presence of poly-N-acetyllactosamine in O-glycans is dependent on the core 2 beta 1,6-N-acetylglucosaminyl transferase. 5) Jacalin-agarose binds to sialylated small oligosaccharides such as NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->6) GalNAc but not the hexasaccharide NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->6) GalNAc. These results indicate that the formation of polylactosaminyl O-glycans and sialyl Le(x) structure in O-glycans is dependent on the core 2 formation.     

Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.     

Glycosyl transferases of baby-hamster-kidney (BHK) cells and ricin-resistant mutants. N-glycan biosynthesis

Five cell lines of ricin-resistant BHK cells have been assayed for gross carbohydrate analysis of cellular glycoproteins, for the activities of several glycosidases and of specific glycosyl transferases active in assembly of N-glycans of glycoproteins. The latter enzymes include sialyl transferase using asialofetuin as glycosyl acceptor, fucosyl transferases using asialofetuin and asialoagalactofetuin acceptors, galactosyl transferases using ovalbumin, ovomucoid and N-acetylglucosamine as acceptors and N-acetylglucosaminyl transferases using ovalbumin and glycopeptides as acceptors. Cell line RicR14, binding less ricin than normal BHK cells, contains reduced amounts of sialic acid, galactose and N-acetylglucosamine in cellular glycoproteins and lacks almost completely N-acetylglucosamine transferase I, an essential enzyme in assembly of ricin-binding carbohydrate sequences of N-glycans. These cells also contain reduced levels of N-acetylglucosamine transferase II active on a product of N-acetylglucosamine transferase I action. Sialyl transferase activity is severely depressed while fucose-(alpha 1 leads to 6)-N-acetylglucosamine fucosyl transferase activity is increased. Cell lines RicR15, 17, 19 and 21 showed partial deficiencies in galactosyl and N-acetylglucosaminyl transferases. A hypothesis is put forward to account for the different carbohydrate compositions and ricin binding properties of glycoproteins synthesised by these cells in terms of the determined enzyme defects, the normal level of sialyl transferases detected in RicR15 and RicR21 cells and the elevated levels of sialyl and fucosyl transferases detected in RicR17 and 19 cells. None of the above changes in glycosyl transfer reactions in the RicR cell lines are due to enhanced glycosidase or sugar nucleotidase activities in the mutant cells.     

The sialoglycoproteins of murine erythrocyte ghosts. A modified periodic acid-Schiff stain procedure staining nonsubstituted and O-acetylated sialyl residues on glycopeptides.

Murine erythrocyte ghosts (from DBA/2, CD-1, and B6D2 strains) contain significant amounts of O-acetylated sialyl residues, which are slowly oxidized by periodate. Sialic acids have been purified from murine erythrocyte ghosts and the existence of O-acetylated sialic acids has been confirmed: 1) by assaying with the Warren procedure before and after de-O-acetylation with 0.1 N NaOH for 45 min at 4 degrees C; 2) by thin layer chromatography on cellulose; and 3) by gas-liquid chromatography. Because these sialyl residues are unevenly distributed on the sialoglycoproteins of murine erythrocyte ghosts, the periodic acid-Schiff staining detects only one major sialoglycoprotein. A modification of the periodic acid-Schiff stain method removes these O-acetyl groups after electrophoresis and reveals two additional sialoglycoproteins which bear the majority of the O-acetylated sialyl residues. Rat erythrocyte ghosts have similar residues on one of their two sialoglycoproteins. Ghosts of human, rabbit, and guinea pig erythrocytes do not contain detectable amounts of O-acetylated sialyl residues.     

Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry

A simple, fast and sensitive method was developed to verify the presence of the sialyl Lewis(x) antigen on an N-linked glycoprotein. High performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was used to identify which of the five N-linked glycosylation sites of human plasma alpha1-acid-glycoprotein (orosomucoid, OMD) contain the sialyl Lewis(x) antigen. OMD was digested with proteolytic enzymes and analyzed by reversed phase chromatography coupled with on-line ESI/MS. A tandem mass spectrometry experiment was designed to detect the presence of the sialyl Lewis(x) antigen based on the observation of an 803 mass to charge ratio ( m/z ) ion produced in the intermediate pressure region of the ESI interface. The ESI/MS signal at m/z 803 is consistent with an oxonium ion for a glycan structure containing NeuAc, Gal, GlcNAc, and Fuc. The identity of the m/z 803 ion was confirmed by ESI/MS/MS analysis of the m/z 803 fragment ion and comparison with a sialyl Lewis(x) standard. The stereochemistry and linkage positions were assigned using previous NMR analysis but could be determined with permethylation analysis if necessary. The analysis of OMD gave a pattern showing signal for the sialyl Lewis(x) antigen coeluting with each of the five N-linked glycopeptides. The ability to monitor sialyl Lewis(x) expression at each of the five sites is of interest in the study of OMD's role in inflammatory diseases.      

Rat-liver lysosomal sialidase. Solubilization, substrate specificity and comparison with the cytosolic sialidase

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity. The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60 000 +/- 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides , those containing (alpha 2----3)sialyl linkages being better substrates than those with (alpha 2----6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin. In contrast to the intralysosomal sialidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60 000. It also hydrolyzed gangliosides but not submaxillary mucin.     

Studies on pig serum lipoproteins. IV. Isolation and characterization of glycopeptides from pig serum low density lipoprotein.

Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.     

Preparation of glycopeptides and oligosaccharides from thyroxine-binding globulin.

Over 99% of thyroxine (T4), the major form of thyroid hormone in plasma, is bound to the plasma glycoprotein thyroxine-binding globulin (TBG). The carbohydrate composition of TBG (14.6% by weight) consists of mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid in the molar ratios of 11:9:16:10 per mol of glycoprotein. No fucose or N-acetylgalactosamine were detected. Amino acid analyses were performed. Glycopeptides, prepared by exhaustive pronase treatment of the glycoprotein, were separated by gel filtration and ion exchange chromatography. All glycopeptides contained the four sugars present in the native glycoprotein. One-fourth of the glycopeptide fraction was resolved into a discrete component, glycopeptide I. The remaining glycopeptides were a mixture termed glycopeptides II and III. Glycopeptides II and III were resolved into two discrete carbohydrate units, termed oligosaccharides A and B, by alkaline-borohydride treatment and DEAE-cellulose chromatography. We propose that TBG contains four oligosaccharide chains as calculated from the molecular weights of the glycopeptides and from compositional data assuming 1 asparagine residue/glycopeptide. The carbohydrate structures of the glycopeptides and relative affinities of TBG, glycopeptides and oligosaccharides for hepatocyte plasma membrane binding are presented in the accompanying paper (Zinn, A.B., Marshall, J.S., and Carlson, D.M. (1978) J. Biol. Chem. 253, 6768-6773.     

500 MHz 1H n.m.r. of oligosaccharides of N-acetyl-lactosamine-type released from human erythrocyte glycopeptides by endo-beta-galactosidase.

500 MHz 1H n.m.r. spectroscopy has been used in structural studies of three linear and five branched oligosaccharides of N-acetyl-lactosamine-type that were released from desialylated blood group O erythrocyte glycopeptides by treatment with the endo-beta-galactosidase of Bacteroides fragilis followed by reduction. The following oligosaccharide alditols were characterized: (formula; see book)     

High-sensitivity FAB-MS strategies for O-GlcNAc characterization.

