Investigation of sialic acids and sialyltransferase activity in blood of patients with systemic scleroderma.

Systemic scleroderma (SSd) is a connective tissue disorder accompanied by generalized fibrosis. A disturbance of the synthesis and production of matrix glycoproteins, such as collagens, fibronectin, and proteoglycans, by connective tissue cells is typical for this disease. We previously demonstrated a decrease in the ganglioside content of cultured skin fibroblasts from patients with SSd. In this work the contents of sialoglycoproteins and sialoglycolipids in blood sera of patients with SSd were estimated. Simultaneously, the level of asialofetuin-sialyltransferase activity in blood plasma of three groups of patients--those with SSd, Raynaud's phenomenon, and with localized scleroderma--was investigated. CMP-5-acetamido-9-deoxy-9-fluoresceinylthioureidoneuraminic acid was used as a substrate for the enzyme assay. It was shown that the concentration of total sialic acid was increased and the concentration of lipid-bound sialic acid was slightly decreased in the blood sera of patients with SSd. A correlation between the lipid-bound sialic acid level and the severity of disease was observed; there was no correlation between severity of disease and total sialic acid. Sialyltransferase assay showed a decrease in the activity level in all three groups of patients. The greatest decrease (2-fold) of this activity was observed in patients with Raynaud's phenomenon. Our data suggest that in SSd and similar diseases the process of glycoconjugate sialylation is disturbed. These changes may considerably affect the mechanisms of regulation of metabolism and cellular interactions.     

Chemotaxonomical investigations on the occurrence of sialic acids in protostomia and deuterostomia

Several tissues of different protostome species were tested in respect to the occurence of sialic acid. There is no sialic acid in the turbellarian Euplanaria gonocephala and the mollusc Helix pomatia. In contrast to other tissues the digestive gland of the decapod crustaceans Astacus leptodactylus and Uca tangeri contains 60–80 μg of sialic acid per gram wet weight. Radioactive tracer experiments showed that Astacus leptodactylus cannot synthesize sialic acid by using the specific precursor N-acetyl-mannosamine but obviously resorbs exogenous sialic acid taken up with the food and stores it in the digestive gland. It is suggested that protostomes in general are devoid of endogenous sialic acid whereas it is abundant in deuterostomes. Contradictory to existing results low amounts of a resorcinol positive substance, probably a sialic acid, were detected in the deuterostome tunicate Phallusia mammilata.     

Matrix sialoprotein of developing bone

Using nondegradative isolation procedures, we purified and characterized a glycoprotein from fetal calf bone that is rich in sialic acid. This bone sialoprotein (BSP) has an apparent Mr = 70,000-80,000 and stains with Alcian blue and Stains All on sodium dodecyl sulfate gels but does not stain with Coomassie blue without prior treatment with neuraminidase. This glycoprotein contains 50% protein, 12% sialic acid, 7% glucosamine, and 6% galactosamine. Fetal calf BSP is rich in glutamate (19%), aspartate (15.4%), and glycine (11.8%) but, in contrast to osteonectin and the bone proteoglycan, has relatively low amounts of leucine (4.3%). Antisera raised against fetal calf BSP localized the glycoprotein by indirect immunofluorescence to developing bone trabeculae with an overall tissue distribution identical with that of osteonectin. On competition enzyme-linked immunosorbent assay analysis, BSP was 11.5% (+/-2.4%, S.E.) of mineral-bound (guanidine-EDTA-soluble) calf bone protein. Immunoreplicas (Western blots) of calf bone extracts suggest that more than 95% of the antigenicity resided in the Mr = 70,000-80,000 region with the remaining cross-reactivity in Alcian blue positive, Mr = approximately 20,000 and approximately 30,000 bands. Brief treatment of the Mr = 70,000-80,000 species with trypsin produced lower molecular weight, Alcian blue-staining products of similar size. No BSP was detected in guanidine extracts of various soft or unmineralized connective tissues, but dentin contained small amounts (0.4%) of the protein. Rat and fetal human bone were also observed to contain a sialoprotein with similar properties and a certain degree of cross-reactivity with the bovine BSP.     

