Cloning, expression, and chromosomal mapping of a human ganglioside sialidase.

Here we report the cDNA sequence of a human ganglioside sialidase. The cDNA was isolated from a human brain cDNA library by screening with a 240 bp probe generated by polymerase chain reaction using primers based on the sequences of rat cytosolic and bovine membrane sialidases which we previously cloned. The 3.0 kb cDNA encodes an open reading frame of 436 amino acids containing a putative transmenbrane domain and an Arg-Ile-Pro and three Asp-box sequences characteristic of sialidases and showing overall 83% and 39% identities to the bovine and rat enzymes, respectively. Northern blot analysis revealed high expression in skeletal muscle and testis, but low level in kidney, placenta, lung, and digestive organs. Transient expression of the cDNA in COS-1 cells resulted in a 130-fold increase in sialidase activity compared to the control level, and the activity was found to be almost specific for gangliosides. Fluorescent in situ hybridization allowed the human sialidase gene localized to chromosome 11 at q 13.5.     

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.     

Molecular cloning and expression of mouse brain sialidase.

Sialidase (EC 3.2.1.18) catalyzes the release of sialic acid from sialo-oligosaccharides, gangliosides, or sialo-glycoproteins. In this investigation, we cloned a novel cDNA for mouse brain sialidase and expressed the cDNA in COS-7 cells. This 1,699 bp cDNA codes for a 41.6 kDa protein consisting of 372 deduced amino acid residues. In COS-7 cells transiently transfected with the cDNA, a 250-fold increase was observed in specific activity toward 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Similarity searches of the nonredundant GenBank peptide sequence database by the PSI-BLAST program identified rat, hamster, human, and bacterial sialidases homologous to this mouse brain sialidase. Amino acid sequence identities to rat and hamster sialidases (84% and 77%, respectively) suggest that this form of sialidase is conserved in rodents. Sequence identities to human and mouse lysosomal sialidases (30% and 28%, respectively) indicate that the mouse brain sialidase is distinct from the lysosomal enzyme. Mouse brain sialidase has two amino acid sequence motifs common to bacterial sialidases: the 'F/YRIP' motif and the 'Asp-box' motif. The 'F/YRIP' motif is present near the N terminus while two 'Asp-box' motifs are present downstream.     

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.     

Differential effect of various inhibitors on four types of rat sialidase

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu²⁺ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-dehydro-N-acetylneuraminic acid - 4MU-Neu5Ac 4-methylumbelliferyl--N-acetyl-d-neuraminic acid     

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.     

Molecular cloning and characterization of NEU4, the fourth member of the human sialidase gene family

Several mammalian sialidases have been cloned so far and here we describe the identification and expression of a new member of the human sialidase gene family. The NEU4 gene, identified by searching sequence databases for entries showing homologies to the human cytosolic sialidase NEU2, maps in 2q37 and encodes a 484-residue protein. The polypeptide contains all the typical sialidase amino acid motifs and, apart from an amino acid stretch that appears unique among mammalian sialidases, shows a high degree of homology for NEU2 and the plasma membrane-associated (NEU3) sialidases. RNA dot-blot analysis showed a low but wide expression pattern, with the highest level in liver. Transient transfection in COS7 cells allowed the detection of a sialidase activity toward the artificial substrate 4MU-NeuAc in the acidic range of pH. Immunofluorescence staining and Western blot analysis demonstrated the association of NEU4 with the inner cell membranes.     