In this paper we report the first application of fast atom bombardment mass spectrometry (FAB-MS) to O-linked N-acetylglucosamine (O-GlcNAc)-bearing glycopeptides. Using N-acetylgalactosamine (GalNAc)- and Gal-GalNAc-containing glycopeptides (isolated from Tn glycophorin and desialylated normal glycophorin, respectively) as readily available model compounds, rapid and sensitive derivatization/FAB-MS strategies applicable to serine/threonine-rich glycopeptides have been devised. Peptides and glycopeptides were propionylated in a 1 min reaction using a mixture of trifluoroacetic anhydride and propionic acid, and the product mixtures were analysed directly by FAB-MS. Glycopeptides and peptides rich in hydroxylated residues afforded characteristic clusters of molecular ions at high sensitivity. Additional sensitivity enhancement was achieved by prior esterification of carboxyl groups. These methods were used in a study of O-GlcNAc glycopeptides produced by purified O-GlcNAc transferase addition of GlcNAc to the synthetic peptides YSDSPSTST and YSGSPSTST in which Y is tyrosine, S is serine, D is aspartic acid, P is proline, T is threonine and G is glycine. The propionyl derivatives afforded high-quality spectra which unequivocally showed that the majority of the glycopeptides were substituted with a single GlcNAc residue. Low pmol quantities of material gave detectable signals. The propionylation/FAB-MS procedure has been combined with gas-phase sequencing strategies and shows promise for defining the sites of glycosylation of O-GlcNAc glycopeptides that are available in limited quantities.     

Structure of glycoproteins of human erythrocytes. Alkali-stable oligosaccharides

Studies have been made on the oligosaccharide residues of the alkali-stable carbohydrate-protein linkage of sialoglycopeptides derived from human erythrocytes. Four glycopeptides were isolated after alkaline borohydride treatment and Pronase digestion of MN-active sialoglycopeptides. The structure of one of these glycopeptides (GPIV) has been studied by sequential hydrolysis with specific glycosidases. Glycopeptide GPIV contained (per mol): 1mol of fucose, 1mol of sialic acid, 3mol of galactose, 3mol of mannose, 4mol of acetylglucosamine, 1mol of aspartic acid and fractional amounts of threonine, serine and glycine. The molecular weight of the glycopeptide was estimated to be 2330 by gel filtration. On the basis of glycosidase-digestion results, a tentative structure is proposed for the oligosaccharide moiety of glycopeptide GPIV.     

Enzymatic synthesis of the core-2 sialyl Lewis X O-glycan on the tumor-associated MUC1a' peptide

Starting from a tumor-associated synthetic MUC1-derived peptide MUC1a' and using a completely enzymatic approach for the synthesis of the core-2 sialyl Lewis X glycopart, the following glycopeptide was synthesized: AHGV[Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-O)]TSAPDTR. First, polypeptide N-acetylgalactosaminyltransferase 3 was used to site-specifically glycosylate MUC1a' to give MUC1a'-GalNAc. Then, in a one-pot reaction employing beta-galactosidase and core-2 beta6-N-acetylglucosaminyltransferase the core-2 O-glycan structure was prepared. The core-2 structure was then sequentially galactosylated, sialylated, and fucosylated by making use of beta4-galactosyltransferase 1, alpha3-sialyltransferase 3, and alpha3-fucosyltransferase 3, respectively, resulting in the sialyl Lewis X glycopeptide. The overall yield of the final compound was 23% (3.2 mg, 1.4 micromol). During the synthesis three intermediate glycopeptides containing O-linked GalNAc, Gal(beta1-4)GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc, and Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc, respectively, were isolated in mg quantities. All products were characterized by mass spectrometry and NMR spectroscopy.     

Enzymatic synthesis of a sialyl Lewis X dimer from egg yolk as an inhibitor of E-selectin

A dimeric sialyl Lewis X (SLe^x) glycopeptide was synthesized enzymatically in three steps from an N-linked oligosaccharide prepared from egg yolk. Treatment of delipidated hen egg yolk with the protease Orientase and neuraminidase gave a dimeric N-acetyllactosamine-containing oligosaccharide linked to asparagine. Addition of sialic acid and fucose catalyzed by -2,3-sialyltransferase and -1,3-fucosyltransferase provided the dimeric SLe^x, which was shown to be as active as monomeric SLe^x as an inhibitor of E-selectin with IC₅₀ 0.75 mM. The synthetic dimeric SLe^x of the mucin type (i.e. SLe^x linked to the 3- and 6-OH groups of Gal) is, however, about five times as active as the monomer. It is suggested that dimeric SLe^x glycopeptides of the mucin type would be effective ligands for E-selectin.     

A comparsion of glycopeptides from the transferrins of several species.