Sialic acid concentrations in plants are in the range of inadvertent contamination

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC–electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-d-manno-octulosonic acid and trace amounts (3–18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.     

Rat colonic mucosal cell sialic acid metabolism in azoxymethane-induced tumours

Colonic tissue was examined from normal (control) rats and azoxymethane- (carcinogen-) treated animals. Tumour-bearing colons from azoxymethane-treated rats were divided into malignant and non-malignant areas. Mucosal cells were prepared from the three types of colonic tissue and then examined for DNA and protein content and for the activities of ten enzymes involved in sialic acid metabolism. Enzyme activities were related to either the protein or the DNA content of fractions. The DNA content of cell homogenates was significantly different between tumour and non-malignant tissue and between both these tissues and normal mucosa. The protein content of the 100000 X g membrane pellet and supernatant fraction did not vary significantly between normal and non-malignant material but both these tissues differed significantly from tumour tissue. Significant variation between normal control and tumour tissue was detected at all levels of sialic acid metabolism, including N-acetylhexosamine interconversion and phosphorylation, sialic acid formation and activation, CMP-NeuAc breakdown and transfer and sialic acid release from glycoconjugates. The results indicate that major changes at all levels of sialic acid metabolism are associated with malignancy in rat colonic mucosa. Some of these changes are apparent in non-malignant mucosa and may reflect a pre-malignant state.     

Changes in ganglioside contents, plasma sialic acid and cAMP levels in experimental hepatoma in mice

The present study was designed to assess whether changes in glycolipids and cyclic AMP contents might serve as markers for the diagnosis of malignancy in the liver. The experimental model was a transplantable murine hepatoma. Experimental mice were divided into three groups: (1) a therapeutic group, which had been transplanted with hepatoma and treated with the antimetabolism drug 5-flurouracil (0.2 mg/day i.p.), (2) a control group, which had been transplanted with hepatoma and treated with 0.2 ml 0.9% NaCl/day and (3) a normal group of mice. The ganglioside and cAMP contents in the hepatoma tissue, plasma cAMP, total- and lipid-bound sialic acid levels and red blood cell membrane sialic acid levels were determined. Results showed that the ganglioside content, total and lipid-bound sialic acid levels in the control group were significantly higher than those in the livers of normal mice (p < 0.01) while these respective values in the therapeutic group were significantly lower than those in the control group (p < 0.01). The cAMP levels of tumor tissues and plasma in the control group were lower than those in normal mice. No significant difference in red blood cell membrane sialic acid content was observed between the therapeutic and control groups though levels for both were higher than those in normal mice. These results indicate that ganglioside content and sialic acid levels in hepatoma tissues were significantly elevated, and cAMP levels in hepatoma tissues were significantly decreased during proliferation and abnormal differentiation.     

Enhanced susceptibility of nasal polyp tissues to avian and human influenza viruses

Background

Influenza viruses bind and infect respiratory epithelial cells through sialic acid on cell surface. Differential preference to sialic acid types contributes to host- and tissue-tropism of avian and seasonal influenza viruses. Although the highly pathogenic avian influenza virus H5N1 can infect and cause severe diseases in humans, it is not efficient in infecting human upper respiratory tract. This is because of the scarcity of its receptor, α2,3-linked sialic acid, in human upper airway. Expression of sialic acid can be influenced by various factors including inflammatory process. Allergic rhinitis and nasal polyp are common inflammatory conditions of nasal mucosa and may affect expression of the sialic acid and susceptibility to influenza infection.

Methodology/Principal Finding

To test this hypothesis, we detected α2,3- and α2,6-linked sialic acid in human nasal polyp and normal nasal mucosal tissues by lectin staining and infected explants of those tissues with avian influenza viruses H5N1 and seasonal influenza viruses. We show here that mucosal surface of nasal polyp expressed higher level of α2,3- and α2,6-linked sialic acid than normal nasal mucosa. Accordingly, both H5N1 avian influenza viruses and seasonal influenza viruses replicated more efficiently in nasal polyp tissues explants.

