Mild Periodate Research Articles

Enhanced interaction of L-selectin with the high endothelial venule ligand via selectively oxidized sialic acids.

The selectins are adhesion receptors that play key roles in leukocyte trafficking. Each has an N-terminal C-type lectin domain that binds to specific carbohydrates in a calcium-dependent manner. L-selectin recognizes sulfated, sialylated ligands on lymph node high endothelial venules. This recognition is abolished by strong periodate oxidation under conditions that destroy oligosaccharides. In contrast, mild periodate oxidation, which selectively oxidizes the side chain of sialic acid residues without affecting the underlying oligosaccharide, markedly enhances this interaction. The enhancement is calcium dependent, indicating that lectin recognition is maintained. Reduction of the sialic acid aldehydes generated by mild periodate to alcohol groups abolishes this effect. Covalent cross-linking of the oxidized ligand to L-selectin can be demonstrated, suggesting Schiff base formation between lysine residues of the selectin and the newly formed aldehydes. Such selectively oxidized sialylated ligands could be used to probe the lectin domains of the selectins and to identify lysine residues near the binding site. Also, this approach could be used to design drugs for disrupting leukocyte-endothelial interactions leading to pathological inflammation.

Specificity of mild periodate oxidation of oligosaccharidealditols: relevance to the analysis of the core-branching pattern of O-linked glycoprotein oligosaccharides

The specificity of mild periodate oxidation of 3- and 6-substituted 2-acetamido-2-deoxy- d-galactitols and 4- and 6-substituted d-glucitols has been investigated. The products were reacted with 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine and the derivatives were analysed by application of liquid secondary-ion mass spectrometry directly to the TLC plates. There was > 95% specificity of cleavage of the C-4C-5 bond ( threo-diol) for the GalNAcol derivatives. The major sites of oxidation for the Glcol derivatives also involved threo-diols. For α-Neu5Ac-(2 → 6)-GalNAcol, ∼ 30% of the products of oxidation involved the sialic acid side chain, and ∼60% were cleaved at the C-4C-5 bond of the GalNacol moiety. The mild periodate oxidation reaction forms part of a strategy for determining the patterns of branching of the cores of O-linked glycoprotein oligosaccharides and other oligosaccharidealditols.

Soil properties associated with alfalfa winter survival at Kamloops, British Columbia

Alfalfa (Medicago sativa L.) survival at Kamloops Agriculture Canada Research Station decreased following the 1985–1986 cold event, when survival varied along a soil gradient. Survival was positively related to the soil's potential available soil-water storage capacity, which may have contributed to a temporary buffering of soil-temperature decline. However, this relationship was not observed following the 1988–1989 cold event, when the air-temperature drop was greater following a prolonged mild period. Key words: Medicago sativa, low-temperature injury, cold hardiness, available water storage capacity, extractable soil potassium

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-.

A cell-type specific ganglioside of glomerular podocytes in rat kidney: an O-acetylated GD3.

We recently described a monoclonal antibody (clone 27A) that detected a membrane antigen specific for glomerular podocytes in adult rat kidney. After binding in vivo, the antibodies induced rapid changes in the foot processes. Here we show that in other rat tissues the antigen is detectable only in cells of adrenal medulla, in some cells of neural or neural crest origin, and in 1 to 5% of the cells of a rat pheochromocytoma cell line PC-12. Attempts to isolate the antigen revealed that it is an acidic, sialic acid containing lipid, as shown by thin layer chromatography and immuno-overlay techniques. Further characterization of the gangliosides extracted from rat glomeruli, bovine kidney, rat adrenal glands, or from PC-12 cells by ion exchange, thin layer, and gas liquid chromatography identified the antigenic lipid as a modified disialosyllactosylceramide (GD3). The results of mild alkaline treatment or periodate oxidation of the antigenic ganglioside, as well as chemical O-acetylation studies of standard gangliosides, showed that the modified ganglioside is O-acetylated, most probably at the 9-carbon of its terminal sialic acid residue. To our knowledge this is the first report of cell-type specific expression of gangliosides in the kidney.

Biochemical characterization of 25 distinct carcinoembryonic antigen (CEA) epitopes recognized by 57 monoclonal antibodies and categorized into seven groups in terms of domain structure of the CEA molecule.

