Separation methods for sialic acids and critical evaluation of their biologic relevance

Sialic acids are biosynthesized by almost all organisms as a 9-carbon carboxylated monosaccharide and are integral components of glycoconjugates. More than 40 naturally occurring sialic acid derivatives of the three main forms of sialic acids, the N-acetyl- and N-glycolylneuraminic acid and 2-keto-3-deoxy-nonulosonic acid have been identified. Due to the great importance of sialic acids as key mediators in a plethora of cellular events, including cell-cell recognition and cell-matrix interactions, their analysis in biologic samples is useful for a deeper understanding of the various (patho)physiological processes and of value in disease diagnosis and monitoring. In this review we summarize the methodology developed to isolate and liberate sialic acids from biologic samples as well as the chromatographic, electromigration and hyphenated techniques available for their separation and analysis. A critical evaluation of the biological relevance of the results obtained by analyzing sialic acids in biologic samples is also presented.     

A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

The synthesis and evaluation of novel sialic acid analogues bound to matrices for the purification of sialic acid-recognising proteins

A novel N-acetylneuraminic acid analogue, 2-S-(5'-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2'-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.     

A comparison of succinyladenylate lyase activity and serum sialic acid as markers of malignancy

A comparison was made of succinyladenylate lyase (SAMP lyase), total serum sialic (TSA), and lipid soluble serum sialic acid (LSA) as early markers of malignancy in three experimentally induced rat tumor models. Elevation of SAMP lyase in 3'-methyl-dimethylaminoazobenzene-induced hepatic tumors at 2 weeks corresponded with microscopic detection of preneoplastic lesions with elevation of LSA occurring 2 weeks later. Elevation of breast SAMP lyase concurred with macroscopic presence of dimethylbenzanthracene involved breast tumors with elevation of LSA occurring 12 weeks later. Neither colon SAMP lyase nor LSA increased in rats bearing colon tumors induced by dimethylhydrazine. The determination of TSA was not a reliable indicator of tumor presence for the three types of tumors investigated. Both SAMP lyase and LSA are very good early indicators of hepatic tumor with SAMP lyase an earlier indicator of breast tumor than LSA.     

A new histochemical method for the selective periodate oxidation of total tissue sialic acids

Summary Evaluation of the intensity of the periodic acid—Schiff (PAS) staining produced following oxidation for 1 h at 4°C with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules orvicinal diols located on hexose, 6-deoxyhexose orN-acetylhexosamine residues (neutral sugars) of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4mm periodic acid in approximately 1m hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure.Studies of the mechanism of the oxidation of neutral sugars with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4mm periodic acid in approximately 1m hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.     

Effects of calcium and bound sialic acid on the viscosity of mucin

A viscosity drop was observed when pig gastric mucin was titrated with HCl below pH 3.0. When EDTA was present, addition of HCl resulted in an increase in viscosity between pH 3.0 and pH 1.0. Previous treatment of the mucin solution with neuraminidase abolished the pH-dependent viscosity rise in the presence of EDTA.     

Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry

The N-acetyl-D-mannosamine (ManNAc) analog Ac5ManNTGc, a non-natural metabolic precursor for the sialic acid biosynthetic pathway, can be used to display thiols on the cell surface. Sugar-expressed cell-surface thiols are readily accessible compared to their protein counterparts, making them ideal for exploitation in cell-adhesion and tissue-engineering applications. This report describes a protocol for the incubation of Jurkat (human acute T-cell leukemia) cells with Ac5ManNTGc and the quantitative estimation of the resulting sialic acid displayed thiols by flow cytometry after a reaction with a water-soluble biotin-conjugated maleimide reagent and fluorescein isothiocyanate-conjugated (FITC) avidin staining. These methods, with minimal optimization, are generally also applicable to other human cell lines. The labeling and flow cytometry steps of this protocol can be performed in five to eight hours.     

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.     

