Attenuated murine cytomegalovirus binds to N-acetylglucosamine, and shift to virulence may involve recognition of sialic acids.

Treatment of cells with lectins specific for N-acetylglucosamine (GlcNAc) blocked infection by mouse cytomegalovirus (MCMV), and GlcNAc pretreatment of the lectin blocked this effect. MCMV failed to infect N-acetylglucosaminidase (GlcNAcase)-treated mouse embryo fibroblasts (MEF). GlcNAc and GlcNAc-containing synthetic oligosaccharides directly inhibited viral infectivity. Ulex lectin inhibition of infection was shown to be due to inhibition of surface adsorption of 35S-labeled virus. Also, GlcNAcase eluted 35S-labeled virus adsorbed to MEF at 4 degrees C and inhibited plaque formation if added after adsorption at this temperature. These findings indicate that GlcNAc binding is involved in attachment rather than in some later step in infection. High-performance thin-layer chromatography overlay of [35S]MCMV indicated that it binds to a GlcNAc-containing asialoglycolipid. Analogous experiments indicated that MCMV made virulent by in vivo salivary gland passage binds to sialic acids in addition to GlcNAc. Treatment of MEF with sialic acid-binding lectins blocked infectivity. Incubation of virus with sialic acids also prevented infection. N-acetylneuraminic acid was 10(3)-fold more potent than N-glycolylneuraminic acid. Sialidase-treated target cells were not efficiently infected by the virus. Thus, MCMV binds to GlcNAc on the cell surface, and the shift to virulence (by in vivo salivary gland passage) correlates with viral recognition of sialic acids.     

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 studies of the carbohydrate chains of human gamma-interferon

Human gamma-interferon (IFN-gamma) was prepared biotechnologically using Chinese hamster ovary cells. These cells were shown to be able to produce glycosylated IFN-gamma. Sugar analysis revealed the presence of Man, Gal, GlcNAc, NeuAc and Fuc residues in a molar ratio of 3.8:2.0:3.5:0.6:0.4 suggesting the occurrence of N-glycosidically linked N-acetyllactosamine type of carbohydrate chains. For structure determination of these chains, the glycoprotein was subjected to the hydrazinolysis procedure, yielding oligosaccharide-alditols. The latter compounds were analysed by 500-MHz 1H-NMR spectroscopy. The carbohydrate material was found to consist of biantennary structures, exhibiting microheterogeneity as to the terminal sialic acids and the core Fuc residue: (Formula: see text). As similar carbohydrates are present on several human secreted proteins, this glycosyl group is not expected to be immunogenic in man. It remains to be established to what extent the carbohydrate chains of this biotechnologically produced IFN-gamma are identical to those of naturally occurring human IFN-gamma.     

Epithelial basement membrane of bovine renal tubuli. Isolation and analysis of the carbohydrate chains.

The carbohydrate chains present in the tubular basement membrane of bovine kidney were studied. Digestion with collagenase followed with pronase resulted in a complete solubilization of the basement membrane. The different glycopeptides were purified by gel filtration and ion-exchange chromatography. Two kinds of carbohydrate chains could be characterized: oligosaccharides composed of glucosamine, mannose, galactose, fucose and sialic acid, and glucosylgalactose disaccharides. A very small portion of the oligosaccharide chains (ca. 4%) appeared to be free of sialic acid. The bulk of these chains contained sialic acid and fucose, although in small amounts. Only traces of galactosamine were found.     

Biosynthesis of a disialylated sequence in N-linked oligosaccharides: identification of an N-acetylglucosaminide (alpha 2----6)-sialyltransferase in Golgi apparatus from rat liver

Rat liver Golgi apparatus are shown to have a CMP-N-acetylneuraminate: N-acetylglucosaminide (alpha 2----6)-sialyltransferase which catalyzes the conversion of the human milk oligosaccharide LS-tetrasaccharide-a (NeuAc alpha 2----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----4Glc) to disialyllacto -N- tetraose containing the terminal sequence: (formula: see text) found in N-linked oligosaccharides of glycoproteins. The N-acetylglucosaminide (alpha 2----6)-sialyltransferase has a marked preference for the sequence NeuAc alpha 2----3-Gal beta 1---- 3GlcNAc as an acceptor substrate. Thus, the order of addition of the two sialic acids in the disialylated structure shown above is proposed to be first the terminal sialic acid in the NeuAc alpha 2----3Gal linkage followed by the internal sialic acid in the NeuAc alpha 2---- 6GlcNAc linkage. Sialylation in vitro of the type 1 branches (Gal beta 1---- 3GlcNAc -) of the N-linked oligosaccharides of asialo prothrombin to produce the same disialylated sequence is also demonstrated.     