Conclusions/Significance

Our data suggest a role of nasal inflammatory conditions in susceptibility to influenza infection, especially by avian influenza viruses, which is generally inefficient in infecting human upper airway. The increased receptor expression may contribute to increased susceptibility in some individuals. This may contribute to the gradual adaptation of the virus to human population.     

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.     

Sialic acid is selectively involved in the interaction of agonists with M2 muscarinic acetylcholine receptors

Neuraminidase and slight acid hydrolysis were used to investigate the role of sialic acid residues in the binding of muscarinic agonists and antagonists to membranes from tissues rich in M1 and M2 receptors. Membranes were pretreated with neuraminidase at pH 5 and the binding parameters were determined from competitive experiments with (3H)-quinuclidinylbenzylate. The removal of sialic acid residues reduced the affinity of muscarinic agonists for cerebellum, heart and lung membranes (M2), in contrast to striatum (M1). The affinity of antagonists was not affected. Thus, sialic acid is selectively involved in the interaction of agonists with M2 muscarinic receptors.     

Differential distribution of sialic acid in alpha2,3 and alpha2,6 linkages in the apical membrane of cultured epithelial cells and tissues.

We used lectin cytochemistry and confocal microscopy to examine the distribution of sialic acid in epithelial cells. Maackia amurensis lectin and Sambuccus nigra agglutinin were used to detect alpha2,3 and alpha2,6 sialic acid, respectively. In Caco-2, HT-29 5M12, and MCF-7 cells, which express sialic acid mainly in one type of linkage, the majority of the signal was observed in the apical membrane. In cells that bound both lectins, alpha2,3 sialic acid was distributed apically, whereas alpha2,6 sialic acid showed a broader distribution. In IMIM-PC-1 cultures, alpha2,3 sialic acid was detected mainly in the apical membrane, whereas alpha2,6 sialic acid was more abundant in the basolateral domain of polarized cells. In these cells, treatment with GalNAc-O-benzyl led to reduced alpha2,3 levels and to an increase and redistribution of alpha2,6 to the apical domain. Similarly, sialic acid was predominantly expressed apically in all epithelial tissues examined. In conclusion, (a) sialic acid is mainly distributed to the apical membrane of epithelial cells, (b) there is a hierarchy in the distribution of sialic acids in polarized epithelial cells, i.e., alpha2,3 is preferred to alpha2,6 in the apical membrane, and (c) IMIM-PC-1 cells are a good model in which to study the regulation of the levels and distribution of sialic acids.     

Gangliosides with O-Acetylated Sialic Acids in Tumors of Neuroectodermal Origin

Gangliosides, carrying an O-acetylated sialic acid in their carbohydrate moiety, are often found in growing and developing tissues, especially of neuro-ectodermal origin. The most prominent one is 9-O-Ac-GD3, which is considered as an oncofetal marker in animal and human tumors like neuronal tumors, melanoma, basalioma or breast cancer, as well as in psoriatic lesions. Also other gangliosides like GD2 or GT3 were found to be O-acetylated in their terminal sialic acid. In this review we are summarising the occurrence of such gangliosides in normal and transformed tissues and delineate a more general theory that O-acetylated sialic acids in gangliosides are a universal marker for growing cells and tissues.     

Developmental sialic acid modifications in rat organs.

The changes in expression of sialic acids in Sprague-Dawley rats in the prenatal and early postnatal time period have been examined in multiple organs, both visceral and non-visceral. In all organs examined, there is a dramatic increase in both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) shortly after birth. The bulk of the sialic acid is present in the ganglioside fraction in all tissues examined. As total amounts of sialic acid present in gangliosides decrease, the proportion present in the low molecular weight cytosolic fraction increases. A curious observation is that Neu5Ac hydroxylase activity is present at the time of the increase in sialic acid, but its activity does not correlate with Neu5Gc expression after the early postnatal period. This implies that Neu5Gc expression has another level of regulation besides CMP-Neu5Ac hydroxylase activity.     

