Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

The inactivation of the bovine J blood group substance by the periodate ion

• 1 Treatment of J-positive (JR) bovine erythrocytes with periodate (0.25 mmol/1 final concentration, 1 hour, room temperature) has no effect on the J activity. Higher periodate concentrations cause spontaneous haemolyses.
• 2 Treatment of the lipids extracted from (and containing all J activity of) J^cs erythrocytes with periodate leads to a decrease of J activity even with lower periodate concentrations.
• 3 Treatment of the stroma prepared from J^cs erythrocytes with periodate demonstrated the relative stability of the J antigen up to 0.25 mmol/l periodate. At the same time the sialic acid concentration of stroma is reduced to about 13 % of the initial concentration.
• 4 Desialylation of J^cs erythrocytes or J^cs stroma with sialidase does not affect the J activity thus confirming previous findings. On the other hand, the J activity of desialylated J^cs stroma is much more susceptible to periodate.
• 5 It is concluded that membrane-bound sialic acid shields the membrane-bound J antigen from being attacked by periodate.

     

A direct evidence for the early membrane desialylation in cobalt-irradiated mouse lymphocytes

Using the sensitive periodate-borotritide radioassay to quantify the membrane sialic acid amount, we show that the $\text{in vitro}$ [⁶⁰Co]-irradiation of mouse splenocytes (10–50 Gy) significantly decrease their membrane sialic acid amount. The results show that the irradiation-induced desialylation is a very early phenomenon since the periodate oxidation is done immediately after the irradiation. A short incubation of cells at 25° C does not increase the extent of the desialylation. This membrane alteration might explain the rapid and drastic decrease in lymphocyte counts in mammals exposed $\text{in vivo}$ to irradiation.     

Histochemical detection of sialic acid residues using periodate oxidation

Synopsis The use of low concentrations of periodate for the detection of sialic acid residues in tissue sections has been investigated. Oxidation of aqueous solutions of sugar glycosides with 0.4mm periodate revealed that sialic acid was oxidized more rapidly than other sugars found in glycoproteins. Sequential treatment of tissue sections with 0.4mm periodate for 30 min followed by Schiff's reagent stained sialic acid residues but other sugar components were not stained under these conditions.     

Modification and introduction of various radioactive labels into the sialic acid moiety of sialoglycoconjugates

A method for modifying and isotopic labeling the sialyl moiety of sialoglycoproteins is described. The basis of the procedure is the reductive amination of the exocyclic aldehyde group, generated on sialic acid by mild periodate oxidation, with a variety of amino compounds and sodium cyanoborohydride. Optimal conditions were selected to obtain maximum modification of sialic acid and minimal non-specific incorporation of the amino compound (glycine). The glycine modified model glycoproteins (α₁-acid glycoprotein, fetuin) yielded single homogenous peaks upon gel filtration and on ion exchange chromatography. On gel electrophoresis a major band accounting for 92–98% of the modified glycoprotein and two minor bands consisting of dimers and trimers of the glycoprotein were observed. The modification did not alter the ability of the sialoglycoproteins to bind to wheat germ agglutinin-Sepharose or to interact with antibodies. The modified sialic acid was only partially released by mild acid hydrolysis suggesting that the introduction of an amino compound into the polyol chain of sialic acid has a stabilizing effect on the ketosidic linkage of the sugar. Interestingly, the modification rendered the sialic acid resistant to a variety of sialidases. The potential uses of this modification procedure include 1) the introduction of different isotopic labels (³H,¹⁴C,³⁵S,¹²⁵I) into the sialic acid moiety of glycoproteins; 2) the preparations of biologically active sialoglycoprotein (hormones, enzymes, co-factors) with increased circulating half-lives in animals; 3) preparation of substrates to search for endoglycosidases; 4) the direct comparison of sialoglycoprotein patterns obtained in small amounts from normal and pathological cells or tissues, and 5) the isolation and purification of cell surface sialoglycoproteins.     