Role of sialic acid in bovine sperm-zona pellucida binding

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.     

Location of the carbohydrate groups of ovomucoid.

Tryptic glycopeptides were purified from the sialic acid-free variant of ovomucoid, O1, and its CNBr fragments. The amino acid sequences adjacent to the four major sites of carbohydrate (Carb.) attachment were: (1), Phe-Pro-Asn(Carb.)-Ala-Thr-Asp-Lys-Glu-Gly-Lys; (2), Ala-Try-Ser-Ile-Glu-Phe-Gly-Thr-Asn (Carb.)-Ile-Ser-Lys; (3), Glu, Thr-Val-Pro-Met-Asn(Carb.)-cys-Ser; (4), Ser-Ser-Tyr-Ala-Asn (Carb.)-Thr-Thr-Ser-Glu-Asp-Gly-Lys, Glycosylated Asn residues were located at position 10, between residues 49 and 60, and at positions 69 and 75, in the primary sequence. All of these carbohydrate groups contained GlcNAc, Man and Gal in the approximate molar proprotions 5:3:0.5. A further glycopeptide containing His was isolated in low yield, suggesting that some carbohydrate is attached at a fifth site. Two of the carbohydrate-attachment sites (Asn-10 and Asn-75) occur in sequences that show internal homologies. These are presumed to have evolved as a consequence of partial gene duplication. Three of the carbohydrate-attachment sites occur in similar positions to the carbohydrate groups in quail ovomucoid [Laskowski (1976) Protides Biol. Fluids Proc. Colloq. 23, in the press]. Prediction of peptide conformation from the sequence data by the method of Chou & Fasman [(1974) Biochemistry 13, 222-225] indicated that four glycosylated Asn residues in hen ovomucoid are very close to groups of amino acids that occur with high frequency in beta-turns. The possible significance of peptide-chain conformation in the attachment of carbohydrate to glycoproteins is briefly discussed.     

Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid

In addition to sialic acid, bacteria produce several other nonulosonic acids, including legionaminic acid (Leg). This has exactly the same stereochemistry as sialic acid, with the added features of 9-deoxy and 7-amino groups. In order to explore the biological effects of replacing sialic acid residues (Neu5Ac) in glycoconjugates with Leg in its diacetylated form, diacetyllegionaminic acid (Leg5Ac7Ac), we tested CMP-Leg5Ac7Ac as a donor substrate with a selection of bacterial and mammalian sialyltransferases. The CMP-Leg5Ac7Ac was synthesized in vitro by means of cloned enzymes from the bacillosamine portion of the Campylobacter jejuni N-glycan pathway and from the Leg pathway of Legionella pneumophila. Using fluorescent derivatives of lactose, Galβ1,4GlcNAcβ and T-antigen (Galβ1,3GalNAcα) as acceptors, we tested eight different sialyltransferases and found that the Pasteurella multocida PM0188h and porcine ST3Gal1 sialyltransferases were significantly active with CMP-Leg5Ac7Ac, showing ～60% activity when compared with CMP-Neu5Ac. The Photobacterium α2,6 sialyltransferase was weakly active, with ～6% relative activity. The Leg5Ac7Ac-α-2,3-lactose product was then tested as a substrate with six sialidases of viral, bacterial and mammalian origin. All showed much lower activities than with the corresponding sialic acid substrate, with the influenza virus N1 being the most active and human NEU2 being the least active. These results show the feasibility of producing glycoconjugates with Leg5Ac7Ac residues as the terminal sugars, which should display novel biological properties.     

Endothelial alpha 2,6-linked sialic acid inhibits VCAM-1-dependent adhesion under flow conditions.

We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal beta 1-4GlcNAc alpha 2, 6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal beta 1-4GlcNAc/Gal beta 1-3GalNAc alpha2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both alpha 2,6- and alpha 2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing alpha 2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with alpha 2,6- but not alpha 2,3-linked sialic acids. However, overexpression of alpha 2,6-sialyltransferase did not increase alpha 2,6-linked sialic acid on VCAM-1 but did increase alpha 2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that alpha 2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions.     