Panagrellus redivivus and Caenorhabditis elegans: evidence for the absence of sialic acids

Complementary experiments were performed to indicate the presence or absence of sialic acids in axenically cultured Panagrellus redivivus and Caenorhabditis elegans. Competitive displacement experiments with radiolabeled Limax flavus agglutinin demonstrated the presence of sialic acid in nematodes grown in medium which contained liver extract as a growth factor but the absence of sialic acid when heme was substituted for liver extract. This finding suggested that sialic acid present in the liver medium was responsible for conflicting results of other studies. Transmission electron microscopy of thin sections from nematodes labeled with an LFA-ferritin conjugate revealed no label to the surface area of the cephalic chemosensilla. Fluorometric analysis with a modification of the thiobarbituric acid assay was negative for sialic acid. Analyses by gas chromatography-mass spectrometry, sensitive to the high picomole range, were also negative for sialic acid. Taken together the results provide evidence for the absence of sialic acid in P. redivivus and C. elegans using the most sensitive and diagnostic technique currently available.     

A method for concentrating organic dyes: colorimetric measurements of nitric oxides and sialic acids

A new method for extraction and concentration of organic dyes that uses a reagent composed of a nonionic detergent mixed with an alcohol is described. We have observed that water-soluble organic dyes are also soluble in nonionic detergents and can be extracted by adding salt, which separates the dye–detergent component from the aqueous phase. We have also found that mixing nonionic detergents with alcohols markedly reduces their viscosity and produces stable, free-flowing, and effective reagents for color extraction. On the basis of these observations, we used a mixture of Triton X-100 and 1-butanol and observed that water-soluble natural and synthetic chromophores, as well as dyes generated in biochemical reactions, can be extracted, concentrated, and analyzed spectrophotometrically. Trypan blue and phenol red are used as examples of synthetic dyes, and red wine is used as an example of phenolic plant pigments. Applications for quantification of nitric oxides and sialic acids are described in more detail and show that as little as 0.15 nmol of nitric oxide and 0.20 nmol of sialic acid can be detected. A major advantage of this method is its ability to concentrate chromophores from dye-containing solutions that otherwise cannot be measured because of their low concentrations.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Conformational relationships between amino acids and their anticodons in the primitive decoding system

Detailed calculations of the conformational characteristics of a primitive decoding system are presented. A penta-nucleotide serves as the primitive tRNA (PIT) with a triplet of primitive anticodon (PAC) in a helical conformation. This molecular moiety has a cleft in the middle. An amino acid can comfortably nestle into the cleft. The conformation of this molecular association is stabilised by a few hydrogen bonds. The stereochemistry of the moiety restricts the conformational possibilities and the sidechain of the amino acid gets oriented at a proper position and in the correct direction to interact intimately with the PAC in the middle of the PIT. The model favours L-amino acids for beta-D-ribonucleotides. The location of the sidechain of the amino acid in the PIT gives a raison d'être for the important features of the organisation of nucleotide triplets for amino acids in the Genetic Code. The interaction of a few key amino acids with the different combinations of bases as PAC sequences has been studied and the stereochemical basis for the selection of the anticodons for amino acids is elucidated.     

O-acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids

We have previously described the preparation and use of 9-O-[acetyl-3H]acetyl-N-acetylneuraminic acid to identify sialic acid O-acetylesterases in tissues and cells (Higa, H. H., Diaz, S., and Varki, A. (1987) Biochem. Biophys. Res. Commun. 144, 1099-1108). All tissues of the adult rat showed these activities, with the exception of plasma. Rat liver contained two major sialic acid esterases: a cytosolic nonglycosylated enzyme and a membrane-associated glycosylated enzyme. The two enzymes were found in similar proportions and specific activities in a buffer extract of rat liver acetone powder. By using the latter as a source, the two enzymes were separated, and the glycosylated enzyme was purified to apparent homogeneity by multiple steps, including ConA-Sepharose affinity chromatography and Procion Red-agarose chromatography (yield, 13%; fold purification, approximately 3000). The homogeneous enzyme is a 61.5-kDa disulfide-linked heterodimeric protein, whose serine active site can be labeled with [3H]diisopropyl fluorophosphate. Upon reduction, two subunits of 36 kDa and 30 kDa are generated, and the 30-kDa subunit carries the [3H]diisopropyl fluorophosphate label. The protein has N-linked oligosaccharides that are cleaved by Peptide N-glycosidase F. These chains are cleaved to a much lesser extent by endo-beta-N-acetylglycosaminidase H, indicating that they are mainly complex-type glycans. The enzyme activity has a broad pH optimum range between 6 and 7.5, has no divalent cation requirements, is unaffected by reduction, and is inhibited by the serine active site inhibitors, diisopropyl fluorophosphate (DFP) and diethyl-p-nitrophenyl phosphate (Paraoxon). Kinetic studies with various substrates show that the enzyme is specific for sialic acids and selectively cleaves acetyl groups in the 9-position. It shows little activity against a variety of other natural compounds bearing O-acetyl esters. It appears to deacetylate di-O-acetyl- and tri-O-acetyl-N-acetylneuraminic acids by first cleaving the O-acetyl ester at the 9-position. The 7- and 8-O-acetyl esters then undergo spontaneous migration to the 9-position, where they can be cleaved, resulting in the production of N-acetylneuraminic acid. In view of its interesting substrate specificity, complex N-linked glycan structure, and neutral pH optimum, it is suggested that this enzyme is involved in the regulation of O-acetylation in membrane-bound sialic acids.     

