Roles of plasma membrane-associated sialidase NEU3 in human cancers

Altered sialylation of glycosphingolipids is observed in cancer as a ubiquitous phenotype, leading to the appearance of tumor-associated antigens, aberrant adhesion and disturbance of transmembrane signaling. To understand the pathological significance of aberrant alterations of gangliosides in cancer, our studies have been focused on sialidase, which is responsible for the removal of sialic acids from glycoproteins and glycolipids. Among human sialidases so far identified, sialidase NEU3 is a key enzyme for ganglioside degradation because of its uniqueness both in its localization in the plasma membrane and in specifically hydrolyzing gangliosides. NEU3 is markedly up-regulated in many types of cancers including colon and renal carcinomas and suppresses apoptosis of cancer cells. The present paper briefly summarizes our recent results on the sialidase alterations and their significance in cancer. NEU3 is indeed closely related to malignancy and thus may be a potential target for cancer diagnosis and therapy.     

Sialidase and malignancy: A minireview

Aberrant sialylation in cancer cells is thought to be a characteristic feature associated with malignant properties including invasiveness and metastatic potential. Sialidase which catalyzes the removal of sialic acid residues from glycoproteins and glycolipids, has been suggested to play important roles in many biological processes through regulation of cellular sialic acid contents. The altered expression of sialidase observed in cancer would, therefore, suggest its involvement in the malignant process. In mammalian cells, three types of sialidase cloned and characterized to date were found to behave in different manners during carcinogenesis. Recent progress in molecular cloning of these sialidases has facilitated elucidation of the molecular mechanisms and significance of these alterations. Herein we briefly describe our own studies on sialidase changes associated with malignant transformation and summarize the topic from both a retrospective and a prospective viewpoint. Sialidases are indeed closely related to malignancy and are thus potential targets for cancer diagnosis and therapy. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sialidase and malignancy: a minireview

Aberrant sialylation in cancer cells is thought to be a characteristic feature associated with malignant properties including invasiveness and metastatic potential. Sialidase which catalyzes the removal of sialic acid residues from glycoproteins and glycolipids, has been suggested to play important roles in many biological processes through regulation of cellular sialic acid contents. The altered expression of sialidase observed in cancer would, therefore, suggest its involvement in the malignant process. In mammalian cells, three types of sialidase cloned and characterized to date were found to behave in different manners during carcinogenesis. Recent progress in molecular cloning of these sialidases has facilitated elucidation of the molecular mechanisms and significance of these alterations. Herein we briefly describe our own studies on sialidase changes associated with malignant transformation and summarize the topic from both a retrospective and a prospective viewpoint. Sialidases are indeed closely related to malignancy and are thus potential targets for cancer diagnosis and therapy.     

Sialidase significance for cancer progression

Aberrant glycosylation is a characteristic feature of cancer cells. In particular, altered sialylation is closely associated with malignant properties, including invasiveness and metastatic potential. To elucidate the molecular mechanisms underlying the aberrancy, our studies have focused on mammalian sialidase, which catalyzes the removal of sialic acid residues from glycoproteins and glycolipids. The four types of mammalian sialidase identified to date show altered expression and behave in different manners during carcinogenesis. The present review briefly summarizes results on altered expression of sialidases and their possible roles in cancer progression. These enzymes are indeed factors defining cancer malignancy and thus potential targets for cancer diagnosis and therapy.     

Visualization of Sialidase Activity in Mammalian Tissues and Cancer Detection with a Novel Fluorescent Sialidase Substrate

Sialidase removes sialic acid from sialoglycoconjugates and plays crucial roles in many physiological and pathological processes. Various human cancers express an abnormally high level of the plasma membrane-associated sialidase isoform.Visualization of sialidase activity in living mammalian tissues would be useful not only for understanding sialidase functions but also for cancer diagnosis. However, since enzyme activity of mammalian sialidase is remarkably weak compared with that of bacterial and viral sialidases, it has been difficult to detect sialidase activity in mammalian tissues. We synthesized a novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. BTP-Neu5Ac can visualize sialidase activities sensitively and selectively in acute rat brain slices. Cancer cells implanted orthotopically in mouse colons and human colon cancers (stages T3-T4) were also clearly detected with BTP-Neu5Ac. The results suggest that BTP-Neu5Ac is useful for histochemical imaging of sialidase activities.     