Cytosolic sialidase in developing rat forebrain

The nature and developmental profile of the soluble sialidase of rat forebrain were studied from birth to 150 days. Forebrain was extracted by two procedures, one (mild) preserving, the other (drastic) destroying nerve endings. The soluble extracts obtained by the mild procedure contained 64–78% of the total tissue cytosol, assayed as lactate-dehydrogenase; those obtained by the drastic procedure 87–94%. These latter extracts were considered as the soluble fraction containing ‘all’ tissue cytosol. The cytosolic origin of the sialidase contained in the soluble extracts at all examined ages was suggested by the following evidence: (a) during extraction sialidase behaved as lactate-dehydrogenase and quite differently from β-hexosaminidase and β-galactosidase, enzymes of lysosomal nature present in the same extracts, (b) the sialidase content of the extract was not influenced by the presence or absence of EDTA in the medium, (c) the sialidase content in the extracts did not diminish even after prolonged centrifugation (2 h) at high speed (150,000 g). The content of cytosolic sialidase referred to g fresh tissue increased from birth to 20 days, and slowly decreased thereafter. Till 20 days the content and the developmental trend of the cytosolic enzyme were similar to that of the better known membrane bound sialidase. This latter enzyme, however, reached its maximum at about 60 days of age. The specific activity of the cytosolic sialidase was lower till 10 days of age, higher from 10 to 30 days, and equalled that of the membrane bound enzyme during adult life. Therefore rat forebrain cytosolic and membrane bound sialidases, also from the developmental point of view, behave as different enzymes.     

Cloning, sequencing and distribution of the Salmonella typhimurlum LT2 siaiidase gene, nanH, provides evidence for interspecies gene transfer

The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli. Sialidase expression was regulated positively by cAMP. In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity. A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S. typhimurium LT2. The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi. Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S. typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S. typhimurium obtained its nanH copy most recently from Salmonella arizonae. S. typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH. These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.     

Low expression of Neu2 sialidase in the thymus of SM/J mice—existence of neuraminidase positive cells “Neu-medullocyte” in the murine thymus

We have already reported that the homogenate of the A/J mouse thymus shows a high sialidase activity at the neutral pH region and that in both soluble and membrane fractions optimal pH was 6.5–7 (Kijimoto-Ochiai et al., Glycoconj. J., 20:375–384, 2004). In the present study, we investigated the level of sialidase activities in the thymus of the SM/J mouse, a mouse strain that we know to have a Neu1^a allele that reveals a low level of sialidase activity in the liver. We found that while in the A/J thymus the soluble sialidase activity at pH 6.5 was high, the SM/J thymus lacked all such activity. A QTL analysis of SMXA recombinant inbred strains showed that soluble sialidase activity correlated well with the D1Mit8/9 marker on chromosome 1. The murine whole DNA-sequence data and the results of our FISH analysis (Kotani et al., Biochem. Biophys. Res. Comm., 286:250–258, 2001) showed that this location is consistent with the position of Neu2 gene. We confirmed that it is hard to detect the Neu2 enzyme of the SM/J mouse thymus by an anti-Neu2 antibody using a Western blot analysis. We also found that while the mRNA expression of Neu2 was quite normal in the SM/J mouse liver, it was very low in the SM/J mouse thymus. We therefore conclude that the lack of soluble sialidase activity in the SM/J mouse thymus is due to the thymus-specific low expression level of the Neu2 gene. We have previously shown that the sialidase positive cell which contains the Mac-1 and immunoglobulin, and which is located sparsely in the corticomedullar region or medullary region of the A/J mouse thymus (Kijimoto-Ochiai et al., Glycoconj. J., 20:375–384, 2004). We showed now in this paper that the detection of this cell in the SM/J mouse thymus at pH 7.0 was difficult. We propose, therefore, to name the cell “Neu-medullocyte”.     

Chemical Synthesis of 4-Trifluoromethylumbelliferyl-α-d-N-acetylneuraminic Acid Glycoside and Its Use for the Fluorometric Detection of Poorly Expressed Natural and Recombinant Sialidases

When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities. The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates. Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-α-d-N-acetylneuraminic acid (CF₃MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources. Kinetic analysis revealed CF₃MU-Neu5Ac to be a very sensitive sialidase substrate. Furthermore, this substance proves to be perfectly suitable for thein vivoexamination of sialidases and for the detection of recombinant sialidase by means of expression cloning.     

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.     