Biomarkers for the lung cancer diagnosis and their advances in proteomics

Over a last decade, intense interest has been focused on biomarker discovery and their clinical uses. This interest is accelerated by the completion of human genome project and the progress of techniques in proteomics. Especially, cancer biomarker discovery is eminent in this field due to its anticipated critical role in early diagnosis, therapy guidance, and prognosis monitoring of cancers. Among cancers, lung cancer, one of the top three major cancers, is the one showing the highest mortality because of failure in early diagnosis. Numerous potential DNA biomarkers such as hypermethylations of the promoters and mutations in K-ras, p53, and protein biomarkers; carcinoembryonic antigen (CEA), CYFRA21-1, plasma kallikrein B1 (KLKB1), Neuron-specific enolase, etc. have been discovered as lung cancer biomarkers. Despite extensive studies thus far, few are turned out to be useful in clinic. Even those used in clinic do not show enough sensitivity, specificity and reproducibility for general use. This review describes what the cancer biomarkers are for, various types of lung cancer biomarkers discovered at present and predicted future advance in lung cancer biomarker discovery with proteomics technology.     

Glial cells of the crayfish and their relationships with neurons. An ultrastructural study

1. Glial cells of the crayfish abdominal ganglia have been studied by transmission electron microscopy. Special attention is paid to the interrelationships between neurons and glial cells. Covers and hemocyte-related elements have also been considered. 2. Glial cells are identified by a common ultrastructure and close relationships with neurons. Four glial classes are considered, depending on their morphology, the compartment of neurons they ensheathe and neuron-glia interface. 3. Four ultrastructural classes of neurons are proposed. They differ in geometry and ultrastructure, as well as in glial covers (complexity and evaginations into the neuron somata). The morphology and organization of glial covers is specific for the neuron type they ensheathe. Specific glial covers do not differ in glia-glia communicatory structures. 4. The morphological and metabolical compartments of neurons are separated from the extracellular matrix or blood by specific glial systems. A system of two cells is interposed between neuron somata and hemolymph or the extracellular matrix. 5. Glial processes are crossed by membraneous tubular systems, at neuron perikarya and axons. Frequent gap junctions of varying area, density and number of IMP are found in the covers of neuron somata. 6. Neuron-glia interface bears numerous communicatory structures for both ionic and macromolecular exchange. They include junctions and transient modifications of membranes. Some of them suggest active transport mechanisms. 7. Modified endocytotic mechanisms seem to be responsible for the glia-to-neuron transfer of macromolecules as well as for the neuron-to-glia transfer of lamellar bodies. 8. The neuropil is divided into glomeruli (electrical or chemical) by glial processes and the trabeculae of the extracellular dense matrix. Neuron-glia membrane appositions have been found in electrical glomeruli. In chemical glomeruli, dense cored vesicles can release their content at neuron-neuron or neuron-glia intercellular cleft, at non-synaptic loci. 9. Neurons of type II contain peripheral complex Golgi systems, associated to subsurface cisternae and neuron-glia gap junctions, suggesting a cooperation of glial cells in specific macromolecular synthesis.