1. The carbohydrate compositions of human, pig and cattle transferrins and duck ovotransferrin have been determined. 2. Glycopeptides have been prepared from these transferrins and their carbohydrate compositions and amino acid sequences determined. One of the glycopeptides from human transferrin carries the C-terminal residue of the protein. 3. Each tranferrrin yielded two glycopeptides that appeared to be identical in carbohydrate composition but different in amino acid sequence. The two glycopeptides have been distinguished as type A, in which the residue following Asn(CHO)(where CHO represents a carbohydrate moiety) is a basic amino acid and type B in which Asn(CHO) is followed by a neutral aliphatic amino acid. Cattle transferrin is exceptional in having two glycopeptides in which this position is occupied by serine. 4. It is suggested that each molecule of human and cattle transferrin and duck ovotransferrin carries an average of two carbohydrate prosthetic groups. Hen and pig transferrins appear to carry only one carbohydrate group per mol of protein. 5. The N-terminal sequences of hen and duck ovotransferrins and of cattle, human and pig transferrins were also determined.     

Negative-ion MALDI-MS2 for discrimination of α2,3- and α2,6-sialylation on glycopeptides labeled with a pyrene derivative

Here, we propose a novel method for the discrimination of α2,3- and α2,6-sialylation on glycopeptides. To stabilize the sialic acids, the carboxyl moiety on the sialic acid as well as the C-terminus and side chain of the peptide backbone were derivatized using 1-pyrenyldiazomethane (PDAM). The derivatization can be performed on the target plate for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), thereby avoiding complicated and time-consuming purification steps. After the on-plate PDAM derivatization, samples were subjected to negative-ion MALDI-MS using 3AQ-CHCA as a matrix. Deprotonated ions of the PDAM-derivatized form were detected as the predominant species without loss of sialic acid. The negative-ion collision-induced dissociation (CID) of PDAM-derivatized isomeric sialylglycopeptides, derived from hen egg yolk, showed characteristic spectral patterns. These data made it possible to discriminate α2,3- and α2,6-sialylation. In addition, sialyl isomers of a glycan with an asparagine could be discriminated based on their CID spectra. In brief, the negative-ion CID of PDAM-derivatized glycopeptides with α2,6-sialylation gave an abundant (0,2)A-type product ion, while that with α2,3-sialylation furnished a series of (2,4)A/Y-type product ions with loss of sialic acids. The unique fragmentation behavior appears to be derived from the difference of pyrene binding positions after ionization, depending on the type of sialylation. Thus, we show that on-plate PDAM derivatization followed by negative-ion MALDI-MS(2) is a simple and robust method for the discrimination of α2,3- and α2,6-sialylation on glycopeptides.     

Identification of N-acetylneuraminyl alpha 2-->3 poly-N-acetyllactosamine glycans as the receptors of sialic acid-binding Streptococcus suis strains.

Streptococcus suis is a common cause of sepsis, meningitis, and other serious infections in young piglets and also causes meningitis in humans. The cell-binding specificity of sialic acid-recognizing strains of Streptococcus suis was investigated. Treatment of human erythrocytes with sialidase or mild periodate abolished hemagglutination. Hemagglutination inhibition experiments with sialyl oligosaccharides indicated that the adhesin preferred the sequence NeuNAc alpha 2-3Gal beta 1-4Glc(NAc). Resialylation of desialylated erythrocytes with Gal beta 1-3(4)GlcNAc alpha 2-3-sialyltransferase induced a strong hemagglutination, whereas no or only weak hemagglutination was obtained with cells resialylated with two other sialyltransferases. Binding of radiolabeled bacteria to blots of erythrocyte membrane proteins revealed binding to the poly-N-acetyllactosamine-containing components Band 3, Band 4.5, and polyglycosyl ceramides and to glycophorin A. The involvement of glycophorin A as a major ligand was excluded by the strong hemagglutination of trypsin-treated erythrocytes and En(a-) erythrocytes defective in glycophorin A. Sensitivity of the hemagglutination toward endo-beta-galactosidase treatment of erythrocytes and inhibition by purified poly-N-acetyllactosaminyl glycopeptides indicated that the adhesin bound to glycans containing the following structure: NeuNAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-.