A study on the regulation of N-glycoloylneuraminic acid biosynthesis and utilization in rat and mouse liver

The relative contribution of N-glycoloyl-beta-D-neuraminic acid (Neu5Gc) to total sialic acids expressed in mouse and rat liver glycoconjugates was found to be 95% and 11%, respectively. This considerable difference in sialic acid composition made these two tissues suitable models for a comparative investigation into the regulation of Neu5Gc biosynthesis and utilization. An examination of the CMP-glycoside specificity of Golgi-associated sialyltransferases using CMP-N-acetyl-beta-D-neuraminic acid (CMP-Neu5Ac) and CMP-Neu5Gc revealed no significant tissue-dependent differences. The Golgi membrane CMP-sialic acid transport system from rat liver did, however, exhibit a slightly higher internalisation rate for CMP-Neu5Ac, though no preferential affinity for this sugar nucleotide over CMP-Neu5Gc was observed. In experiments, where Golgi membrane preparations were incubated with an equimolar mixture of labelled CMP-Neu5Ac and CMP-Neu5Gc, no significant tissue-dependent differences in [14C]sialic acid composition were observed, either in the luminal soluble sialic acid fraction or in the precipitable sialic acid fraction, results which are consistent with the above observations. From this experiment, evidence was also obtained for the presence of a Golgi-lumen-associated CMP--sialic acid hydrolase which exhibited no apparent specificity for either CMP-Neu5Ac or CMP-Neu5Gc. The specific activity of the CMP-Neu5Ac hydroxylase, the enzyme responsible for the biosynthesis of Neu5Gc, was found to be 28-fold greater in high-speed supernatants of mouse liver than of rat liver. No hydroxylase activity was detected in the Golgi membrane preparations. It is therefore proposed that the cytoplasmic ratio of CMP-Neu5Ac and CMP-Neu5Gc produced by the hydroxylase, remains largely unmodified after CMP-glycoside uptake into the Golgi apparatus and transfer on to growing glycoconjugate glycan chains. The close relationship between the total sialic acid composition and the sialic acid pattern in the CMP-glycoside pools of the tissues lends considerable weight to this hypothesis.     

Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool

N-Glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans and is developmentally regulated in rodents. We have explored the biology of N-acetylneuraminic acid hydroxylase, the enzyme responsible for conversion of the parent sialic acid, N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. We show that the major sialic acid in all compartments of murine myeloma cell lines is Neu5Gc. Pulse-chase analysis in these cells with the sialic acid precursor [6-3H]N-acetylmannosamine demonstrates that most of the newly synthesized Neu5Gc appears initially in the cytosolic low-molecular weight pool bound to CMP. The percentage of Neu5Gc on membrane-bound sialic acids closely parallels that in the CMP-bound pool at various times of chase, whereas that in the free sialic acid pool is very low initially, and rises only later during the chase. This implies that conversion from Neu5Ac to Neu5Gc occurs primarily while Neu5Ac is in its sugar nucleotide form. In support of this, the hydroxylase enzyme from a variety of tissues and cells converted CMP-Neu5Ac to CMP-Neu5Gc, but showed no activity towards free or alpha-glycosidically bound Neu5Ac. Furthermore, the majority of the enzyme activity is found in the cytosol. Studies with isolated intact Golgi vesicles indicate that CMP-Neu5Gc can be transported and utilized for transfer of Neu5Gc to glycoconjugates. The general properties of the enzyme have also been investigated. The Km for CMP-Neu5Ac is in the range of 0.6-2.5 microM. No activity can be detected against the beta-methylglycoside of Neu5Ac. On the other hand, inhibition studies suggest that the enzyme recognizes both the 5'-phosphate group and the pyrimidine base of the substrate. Taken together, the data allow us to propose pathways for the biosynthesis and reutilization of Neu5Gc, with initial conversion from Neu5Ac occurring primarily at the level of the sugar nucleotide. Subsequent release and reutilization of Neu5Gc could then account for the higher steady-state level of Neu5Gc found in all of the sialic acid pools of the cell.     

The use of peroxidase-labelled Limulus polyphemus agglutinin for the histochemistry of sialic acid-containing glycoproteins in light microscopy.