Biochemical engineering of the N-acyl side chain of sialic acid: biological implications

N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications.     

Binding of the wheat germ lectin to Cryptococcus neoformans suggests an association of chitinlike structures with yeast budding and capsular glucuronoxylomannan

The capsule of Cryptococcus neoformans is a complex structure whose assembly requires intermolecular interactions to connect its components into an organized structure. In this study, we demonstrated that the wheat germ agglutinin (WGA), which binds to sialic acids and beta-1,4-N-acetylglucosamine (GlcNAc) oligomers, can also bind to cryptococcal capsular structures. Confocal microscopy demonstrated that these structures form round or hooklike projections linking the capsule to the cell wall, as well as capsule-associated structures during yeast budding. Chemical analysis of capsular extracts by gas chromatography coupled to mass spectrometry and high-pH anion-exchange chromatography suggested that the molecules recognized by WGA were firmly associated with the cell wall. Enzymatic treatment, competition assays, and staining with chemically modified WGA revealed that GlcNAc oligomers, but not sialic acids, were the molecules recognized by the lectin. Accordingly, treatment of C. neoformans cells with chitinase released glucuronoxylomannan (GXM) from the cell surface and reduced the capsule size. Chitinase-treated acapsular cells bound soluble GXM in a modified pattern. These results indicate an association of chitin-derived structures with GXM and budding in C. neoformans, which may represent a new mechanism by which the capsular polysaccharide interacts with the cell wall and is rearranged during replication.     

Structural studies on the carbohydrate moieties of human liver alpha-L-fucosidase

alpha-L-Fucosidase was purified from human liver to apparent homogeneity and subjected to exhaustive digestion with Pronase. The resulting glycopeptides were isolated by gel filtration on Sephadex G-50 and further fractionated by Bio-Gel P-4 chromatography. Five glycopeptide fractions were obtained. The structures of the carbohydrate portions of all glycopeptide components were fully characterized by a combination of 500-MHz 1H NMR spectroscopy and carbohydrate composition analysis. Fraction I contained disialyl diantennary glycopeptides of the N-acetyllactosamine type. Fractions II and III contained predominantly mono(sialyl-N-acetyllactosaminyl) diantennary glycopeptides with the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----2) branch attached to alpha(1----3)-linked Man in II and to alpha(1----6)-linked Man in III. The N-acetyllactosamine-type glycopeptides in fractions I to III have a small portion (10-15%) of their Asn-linked GlcNAc residues substituted by additional alpha(1----6)-linked Fuc. Also, a minor portion of the NeuAc residues appeared to be attached to Gal in alpha(2----3) rather than alpha(2----6) linkage. Fraction IV contained a mixture of larger-size oligomannoside-type glycopeptides with a variable number (6 to 9) of Man residues. Smaller-size oligomannoside-type glycopeptides were found in fraction V, containing 3 or 5 Man residues; a small portion (10%) of the Man3GlcNAc2Asn component appeared to contain in addition a Fuc residue in alpha(1----6) linkage to the Asn-bound GlcNAc. The overall ratio of oligomannoside-type to N-acetyllactosamine-type carbohydrate structures was found to be 5:4. This article is the first account of the complete characterization of the oligomannoside-type structures in alpha-L-fucosidase; furthermore, the occurrence in alpha-L-fucosidase of mono(sialyl-N-acetyllactosaminyl) structures, Fuc-containing oligosaccharides, and NeuAc alpha(2----3) linked to Gal are reported for the first time.     

Effect of the carbohydrate moiety on the secondary structure of beta 2-glycoprotein. I. Implications for the biosynthesis and folding of glycoproteins

By use of six highly purified exoglycosidases with well-defined specificity, the oligosaccharide units of human plasma beta 2-glycoprotein I (beta 2I) were modified by sequential enzymatic degradation. The released monosaccharides (NeuAc, Gal, GlcNAc, and Man) were quantified, and the carbohydrate compositions of the resulting glycoprotein (gp) derivatives were determined. The gp was found to be both partially sialylated and galactosylated. These findings which are in agreement with earlier reports suggest that the carbohydrate moiety of beta 2I possesses more bi- than tri-antennas, probably three of the former and two of the latter carbohydrate units. Circular dichroic (CD) spectra of native beta 2I and its derivatives were measured in aqueous buffer and 2-chloroethanol (2-CE). Analysis of these spectra for elements of secondary structure showed beta 2I and most of the derivatives to contain predominantly beta-sheet and beta-turn structures. The lack of alpha-helical structures in aqueous buffer was noted. Removal of a large portion of the carbohydrate moiety did not alter the CD spectra or secondary structure of beta 2I in either aqueous buffer or in 2-CE. However, after enzymatic removal of approximately 96% of the carbohydrate moiety, large significant changes in the spectra and secondary structures were observed. In aqueous buffer a shift in the wavelength minimum occurred, accompanied by an increase in the magnitude of the molar ellipticity and the amount of beta-turn, with a reduction in random coil. One-third of the amino acids which were originally in random coil conformation assumed beta-turns after removal of 96% of the carbohydrate moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycoprofile of the different cell types present in the mucosa of the horse guttural pouches