The chemical nature of 25 distinct carcinoembryonic antigen (CEA) epitopes, which were recognized by 57 different monoclonal antibodies and categorized into 7 groups (Groups A to G) in terms of domain structure of the CEA molecule, was analyzed and the findings obtained were compared with the results of our previous studies using recombinant CEA proteins. All 21 epitopes of Groups A to F defined by 48 MAbs were resistant to periodate oxidation and were to a greater or lesser extent retained after deglycosylation of CEA, indicating that they are all protein in nature. The 21 epitopes were detected in recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Seven of the 21 epitopes of protein nature were partially or completely sensitive to reduction and alkylation of CEA and not detected or only slightly revealed in the recombinant CEA proteins expressed in E. coli, indicating that those epitopes are dependent on the tertiary structure of the peptide chain, which is formed by disulfide bonds. All 4 epitopes of Group G defined by 9 MAbs were sensitive to mild periodate oxidation and deglycosylation, but resistant to reduction and alkylation and to digestion with pepsin or pronase, indicating that those 4 epitopes are carbohydrate in nature. Although none of the 4 epitopes of Group G were detected in the recombinant CEA proteins expressed in E. coli, two were detected in those expressed in CHO cells. The biochemical studies reported here thus provide information as to the nature of the epitopes on the CEA molecule and help form the basis for selecting the anti-CEA MAbs for use in biological study and potential clinical applications of CEA.

A periodate-sensitive anti-phagocytic surface structure, induced by growth in milk whey, on Staphylococcus aureus isolated from bovine mastitis

The phagocytic and chemiluminescent activity of purified bovine neutrophils in response to two Staphylococcus aureus strains isolated from mastitic bovine milk and grown in milk whey was studied. The activity was significantly reduced compared with the response elicited by the same strains grown in tryptic soy broth (TSB). A mild periodate treatment of the milk whey-grown strains resulted in a significant increase of both chemiluminescence and phagocytosis, whereas trypsin, subtilisin or papain treatment had no effect. The decreased binding of complement factor C3 to milk-whey-grown bacteria was restored to the level of TSB-grown homologous organisms by periodate treatment. Moreover, this treatment, but not treatment with trypsin, increased the surface hydrophobicity of milk-whey-grown bacteria. The chemiluminescent activity was as high towards heat-killed as towards live bacteria. Also, incubation of heat-killed TSB-grown bacteria in milk whey did not alter the chemiluminescent response, indicating that the reduced neutrophil activity towards milk-whey-grown bacteria was not due to binding of milk components to the microorganisms. These results strongly suggest that bovine mastitis S. aureus strains grown in milk whey produce an anti-phagocytic surface structure. This structure is heat- and protease-resistant and renders the bacterial surface hydrophilic. The anti-phagocytic material is altered or, more likely, released from the bacterial surface on periodate treatment and is probably of carbohydrate nature.

Isolation and partial characterization of a soluble oncofetal antigen from murine and human amniotic fluids

A soluble (cell-free) oncofetal antigen (OFA) was detected in murine and human amniotic fluids by immunostaining with the murine monoclonal antibody (MAb 115) produced by syngeneic immunization with mid-gestational mouse fetal cells. OFA was purified from the amniotic fluids by ammonium sulfate precipitation at 30-70% saturation, followed by successive gel chromatography of the OFA-containing fraction on Sephacryl-S300 HR, Q- and S-Sepharoses and lentil lectin agarose. The fraction eluted from the lentil lectin column gave a single band on SDS-PAGE of the same molecular weight as the membrane-bound OFA found on both fetal and tumor tissues of humans and several rodents. Both soluble and membrane-bound OFAs share several chemical characteristics, including binding to lentil lectin and wheat-germ agglutinin, molecular weight (44 kDa) and pI (6.8). Mild periodate oxidation of OFA did not affect its binding to MAb 115 in an enzyme-linked immunosorbent assay, indicating that the reactive epitope is a peptide.

Establishment and characterization of monoclonal antibodies against Chuzan virus K-47.

We established sixteen mouse monoclonal antibodies reactive to Chuzan virus K-47 strain using P3-X63-Ag8-U1 cells as fusion partner cells. Among them, CG53/2/4 recognized a 100K structural protein of the virus. The 100K antigen lost it's antigenicity for CG53/2/4 after mild periodate oxidation treatment, suggesting that the 100K viral antigen is a glycoprotein. In addition, CG53/2/4 neutralized the viral infectivity. This indicates that the 100K glycoprotein is essential for the infection of the virus. The other monoclonal antibodies reacted with a 41K antigen of the virus. Especially CG1/1 showed the highest reactivity to the virus. Forward step sandwich assay using CG1/1 and biotinylated CG53/2/4 could detect the virus at 10TCID50/ml. Therefore, these monoclonal antibodies can eventually predict the virus infection to the animals before their sideration.