Content and accessibility of sialic acid on the surface of rat hepatocytes during primary culture

The content and accessibility of terminal sialic acid and galactose residues of rat hepatocytes in primary culture were determined by in situ labeling using either periodate or sialidase/galactose oxidase treatment followed by sodium borotritiide reduction. Rat erythrocytes which were used for comparison showed a strongly enhanced tritium incorporation into galactose after sialidase treatment. In contrast, with freshly prepared rat hepatocytes only a small amount of galactose labeling was achieved after sialidase treatment. The amount of galactose labeled following sialidase treatment increased with time in culture up to day 6 and roughly paralleled the increase of the total sialic acid content. Major changes of sialic acid-containing glycoconjugates were restricted to the gangliosides. There was a transient drop in surface labeling of ganglioside-associated sialic acid on the first day in culture. The specific radioactivity of the in situ-tritiated ganglioside-sialic acid also fell by 50% in this period. Between day 2 and 4, there was an increase in gangliosidesialic acid labeling but the specific radioactivity of the sialic acid remained constant. This indicates that newly synthesized gangliosides but not the preexisting ones were accessible to periodate oxidation. The data allow conclusions about turnover and topology of the sialic acid-containing glycolipids.     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulphate ester. I. Methods based upon the selective periodate oxidation of sialic acids

Summary Five new methods, based upon the selective oxidation of sialic acid residues with 0.4mm periodic acid in approximately 1m hydrochloric acid at 4°C for 1 h (PA^*), have been devised for the simultaneous visualization of neutral sugars and either sialic acid and its side chainO-acyl variants orO-sulphate ester. In the first of these, the selective periodate oxidation—borohydride reduction—saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (PA^*—Bh—KOH—PA^*—T—KOH—Bh—PAS) technique, sialic acids withO-acyl substituents at C7, C8 or C9 (or which have two of three side chainO-acyl substituents) stain blue while neutral sugars with periodate-sensitivevicinal diols (hexose, 6-deoxyhexose, andN-acetylhexosamine) stain magenta. The second method, the saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (KOH—PA^*—T—KOH—Bh—PAS), stains all sialic acids blue and neutral sugars magenta. In the third procedure, the selective periodate oxidation—Thionin Schiff—borohydride reduction—periodic acid—Schiff—saponification (PA^*—T—Bh—PAS—KOH) method, sialic acids without side chain substituents (or which have anO-acyl substituent at C7) stain blue and neutral sugars stain magenta. In the fourth method, the saponification-selective periodate oxidation—borohydride reduction—Alcian Blue pH 1.0—periodic acid—Schiff (KOH—PA^*—Bh—AB1.0—PAS) technique,O-sulphate esters stain aquamarine blue and neutral sugars stain magenta. In all of these techniques mixtures of the components stain in various shades of purple. Performance of the KOH—PA^*—Bh—AB1.0—PAS technique without the Alcian Blue pH 1.0 step provides a method for the selective identification of neutral sugars in macromolecules that also contain sialic acids.     

A micromethod for the quantitation of sialoglycoconjugate immobilization on insoluble supports: Its use in the preparation of supports with varying ligand concentration

A micromethod is described for the evaluation of immobilization of sialoglycoconjugates on insoluble supports. Ligands were radioactively labeled in their sialic acid moieties after mild periodate oxidation and borotritide reduction, or in the glycosylamino residue after borotritide reduction of the Schiff's base formed between reducing sialooligosaccharides and β-(p-aminophenyl)-ethylamine. Sephadex G-25, Sepharose 4B, and Cellulose MN 2100 were activated by CNBr or periodate oxidation. The hydrazido derivatives of these supports were prepared using both activation methods, and activated to azido-supports using nitrous acid. Controlled Pore Glass-glycophase activated by periodate oxidation was also studied. The investigation of conditions for the binding of the radioactive ligands was carried out in the microassay using 0.5-ml aliquots of the activated supports. The stability of the bound ligands in dependence on various parameters was investigated using the immobilized radioactive ligands. Multivalent linkages formed between ligand and support gave increased stability to release compared to monovalent attachment, for cyanogen bromide activation. The use of periodate activation yielded ligands with much greater stability even for monovalent linkages.The microassay was used successfully to predict conditions for the batchwise preparation of immobilized ligands.     