Characterization of the major sialidases of various types of rat blood cells: their comparison with rat liver sialidases

The substrate specificity and subcellular location of the major sialidases of three types of rat blood cells were characterized and compared with those of the known three types of rat liver sialidase, which have been designated intralysosomal, cytosolic, and plasma membrane-associated sialidases. Platelets and leucocytes contain mainly an acid sialidase, which is highly active towards oligosaccharides and 4MU-NeuAc, and erythrocytes possess a high level of a sialidase acting on gangliosides. A Percoll gradient centrifugation study showed that the former is located in lysosomes and the latter in plasma membrane. When the sialidase was solubilized and partially purified from erythrocyte ghosts, the enzyme was found to hydrolyze actively gangliosides but only poorly other substrates such as 4MU-NeuAc, oligosaccharides, and glycoproteins. The sialidase partially purified from rat liver membrane fraction exhibited the same substrate specificity. It is concluded that the major sialidase of platelets and leucocytes corresponds to hepatic intralysosomal sialidase while erythrocytes contain almost exclusively a ganglioside sialidase which corresponds to hepatic plasma membrane sialidase.     

Fluorogenic sialic acid glycosides for quantification of sialidase activity upon unnatural substrates

Herein we report the synthesis of N-acetyl neuraminic acid derivatives as 4-methylumbelliferyl glycosides and their use in fluorometrically quantifying human and bacterial sialidase activity and substrate specificities. We found that sialidases in the human promyelocytic leukemic cell line HL60 were able to cleave sialic acid substrates with fluorinated C-5 modifications, in some cases to a greater degree than the natural N-acetyl functionality. Human sialidases isoforms were also able to cleave unnatural substrates with bulky and hydrophobic C-5 modifications. In contrast, we found that a bacterial sialidase isolated from Clostridium perfringens to be less tolerant of sialic acid derivatization at this position, with virtually no cleavage of these glycosides observed. From our results, we conclude that human sialidase activity is a significant factor in sialic acid metabolic glycoengineering efforts utilizing unnatural sialic acid derivatives. Our fluorogenic probes have enabled further understanding of the activities and substrate specificities of human sialidases in a cellular context.     

Plasma membrane-associated sialidase as a crucial regulator of transmembrane signalling

Mammalian sialidases, glycosidases responsible for the removal of sialic acids from glycoproteins and glycolipids, has been implicated to participate in many biological processes as well as in lysosomal catabolism. Among those forms identified to date, plasma membrane-associated sialidase, Neu3, is a key enzyme in degradation of gangliosides, for which it exhibits a special substrate preference. This sialidase has been shown to control transmembrane signalling for many cellular processes, including cell differentiation, cell growth and apoptosis, and human orthologue NEU3 is markedly up-regulated in various cancers. It is known to suppress apoptosis in cancer cells. Furthermore, its overexpression causes impaired glucose tolerance and hyper-insulinaemia together with overproduction of insulin in enlarged islets in the transgenic mice. The present review primarily summarizes our recent results, focusing on Neu3 as a regulator of transmembrane signalling.     

Lysosomal and plasma membrane ganglioside GM3 sialidases of cultured human fibroblasts. Differentiation by detergents and inhibitors

Cultured human fibroblasts contain two sialidases that degrade gangliosides such as GM3: a lysosomal activity that appears identical with the activity towards water-soluble substrates and that is deficient in the genetic lysosomal disorder sialidosis, and another enzyme that seems localized on the external surface of the plasma membrane. In this report we show that both enzymes can be differentiated in the presence of each other by choice of the detergent used for activation, and also by the inhibitory action of some polyanionic compounds such as sulphated glycosaminoglycans. The lysosomal ganglioside GM3 sialidase is greatly stimulated by sodium glycodeoxycholate and, to lesser degrees, by sodium glycocholate and sodium cholate. The ganglioside GM3 sialidase of the plasma membrane is not measurably active under the conditions of the lysosomal enzyme but is specifically activated by the non-ionic detergent Triton X-100. The glycodeoxycholate-stimulated, but not the Triton-activated, ganglioside GM3 sialidase activity was profoundly diminished in cell lines from patients with the lysosomal disorders sialidosis and galactosialidosis; however, both activities were normal in fibroblasts from patients with mucolipidosis IV, previously thought to be a ganglioside sialidase deficiency disorder. Both the lysosomal and the plasma membrane ganglioside GM3 sialidases were inhibited by sialic acids, suramin, dextran sulphate and sulphated glycosaminoglycans. Among the latter, heparin and heparan sulphate showed a much higher inhibitory potency towards the plasma membrane ganglioside GM3 sialidase than towards the lysosomal onw.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of endogenous,membrane-associated sialidase activity (N-acetylneuraminidase) of the goldfish visual system