Selection and exploitation of feeding areas by staging and wintering geese in southmost Sweden

Bean Geese and Canada Geese preferred high-energy food (spilled grain, sugar-beet spill, potatoes and carrots) in autumn. When harvested fields were ploughed or when the ground was frozen, the geese grazed winter cereals. During periods with deep snow most of the Canada Geese utilized rape sticking up through the snow. In spring, both species fed on sprouting grass on permanent pastures. Such pastures were also heavily utilized by Bean Geese during mild periods in late winter. White-fronted Geese primarily used winter cereals and grassland. Marked changes in agricultural practices have occurred in southern Sweden contemporarily with the population increase in the Bean Goose. Mechanical harvesting has provided abundant spill of sugar beet, potatoes and carrots, serving as a high-rated food source for geese in autumn. Distance to the roost was also an important factor influencing field choice. Flying distances of up to 14 km were noted both for Bean and Canada Geese, although mean distances were much shorter. SW Skåne could probably support larger staging populations. During the last 10 years only a small proportion of suitable areas (36% of 1 X 1 km squares within flying distance of 14 km of a roost) was used by feeding Bean Geese.

A carbohydrate-directed heterobifunctional cross-linking reagent for the synthesis of immunoconjugates

A novel, highly water-soluble, heterobifunctional cross-linking reagent, S-(2-thiopyridyl)- l-cysteine hydrazide (TPCH), was synthesized which contains a hydrazide moiety for coupling to aldehyde groups generated in the carbohydrate residues of antibodies by mild periodate oxidation, and a pyridyl disulfide moiety for coupling to molecules with a free sulfhydryl group. Since the carbohydrate moieties are distal to the antigen binding region of antibodies, derivatization with this cross-linker minimizes impairment of the antigen binding function. Derivatization of the human monoclonal IgM antibody 16–88 against human colon carcinoma cells with as many as 16 TPCH cross-linker molecules did not impair its antigen binding capability. Using mild oxidation conditions for antibody derivatization, sialic acid residues were identified as attachment sites for the cross-linker molecules, since after desialylation of antibody 16–88 by neuraminidase virtually no cross-linker molecules could be incorporated. Comparison of TPCH with S-(2-thiopyridyl)mercaptopropionic acid hydrazide and S-(2-thiopyridyl)- l-cysteine, two related cross-linking reagents, revealed that TPCH is most efficiently incorporated into periodate-treated antibody. Based on the structural differences of the cross-linkers, the more efficient incorporation of TPCH appears to be a function of the presence of a hydrazide moiety with an adjacent amino group. When three to four molecules of pyridyl disulfide-derivatized barley toxin were coupled to TPCH-derivatized antibody 16–88, the antigen binding capability remained uncompromised. In addition, no significant impairment of toxin activity upon coupling to the antibody was observed. Based on these data, TPCH may be very useful for the synthesis of immunoconjugates with no or only minimal impairment of the antigen binding function.

The Structure of an Extracellular Polysaccharide from a Marine Strain of Enterobacter.

An extracellular, acidic polysaccharide was isolated from a marine strain of Enterobacter sp., designed MU-3, which was tentatively identified as E. agglomerans. The structure of this polysaccharide was determined by techniques of methylation, mild acid hydrolysis, periodate oxidation, Smith hydrolysis, and β-elimination. 1H and 13C NMR spectroscopy was used to establish the nature of the anomeric linkages of the polysaccharide and of oligosaccharides derived through degradative procedures. The polysaccharide has a branched structure containing a backbone chain of →3)-β-D-Galp (1→3)-α-D-Galp (1→6)-β-D-Glcp (1→, and a side chain of α-D-Galp (1→4)-β-D-GlcUAp (l→. The glucuronic acid residue in the side chain joins through C-4, to β-D-galactose residue in the main chain. The terminal galactose in the side chain bears a pyruvic acetal substituent at positions C-4 and C-6.

Effect of plasma on composition of human enamel and cementum pellicle

Slabs of human enamel and cementum were incubated with plasma alone or with various mixtures of plasma and saliva. Proteins and glycoproteins that adsorbed to the surface of the slabs in 0 to 60 min were labeled by lactoperoxidase-catalyzed 125I-iodination and by mild periodate oxidation followed by NaB3H4 reduction. The labeled components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography or fluorography. From plasma alone, a 58 and a 66 kDa protein (probably albumin) were adsorbed to the enamel surface in relatively equal amounts, but no 125I-labeled components were detected on the cementum surface in the absence of saliva. Adding 10% saliva to the incubation mixture promoted the adsorption of the 58 and 66 kDa components to cementum. In addition, another set of proteins, including components of 44, 47, 29, and 25 kDa, was adsorbed to both cementum and enamel in the presence of saliva. These six proteins were the major 125I-labeled species in all of the pellicles formed from mixtures of plasma and saliva. The electrophoretic mobility of the major 120 and 140 kDa 3H-labeled sialoglycoproteins adsorbed to both cementum and enamel was similar to that of the low-molecular-weight mucin of submandibular/sublingual saliva.