Effects of chemical disruption on the biological activities of sea urchin egg jelly

In an attempt to relate the biological activities of sea urchin egg jelly to the structural characteristics of the acid glycoprotein molecule, the jelly was oxidized with H₂O₂ and sodium periodate, and digested with trypsin and pronase. The non-dialyzable products of H₂O₂ and periodate oxidation, and a fucose-rich fraction isolated from enzyme-digested jelly by column chromatography, were tested for their capacity to induce sperm agglutination and acrosome reaction in Hemicentrotus pulcherrimus. It was found that a degree of enzyme digestion sufficient to remove about 80% of the amino acids reduced, but failed to eliminate, the capacity of the jelly to elicit agglutination and acrosomal reaction. Mild oxidation with H₂O₂ suppressed sperm agglutination, but more drastic treatment was required to destroy the capacity of the jelly to induce the acrosome reaction. The loss of both these biological activities after periodate oxidation was found to parallel the release of sialic acid.     

Histochemical studies of the mechanism of the periodic acid-phenylhydrazine-Schiff (PAPS) procedure

Summary Histochemical investigations of the periodic acid-phenylhydrazine-Schiff (PAPS) procedure were carried out on tissues containing carbohydrate macromolecules known to produce on periodate oxidation, only sialic acid monoaldehydes or hexosedialdehydes or mixtures of the two. The results indicated that the PAPS reaction is a generalized phenomenon independent of the hydrazine or hydrazide used, the nature of the Schiff reagent or the presence of anionic groups. It is proposed that phenylhydrazine condenses with periodate-engendered sialic acid monoaldehydes to yield the corresponding phenylhydrazone and with periodate-engendered dialdehydes to yield the corresponding morpholine or azido derivatives. Subsequent Schiff treatment results in the reversal of the blockade of sialic acid monoaldehydes but not of the dialdehydes, thus leading to selective Schiff staining of sialic acid residues.     

Panagrellus redivivus: failure to find evidence for the occurrence and biosynthesis of sialic acids.

The occurrence of sialic acids in the free-living nematode Panagrellus redivivus was studied by periodate oxidation/[3H]sodium borohydride reduction of about 10(7) nematodes. In parallel, the capability of sialic acid biosynthesis was examined by metabolic labeling of the same number of nematodes with N-[3H]acetylmannosamine. In both experiments, radioactivity was incorporated into the nematodes. Mild acid hydrolysis, however, did not release radioactively labeled sialic acids or derivatives as tested by radio thin-layer chromatography, suggesting that P. redivivus does not contain or synthesize sialic acids.     

Glucose suppression of deoxyribose interference in the thiobarbituric acid determination of sialic acid.

Glucose has been demonstrated to suppress the reaction of deoxyribose in the thiobarbituric acid determination of sialic acid. Suppression occurs at the periodate oxidation step in which glucose apparently competes with deoxyribose. This suppression is augmented by sodium chloride and trichloroacetic acid. With these three substances, deoxyribose reactivity can be completely eliminated at concentrations up to 75 μm with only a 25% decrease in sialic acid reactivity.Mixtures of deoxyribose and sialic acid and hydrolyzed extracts of rat organs gave reaction products with an absorption spectrum closely resembling that given by a sialic acid standard. Provided that the spectral characteristics of the colored product from an unknown sample are verified, sialic acid can be determined directly from absorbance at 550 nm without interference by deoxyribose.     

Behavior of aldehyde moieties involved in the activation of suppressor cells by sodium periodate

The treatment of mouse spleen cells with periodate at the optimal mitogenic concentration (1 mM) induces the activation of suppressor cells of the in vitro antibody response and leads to the formation of aldehydes on the carbohydrate termini of the surface sialoglycoconjugates. These aldehyde moieties are found on the C8 (N-AN 8) and the C7 (N-AN 7) derivatives of sialic acid. Immediate borohydride reduction prevents the activation of the suppressor cells. Data from this work show that borohydride reduction must be performed within the first 6 hr to prevent the generation of suppressor cells; 18 hr after the initial periodate oxidation, borohydride treatment did not reverse the in vitro suppressive activity of periodate-treated cells. The kinetics of the disappearance of aldehydes from the cell surface were studied by using [3H]borohydride labeling and chromatographic analysis of sialic acid derivatives. About 70 to 80% of the aldehyde moieties were found to be present 6 hr after periodate oxidation. After 18 hr, 50 to 70% of the aldehyde had disappeared from the lymphocyte membrane. Oxidized sialyl residues disappear completely after 60 hr of culture. This period corresponds to the de novo synthesis of sialic acid residues on the surface of periodate-activated cells. The two classes of oxidized sialyl-glycoconjugates were found to behave in different ways. In effect, our data showed that the aldehydes remaining at 18 hr are mainly located on the gangliosides, whereas the aldehyde moieties located on high m.w. glycoproteins disappear from the cell surface between 9 and 18 hr. This would suggest that the remaining aldehydes located on gangliosides are not directly involved in the expression of suppressive activity.     