Synopsis The usefulness of a lectin,Limulus polyphemus agglutinin (LPA) has been tested in a series of mammalian tissues with sialic acid-containing glycoproteins. In nearly all the tissues employed, the positive peroxidase-labelled LPA diaminobenzidine (LPA-PO-DAB) reaction of various histological structures was markedly diminished in intensity or abolished, following digestion with neuraminidase. In the same tissues, sialic acid added with LPA-PO abolished the LPA-PO-DAB reaction or notably suppressed its intensity. In the majority of the tissues tested, the LPA-PO-DAB-Alcian Blue (AB) (pH 1.0 or 2.5) procedures appear to be useful dual staining methods which enable one to colour selectively sialic acid-containing and other acidic carbohydrates. In view of the endogenous peroxidase activity in particular histological structures, however, appropriate control staining procedures should be performed when the LPA-PO-DAB procedure is employed, either alone or in combination with AB procedures, to determine the histochemical properties of sialic acid-containing glycoproteins.     

N-glycolylneuraminic acid conjugates: implications of their absence in mammalian biochemistry

N-glycolylneuraminic acid (Neu5Gc) is one of the two most common forms of sialic acids present in glycoproteins and glycolipids of mammalian tissues. It is synthesized from the most ubiquitous sialic acid, N-acetylneuraminic acid (Neu5Ac) in a hydroxylation reaction catalysed by the enzyme Neu5Ac hydroxylase. Though Neu5Gc conjugates are prevalent in many tissues of mammals, they are absent in glycolipids and only trace amounts are present in glycoproteins of the brain and central nervous system. In humans Neu5Ac is the main sialic acid as Neu5Ac hydroxylase is inactive due to mutation of its gene. The importance of sialic acids in biochemical phenomena and the distinct roles played by specific forms of these amino sugars is adequately reflected in functional studies of selectin and sialoadhesin families of adhesion molecules. The absence of Neu5Gc, therefore, in tissues of humans and brain of mammals has raised interest, especially with regard to its impact on biochemical differences evident between humans and other mammals. It is suggested that though Neu5Gc conjugates are important in cellular interactions, their presence in brain and the central nervous system is deleterious to the latter's normal functions. Their interaction with other cellular components to form supramolecular associations is indicated that may have a bearing on major biochemical differences, a few of which are presently evident between humans and other mammals.     

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.     

Higher reactivity of apolipoprotein B-100 and alpha-tocopherol compared to sialic acid moiety of low-density lipoprotein (LDL) in radical reaction

Radical reaction of low-density lipoprotein (LDL) is a key step in atherogenesis and causes both a decrease in the sialic acid moiety and modification of apolipoprotein B-100 (apoB). Although apoB modification (cross-link and fragmentation) increases in atherosclerosis, the change in apoB-bound sialic acid in atherosclerosis is controversial. To elucidate the physiological implications of desialylation of LDL by radical reaction, the reactivity of sialic acid of LDL was compared with that of apoB, which underwent facile fragmentation in radical reactions. ApoB was determined by immunoblot analysis with anti-apoB antiserum, and the sialic acid moiety was measured by blot analysis with a biotin-bound lectin [biotin-SSA from Japanese elderberry (Sambucus sieboldiana)] specific to sialic acid. When human LDL was oxidized with Cu(2+) at 37 degrees C, apoB and apoB-attached sialic acid decreased simultaneously. Comparison of the staining bands with anti-apoB and with biotin-SSA shows that sialic acid moieties still remain on fragmented apoB proteins, indicating that the decrease in sialic acid is much slower than that of apoB fragmentation. In addition, human plasma was oxidized with 400 microM of Cu(2+) at 37 degrees C. Similar analysis indicates that the decrease in sialic acid attached to apoB also results from the fragmentation of apoB. This study indicates that the fragmentation of apoB proceeds at a much faster rate than the decrease in sialic acid content when a free radical reaction is induced in isolated LDL as well as in plasma LDL exposed to Cu(2+)-induced oxidative stress. On the basis of these results, the modification of apoB is much more sensitive than the decrease in sialic acid as an indicator of oxidative stress.