N-linked oligosaccharides of human transferrin are not required for binding to bacterial transferrin receptors

Derivatives of human transferrin (hTf) with removed or modified N-linked oligosaccharides were compared with native hTf with respect to their binding to bacterial hTf receptors from Neisseria meningitidis, N. gonorrhoeae, and Haemophilus influenzae. Partially and fully deglycosylated hTf were prepared by enzymatic deglycosylation with glycopeptidase F and isolated by concanavalin A-Sepharose affinity chromatography. Oligosaccharide-modified hTf was prepared via mild periodate oxidation. Competition and direct binding experiments with the hTf derivatives demonstrated that the hTf oligosaccharides are not essential for binding to the bacterial hTf receptors.

Generation and characterization of hamster-mouse hybridomas secreting monoclonal antibodies with specificity for lipopolysaccharide receptor.

Experiments are described for the partial purification of the 80-kDa LPS binding protein expressed on macrophages and lymphocytes. This partially purified Ag was used to immunize adult Armenian hamsters and splenocytes from immunized animals were fused with murine myeloma cell lines. Hybridoma cell culture supernatants containing mAb were screened by ELISA for positive binding to the immunizing Ag, murine splenocytes and the murine 70Z/3 pre B cell and for an absence of binding to sheep E. Positive clones were further screened for reciprocal competitive binding with LPS on spleen cells and ability to modulate B lymphocyte mitogenic activity. Two hybridoma cell lines secreting IgM monoclonals, termed mAb3D7 and mAb5D3, were identified that satisfied all of the selection criteria. These hybridoma cell lines were subcloned and expanded. Binding of one (mAb3D7) was abrogated by treatment of Ag with mild periodate; binding of the second (mAb5D3) was destroyed by digestion of Ag with proteinase K. Binding specificity for mAb5D3 has been confirmed by ELISA using highly purified 80-kDa protein. These mAb have been of value in establishing that the 80-kDa LPS binding protein previously identified may serve as a specific functional receptor for LPS.

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to β-galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.

Lipophosphoglycan expression and virulence in Ricin-resistant variants of Leishmania major

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats joined by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the M r 63 000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies. All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.

Microscale sequencing of O‐linked oligosaccharides using mild periodate oxidation of alditols, coupling to phospholipid and TLC‐MS analysis of the resulting neoglycolipids

In the course of characterising neoglycolipid products derived from mucin oligosaccharide alditols after periodate oxidation and coupling by reductive amination to the aminolipid dipalmitoylglycerophosphoethanolamine, we have obtained evidence that oxidative cleavage occurs specifically at the C4-C5 bond of core N-acetylgalactosaminitol. Two lipid-linked fragments thus obtained from each oligosaccharide alditol are well resolved on thin-layer chromatography and can be sensitively analysed by liquid-secondary-ion mass spectrometry to assign the sequence and branching patterns of oligosaccharides linked at C6 and C3 to the N-acetylgalactosamitol. These conclusions have been reached from detailed studies of the neoglycolipid derivatives of several oligosaccharides (di- to hexasaccharides) which were isolated from human meconium and characterised previously by MS and NMR studies as the free alditols.

Partial structure of an extracellular mucopolysaccharide produced by a marine bacterium.

The alanine associated with the extracellular mucopolysaccharide produced by a marine Pseudomonas (strain No. 9-12) was determined to be attached covalently to the carboxyl group of galacturonic acid and to have the D-absolute configuration. Depolymerization of this mucopolysaccharide was performed using techniques of mild acid hydrolysis, deamination, and periodate oxidation. Mild acid hydrolysis provided two polymers free of pyruvic acid with molecular weights of 5.5×104 and 8.5×104. Although the native polysaccharide was resistant to both deamination and periodate oxidation, partially N-deacetylated polysaccharide (M.W.=2.4×105) was sensitive to both reactions. Thus, some galactosamine residues were deaminated to 2, 5-anhydrotalitol and some galacturonic acid and galactosamine residues were oxidized by the peri-odate. Deamination and Smith hydrolysis yielded polysaccharides with molecular weights of 1.4×104 and 5.0×104, respectively.

Demonstration of a Surface Antigen of Clostridium tyrobutyricum by Use of Immunoblotting with a Monoclonal Antibody

A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.