Histochemical characterization of the equine guttural pouches was performed using lectins combined with sialidase digestion and deglycosylation pre-treatments.The goblet cells contained O- and N-linked oligosaccharides with α-Fuc, GlcNAc moieties whereas β-GalNAc, β-Gal-(1–3)-GalNAc, β-Gal-(1–4)-GlcNAc and α-Gal residues belonged only to O-linked glycoproteins. The acinar and ductal cells expressed α-Man/α-Glc in N-linked oligosaccharides, GlcNAc in both O- and N-glycoproteins and β-GalNAc, β-Gal-(1–3)-GalNAc, β-Gal-(1–4)-GlcNAc and α-Gal residues included in O-linked glycoproteins. The Golgi area of the epithelial lining expressed α-Fuc in O-linked glycoproteins, internal GlcNAc in N-linked glycoproteins and large amounts of sialic acid residues linked to subterminal β-GalNAc, Galβ1,4GlcNAc and Galβ1,3GalNAc. High amounts of sulpho-carbohydrates and of sialic acids (α2,3–6), linked to-α/β-Gal and sialic acids (α2–6) linked to β-GalNAc, were also demonstrated.Such diversity of the mucin saccharide residues may be implicated in the binding of macromolecules such as those of bacterial or viral etiology, thus playing a role in the organism's host-defense mechanism in the guttural pouches.     

Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction

The Ia+ B cell lymphoma, AKTB-1b, fails to stimulate thymic lymphocytes in a one-way mixed lymphocyte reaction unless pretreated with sialidase or inhibitors of N-linked oligosaccharide processing. A comparison of different sialidases and sialyltransferases suggests that the removal of only a subset of total surface sialic acid, rather than net desialylation of the cell surface, is required. Three sialidases were compared, including Vibrio cholerae (VC) and Clostridium perfringens (CP), which will cleave alpha 2-3, alpha 2-6, and alpha 2-8, sialic acid linkages, and Newcastle Disease virus (NDV), which will remove only alpha 2-3 and alpha 2-8 linked sialic acid. When treated with equivalent units of sialidase, CP-, VC-, and NDV-treated cells were 24-fold, sixfold, and threefold better stimulators than untreated cells. In contrast, VC released 1.3-fold and 2.5-fold more sialic acid per cell than did CP or NDV, respectively. Furthermore, VC was superior in reducing the levels of binding of the sialic acid-specific lectin, Limulus polyphemus agglutinin, in exposing Gal beta 1-3GalNAc and Gal beta 1-4GlcNAc residues, and in desialylating gangliosides. Two-dimensional gel analysis indicated that VC and CP were both equal and superior to NDV in the desialylation of iodinatable cell-surface proteins, including H-2Kk, I-A beta k, and a highly sialylated 65,000 dalton protein of unknown identity. Maximal resialylation of CP-treated cells with exogenously added CMP-NANA and either the alpha 2-3(Gal beta 1-3GalNAc) or alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase did not reduce the stimulatory capacity of these cells. However, resialylation of VC-treated cells with just CMP-NANA alone resulted in 49% reversal of their stimulatory capacity, and no additional reversal could be achieved with either of the sialyltransferases. Although the alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase was capable of adding back approximately 10% of the sialic acid removed, the endogenous activity added back approximately 0.1% of the total sialic acid removed. SDS-PAGE gels of the sialylated cells indicated that the exogenously added sialyltransferase labeled many different proteins, whereas the endogenous activity labeled far fewer proteins, predominantly in 46,000 and 25,000 m.w. range. Both the desialylation and resialylation data suggest that the sialidase-dependent stimulation is due to the desialylation of specific membrane structures. Together with previous studies, these data suggest that the sialic acids involved are probably alpha 2-6 linked to N-linked glycosyl moieties.     