The structure of a glycopeptide purified from porcine thyroglobulin

1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, alpha-l-fucosidase, beta-d-galactosidase, beta-N-acetyl-d-glucosaminidase and alpha-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid-->galactose-->N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose-->N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, alpha-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide.     

Modification of sialyl residues of glycoconjugates by reductive amination. Characterization of the modified sialic acids

The sialic acid residues of alpha 1-acid glycoprotein and fetuin were modified by introduction of an amino residue, such as glycine and [3H]glycine. This modification involved (a) the selective periodate oxidation of the exocyclic carbon atoms of the sialic acid residue generating an aldehyde group at C-7, and (b) the reduction of the Schiff base formed with an amino compound by use of sodium cyanoborohydride. Thin layer chromatography, high pressure liquid chromatography, and amino acid composition data of the modified glycoprotein showed that the conversion was essentially quantitative. The glycine-modified sialic acids were isolated by mild acid hydrolysis and identified by g.l.c.-m.s. and n.m.r. spectroscopy, thus confirming that the quantitative modification produced a glycine-aminated C-7 sialic acid analog. Strong acid hydrolysis of the glycine-modified sialic acid yielded a fragment that had chromatographic characteristics similar to those of glycine.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

The histochemical application of dansylhydrazine as a fluorescent labeling reagent for sialic acid in cellular glycoconjugates.

A new method for the fluorescent staining of stalic acid-containing glycoconjugates in fixed tissues is described. The procedure uses mild periodate oxidation, followed by condensation with dansylhydrazine and reduction of the hydrazones to hydrazines. The specificity of the reaction for sialic acid is tested on model glycoconjugates. The procedure gives superior resolution in comparison to the standard periodate Schiff procedure for cellular carbohydrates.     

Direct evidence for sialic acid oxidation in periodate-induced lymphocyte proliferation.

Normal mouse spleen cells treated with periodate were stimulated to undergo blastogenesis. In contrast spleen cells from nude mice did not respond to periodate. Such a treatment of normal and nude spleen cells led to the oxidation of the cell surface sialic acid residues with formation of N-AN8. Prior treatment with neuraminidase of normal spleen cells greatly impaired their capacity to respond to periodate activation with a decrease in the amount of N-AN8 formed.     

Gangliosides of the starfish Aphelasterias japonica, evidence for a new linkage between two N-glycolylneuraminic acid residues through the hydroxy group of the glycolic acid residue

Mono- and disialogangliosides containing glucose, galactose and sialic acids were isolated from the total lipid extract of hepatopancreas of the starfish Aphelasterias japonica. Their structures were elucidated by total and partial acid hydrolysis, trideuteriomethylation analysis, neuraminidase treatment, chromium trioxide oxidation, methanolysis and periodate oxidation. The monosialoganglioside was identified as 8-O-methyl-N-glycolylneuraminosyl-alpha-(2-3)-galactosyl-beta-(1- 4)-glucosyl-beta-(1-1)-ceramide. The disialoganglioside has the additional N-glycolylneuraminic acid or its 8-O-methyl derivative residue at the subterminal position to which the terminal sialic acid residue is linked through the hydroxy group of the glycolic acid unit. The long-chain bases were found to be mixtures of phytosphingosines with both branched and linear chains, and the fatty acids were shown to be mixtures of normal and alpha-hydroxy fatty acids, the latter amounted to about 90% of the fatty-acid mixtures. The composition of the lipid moieties of the gangliosides was determined by GLC and GLC-MS.     

Preparation of neuraminidase-resistant human alpha 1-protease inhibitor and its clearance in rat blood circulation

The sialic acid residues of human alpha 1-protease inhibitor were modified by periodate oxidation and subsequent reductive amination with ethanolamine and sodium cyanoborohydride. The modified inhibitor retained its original trypsin inhibitory activity and was not digested by neuraminidase from Clostridium perfringens. The modified inhibitor disappeared from rat blood circulation at the same rate as the native inhibitor.