The endogenous sialidase (N-acetylneuraminidase) activity of membranes prepared from goldfish retina and optic tectum displays characteristics similar to those reported for neural plasma membrane sialidases of other organisms. Endogenous membrane sialidase activity was found to be optimal at ph 4.0, and maximal release was obtained at 37-50 degrees C, above which temperature thermal instability of the preparations was observed. Optic nerve crush, which results in regeneration of retinal ganglion cell axons, did not result in significant changes in measured endogenous membrane sialidase activity in either the retina or the optic tectum. Enzymatic hydrolysis of membrane sialoglycolipid (ganglioside) accounted for about 70% of the total sialic acid released. Ganglioside GM1 accumulated as the major lipid product in both retina and tectum, indicating that the inner sialosylgalactosyl linkage in the ganglio oligosaccharide series was resistant to hydrolysis by the endogenous enzyme.     

Recent Development in Mammalian Sialidase Molecular Biology

This review summarizes the recent research development on mammalian sialidase molecular cloning. Sialic acid–containing compounds are involved in several physiological processes, and sialidases, as glycohydrolytic enzymes that remove sialic acid residues, play a pivotal role as well. Sialidases hydrolyze the nonreducing, terminal sialic acid linkage in various natural substrates, such as glycoproteins, glycolipids, gangliosides, and polysaccharides. Mammalian sialidases are present in several tissues/organs and cells with a typical subcellular distribution: they are the lysosomal, the cytosolic, and the plasma membrane–associated sialidases. Starting in 1993, 12 different mammalian sialidases have been cloned and sequenced. A comparison of their amino acid sequences revealed the presence of highly conserved regions. These conserved regions are shared with viral and microbial sialidases that have been characterized at three-dimensional structural level, allowing us to perform the molecular modeling of the mammalian proteins and suggesting a monophyletic origin of the sialidase enzymes. Overall, the availability of the cDNA species encoding mammalian sialidases is an important step leading toward a comprehensive picture of the relationships between the structure and biological function of these enzymes.     

The action of sialidases on substrates containing O-acetylsialic acids

O-Acetyl substitution of sialic acids in glycoconjugates reduces the rate of action of sialidases on these substrates. A plasma glycoprotein fraction and an erythrocyte ganglioside containing 4-O-acetylsialic acids were isolated and characterized from equine blood, and a sialyllactose preparation with Neu5,9Ac2 was purified from rat urine. Using the novel substrates II3Neu4Ac5Gc-LacCer and II3Neu5,9Ac2-Lac the influence of individual mono-O-acetylated sialic acids on bacterial and viral sialidases could be clearly shown. This extends and clarifies observations with glycoproteins containing mixtures of mono-, di- and higher O-acetylated sialic acids with substitution at the hydroxyls on carbons 4, 7, 8 and 9. A 4-O-acetyl substitution in sialic acids blocks the action of bacterial sialidases for substrates containing these derivatives, while viral enzymes show low but significant activity, reflected in Km and Vmax values. A small reduction in bacterial sialidase activity was observed for II3Neu5,9Ac2-Lac relative to II3Neu5Ac-Lac in agreement with kinetic analysis. Newcastle disease virus sialidase showed a 50% reduction in hydrolysis rate for the 9-O-acetylated substrate and ten-fold reductions of both Km and Vmax values.     

Cloning and characterization of a sialidase from the filamentous fungus, <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed β-propeller structure with the model’s highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl α-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl α-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred α2-3-linked sialic acids over the α2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A. fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes.     

Immunological discrimination of intralysosomal, cytosolic, and two membrane sialidases present in rat tissues

Cytosolic sialidase was purified from rat skeletal muscle, and the purified enzyme migrated as a single band of Mr 43,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the enzyme inhibited and immunoprecipitated rat liver cytosolic sialidase as well as the muscle enzyme but failed to cross-react with the intralysosomal sialidase of rat liver and membrane sialidases I (synaptosomal) and II (lysosomal) of rat brain. The antibody against brain membrane sialidase I (anti-I) and that against sialidase II (anti-II), which could be useful to discriminate the two enzymes, did not cross-react with the intralysosomal and cytosolic sialidases of liver. Although more than 90% of liver plasma membrane sialidase was immunoprecipitated with anti-I, only 60% of liver lysosomal membrane sialidase was immunoprecipitated with anti-II, the remainder being immunoprecipitated with anti-I. In confirmation of these data, liver lysosomal membrane exhibited two peaks of ganglioside sialidase corresponding to the membrane sialidases I and II on Aminohexyl-Sepharose chromatography while only one peak of ganglioside sialidase corresponding to sialidase I was observed for liver plasma membrane. These results indicate that the four types of rat sialidase are proteins distinct from one another and that the three kinds of antisera described above are useful for discriminating these sialidases qualitatively and probably quantitatively.     