A lectin histochemical study on carbohydrate moieties of the gonadotropin-like substance in the epithelial cells of Hatschek's pit ofBranchiostoma belcheri

The present light microscopic lectin, histochemical study suggests for the first time that the vertebrate gonadotropin-like substance in the basal part of the epithelial cells of Hatschek's pit is a sialic acid-containing glycoprotein. The binding intensity of the epithelial cells in Hatschek's pit to 6 lectins (Limulus polyphemus agglutinin (LPA), Wheat germ agglutinin (WGA),Helix pomatia agglutinin (HPA), Concanavalin A (Con A),Ulex europaeus agglutinin I (UEA I) andRicinus communis agglutinin I (RCA I)) indicate that the carbohydrate composition of the gonadotrophic glycoprotein is similar to that of mammals and fish, and that N-acetyl-D-galactosamine, sialic acid, glucosamine, D-mannose and L-fucose are components of the carbohydrate portion.     

Comparative study of chicken ovalbumin subfractions having different carbohydrate chain from each other by high performance anion exchange chromatography

1. Ovalbumin was fractionated to six fractions according to their phosphate content by high performance anion exchange chromatography. 2. This method was applied to analyze the phosphate content of ovalbumin subfractions having different carbohydrate chain from each other which were prepared by concanavalin A/Sepharose chromatography from oviduct slices incubated with [2-3H]mannose. 3. Most biosynthetic intermediates bearing a carbohydrate chain of Man8 or 9 GlcNAc2 was not phosphorylated while other fractions bearing differently processed carbohydrate chains such as Man5 or 6 GlcNAc2 or hybrid type carbohydrate chain was phosphorylated at their peptide portion.     

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.     

The crystal structure of staphylococcal superantigen-like protein 11 in complex with sialyl Lewis X reveals the mechanism for cell binding and immune inhibition

Staphylococcus aureus is a major pathogen that produces a family of 14 staphylococcal superantigen-like (SSL) proteins, which are structurally similar to superantigens but do not stimulate T cells. SSL11 is one member of the family that is found in all staphylococcal strains. Recombinant SSL11 bound to granulocytes and monocytes through a sialic acid-dependent mechanism and was rapidly internalized. SSL11 also bound to sialic acid-containing glycoproteins, such as the Fc receptor for IgA (FcalphaRI) and P-selectin glycoprotein ligand-1 (PSGL-1), and inhibited neutrophil attachment to a P-selectin-coated surface. Biosensor analysis of two SSL11 alleles binding to sialyl Lewis X [sLe(x)- Neu5Acalpha2-3Galbeta1-4(Fuc1-3)GlcNAc] coupled to bovine serum albumin gave dissociation constants of 0.7 and 7 mum respectively. Binding of SSL11 to a glycan array revealed specificity for glycans containing the trisaccharide sialyllactosamine (sLacNac - Neu5Acalpha2-3Galbeta1-4GlcNAc). A 1.6 A resolution crystal structure of SSL11 complexed with sLe(x) revealed a discrete binding site in the C-terminal beta-grasp domain, with predominant interactions with the sialic acid and galactose residues. A single amino acid mutation in the carbohydrate binding site abolished all SSL11 binding. Thus, SSL11 is a staphylococcal protein that targets myeloid cells by binding sialyllactosamine-containing glycoproteins.     

Histochemical properties of sialic acids and antimicrobial substances in canine anal glands

The functional properties of sialic acids appear to be manifold. Additionally, antimicrobial substances serve as a non-specific defense against microorganisms. In this study, therefore, the localization of sialic acids and antimicrobial substances in the anal glands of dog was studied by sialoglycoconjugate histochemistry and immunohistochemistry. The secretory epithelium, luminal secretions and excretory ducts exhibited high levels of sialoglycoconjugates that terminated in Siaα2-6Gal/GalNAc or Siaα2-3Gal1-4GlcNAc. Additionally, O-acetylated sialic acids were detectable in these glandular structures. Antimicrobial substances, such as lysozyme, immunoglobulin A (IgA), lactoferrin and the peptide group of β-defensins, were also demonstrated as products of the anal glands. The results obtained are discussed with regard to the functional significance of the anal glands. These secretory products may create a defensive barrier against microbial invasion at the anal mucosa.