Comparative enzymology, biochemistry and pathophysiology of human exo-alpha-sialidases (neuraminidases)

This review summarizes the current research on human exo-alpha-sialidase (sialidase, neuraminidase). Where appropriate, the properties of viral, bacterial, and human sialidases have been compared. Sialic acids are implicated in diverse physiological processes. Sialidases, as enzymes acting upon sialic acids, assume importance as well. Sialidases hydrolyze the terminal, non-reducing, sialic acid linkage in glycoproteins, glycolipids, gangliosides, polysaccharides, and synthetic molecules. Therefore, a variety of assays are available to measure sialidase activity. Human sialidase is present in several organs and cells. Its cellular distribution could be cytosolic, lysosomal, or in the membrane. Human sialidase occurs in a high molecular-mass complex with several other proteins, including cathepsin A and beta-galactosidase. Multi-protein complexation is important for the in vivo integrity and catalytic activity of the sialidase. However, multi-protein complexation, the occurrence of isoenzymes, diverse subcellular localization, thermal instability, and membrane association have all contributed to difficulties in purifying and characterizing human sialidases. Human sialidase isoenzymes have recently been cloned and sequenced. Even though crystal structures for the human sialidases are not available, the highly conserved regions of the sialidase from various organisms have facilitated molecular modeling of the human enzyme and raise interesting evolutionary questions. While the molecular mechanisms vary, genetic defects leading to human sialidase deficiency are closely associated with at least two well-known human diseases, namely sialidosis and galactosialidosis. No therapy is currently available for either disease. A thorough investigation of human sialidases is therefore crucial to human health.     

Sialidase specificity determined by chemoselective modification of complex sialylated glycans

Sialidases hydrolytically remove sialic acids from sialylated glycoproteins and glycolipids. Sialidases are widely distributed in nature and sialidase-mediated desialylation is implicated in normal and pathological processes. However, mechanisms by which sialidases exert their biological effects remain obscure, in part because sialidase substrate preferences are poorly defined. Here we report the design and implementation of a sialidase substrate specificity assay based on chemoselective labeling of sialosides. We show that this assay identifies components of glycosylated substrates that contribute to sialidase specificity. We demonstrate that specificity of sialidases can depend on structure of the underlying glycan, a characteristic difficult to discern using typical sialidase assays. Moreover, we discovered that Streptococcus pneumoniae sialidase NanC strongly prefers sialosides containing the Neu5Ac form of sialic acid versus those that contain Neu5Gc. We propose using this approach to evaluate sialidase preferences for diverse potential substrates.     

Edwardsiella tarda sialidase: Pathogenicity involvement and vaccine potential

Bacterial sialidases are a group of glycohydrolases that are known to play an important role in invasion of host cells and tissues. In this study, we examined in a model of Japanese flounder (Paralichthys olivaceus) the potential function of NanA, a sialidase from the fish pathogen Edwardsiella tarda. NanA is composed of 670 residues and shares low sequence identities with known bacterial sialidases. In silico analysis indicated that NanA possesses a sialidase domain and an autotransporter domain, the former containing five Asp-boxes, a RIP motif, and the conserved catalytic site of bacterial sialidases. Purified recombinant NanA (rNanA) corresponding to the sialidase domain exhibited glycohydrolase activity against sialic acid substrate in a manner that is pH and temperature dependent. Immunofluorescence microscopy showed binding of anti-rNanA antibodies to E.?tarda, suggesting that NanA was localized on cell surface. Mutation of nanA caused drastic attenuation in the ability of E.?tarda to disseminate into and colonize fish tissues and to induce mortality in infected fish. Likewise, cellular study showed that the nanA mutant was significantly impaired in the infectivity against cultured flounder cells. Immunoprotective analysis showed that rNanA in the form of a subunit vaccine conferred effective protection upon flounder against lethal E.?tarda challenge. rNanA vaccination induced the production of specific serum antibodies, which enhanced complement-mediated bactericidal activity and reduced infection of E.?tarda into flounder cells. Together these results indicate that NanA plays an important role in the pathogenesis of E.?tarda and may be exploited for the control of E.?tarda infection in aquaculture.     

Recovery of function and mass of endogenous beta-cells in streptozotocin-induced diabetic rats treated with islet transplantation

We found that the hepatopancreas of oyster, Crassostrea virginica, contained a sialidase capable of releasing Neu5Gc from the novel polysialic acid chain (-->5-O(glycolyl)Neu5Gcalpha2-->)n more efficiently than from the conventional type of polysialic acid chains, (-->8Neu5Acalpha2-->)n, or (-->8Neu5Gcalpha2-->)n. We have partially purified this novel sialidase and compared its reactivity with that of microbial sialidases using four different sialic acid dimers, Neu5Gcalpha2-->5-O(glycolyl)Neu5Gc (Gg2), Neu5Acalpha2-->8Neu5Ac (A2), Neu5Gcalpha2-->8Neu5Gc (G2), and KDNalpha2-->8KDN (K2) as substrates. Hydrolysis was monitored by high performance anion-exchange chromatography with a CarboPac PA-100 column and pulsed amperometric detection, the method by which we can accurately quantitate both the substrate (sialiac acid dimers) and the product (sialic acid monomers). The oyster sialidase effectively hydrolyzed Gg2 and K2, whereas A2 and G2 were poor substrates. Neu5Ac2en but not KDN2en effectively inhibited the hydrolysis of Gg2 by the oyster sialidase. Likewise, the hydrolysis of K2 by the oyster sialidase was inhibited by a cognate inhibitor, KDN2en, but not by Neu5Ac2en. Using the new analytical method we found that Gg2 was hydrolyzed less efficiently than A2 but much more readily than G2 by Arthrobacter ureafaciens sialidase. This result was at variance with the previous report using the thiobarbituric acid method to detect the released free sialic acid [Kitazume, S., et al. (1994) Biochem. Biophys. Res. Commun. 205, 893-898]. In agreement with previous results, Gg2 was a poor substrate for Clostridium perfringens sialidase, while K2 was refractory to all microbial sialidases tested. Thus, the oyster sialidase is novel and distinct from microbial sialidases with regards to glycon- and linkage-specificity. This finding adds an example of the presence of diverse sialidases, in line with the diverse sialic acids and sialic acid linkages that exist in nature. The new sialidase should become useful for both structural and functional studies of sialoglycoconjugates.     

Arthrobacter ureafaciens sialidase isoenzymes, L, M1 and M2, cleave fucosyl GM1

Among bacterial, fungal and viral sialidases, the sialidase from Arthrobacter ureafaciens has the unique property of cleaving sialic acids linked to the internal galactose of gangliotetraose. In this study, we examined the ability to cleave the internal sialic acids of GM1 and fucosyl GM1 of sialidases from several bacterial and fungal origins, including Clostridium perfringens and Vibrio cholerae. We found that A. ureafaciens sialidase could liberate the sialic acid of GM1 at the highest rate, and was the only enzyme which could cleave fucosyl GM1 among the sialidases examined.The affinity-purified sialidase derived from the culture medium of A. ureafaciens was comprised of four isoenzymes with different molecular weights and isoelectric points, the isoenzymes that cleaved fucosyl GM1 being L (88 kDa, pI 5.0), M1 (66 kDa, pI 6.2) and M2 (66 kDa, pI 5.5), but not S (52 kDa, pI 6.2) which showed the highest specific activity toward colominic acid among the four isoenzymes. Abbreviations: SA, sialic acid; PBS, phosphate-buffered saline; PVP, polyvinylpyrrolidone; FABMS, fast atom bombardment mass spectrometry; Galint, internal galactose of Gg4Cer; Galext, external galactose of Gg4Cer     

Changes in sialic acid content of the plasma membrane in hepatocellular proliferation

The content of sialic acid bound to the sinusoidal region of plasma membrane during the prereplicative phase after the intravenous injection of a solution containing triiodothyronine, amino acids, glucagon and heparin (T.A.G.H. solution) has been measured. The results obtained show that an important decrease in sialic acid content is produced as it occurs in the hepatic cells of hepatectomized animals. In order to know if sialidase activity is involved in the decrease of sialic acid content during liver regeneration, the activity of sinusoidal plasma membrane sialidases during the prereplicative phase after the partial hepatectomy has been studied. No modifications of sialidase activity were detected during this period of time indicating that this decrease in sialic acid content has to be produced by other mechanisms such as diminution in the synthesis of precursor molecules. On the other hand due to the importance of Ca2+-calmodulin complexes in the activation of the hepatic cell proliferation the possible implication of this complex on the loss of sialic acid, observing the effect of trifluoperazine (inhibitor of Ca2+-calmodulin complexes) during the prereplicative phase of liver regeneration has been studied. The results show a delay in the decrease of the amount of sugar studied from 10 to 12 hours compared to the results obtained with the hepatectomized rats that have not received trifluoperazine.