Differential expression of endogenous sialidases of human monocytes during cellular differentiation into macrophages

Sialidases are enzymes that influence cellular activity by removing terminal sialic acid from glycolipids and glycoproteins. Four genetically distinct sialidases have been identified in mammalian cells. In this study, we demonstrate that three of these sialidases, lysosomal Neu1 and Neu4 and plasma membrane-associated Neu3, are expressed in human monocytes. When measured using the artificial substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (4-MU-NANA), sialidase activity of monocytes increased up to 14-fold per milligram of total protein after cells had differentiated into macrophages. In these same cells, the specific activity of other cellular proteins (e.g. beta-galactosidase, cathepsin A and alkaline phosphatase) increased only two- to fourfold during differentiation of monocytes. Sialidase activity measured with 4-MU-NANA resulted from increased expression of Neu1, as removal of Neu1 from the cell lysate by immunoprecipitation eliminated more than 99% of detectable sialidase activity. When exogenous mixed bovine gangliosides were used as substrates, there was a twofold increase in sialidase activity per milligram of total protein in monocyte-derived macrophages in comparison to monocytes. The increased activity measured with mixed gangliosides was not affected by removal of Neu1, suggesting that the expression of a sialidase other than Neu1 was present in macrophages. The amount of Neu1 and Neu3 RNAs detected by real time RT-PCR increased as monocytes differentiated into macrophages, whereas the amount of Neu4 RNA decreased. No RNA encoding the cytosolic sialidase (Neu2) was detected in monocytes or macrophages. Western blot analysis using specific antibodies showed that the amount of Neu1 and Neu3 proteins increased during monocyte differentiation. Thus, the differentiation of monocytes into macrophages is associated with regulation of the expression of at least three distinct cellular sialidases, with specific up-regulation of the enzyme activity of only Neu1.     

Chemical Synthesis of 4-Trifluoromethylumbelliferyl-α- -N-acetylneuraminic Acid Glycoside and Its Use for the Fluorometric Detection of Poorly Expressed Natural and Recombinant Sialidases

When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities. The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates. Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-α- -N-acetylneuraminic acid (CF₃MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources. Kinetic analysis revealed CF₃MU-Neu5Ac to be a very sensitive sialidase substrate. Furthermore, this substance proves to be perfectly suitable for thein vivoexamination of sialidases and for the detection of recombinant sialidase by means of expression cloning.     

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.     

Distinguishing mammalian sialidases by inhibition kinetics with novel derivatives of 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic acid, an unsaturated derivative of N-acetylneuraminic acid.

Kinetic analysis of mammalian sialidases was carried out using analogs of the potent sialidase inhibitor, 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic+ ++ acid (1). Substitutents at C-9 in place of the terminal hydroxyl group included a, 4-azido-2-nitrophenylthio group to give 5-acetamido-2,6-anhydro-9-S-(4-azido-2-nitrophenyl)-3,5, 9-trideoxy-9-thio-D-glycero-D-galacto-non-2-enonic acid (2), and an azide group to give 5-acetamido-2,6-anhydro-9-azido-3,5,9-trideoxy-D-glycero-D-galacto-non-2 -enonic acid (3). Competitive inhibition kinetics were observed when 1,2, and 3 were tested with the lysosomal sialidase (cultured fibroblasts) and the plasma membrane sialidase (adenovirus DNA-transformed, human embryonic kidney cells), giving a Ki of about 10 microM for both enzymes with all three compounds. In contrast, only 1 was a potent inhibitor of the microsomal sialidase (rat muscle).     

Biochemical and immunological studies on two distinct ganglioside-hydrolyzing sialidases from the particulate fraction of rat brain

Ganglioside-hydrolyzing sialidase activity was solubilized from rat brain particulate fraction by using Triton X-100 plus sodium deoxycholate. When chromatographed on AH-Sepharose 4B, the solubilized activity was resolved into two peaks, which were designated sialidases I and II in order of elution. The two sialidases were purified by using sequential chromatographies on Octyl-Sepharose CL-4B, Phenyl-Sepharose CL-4B, and Sephadex G-200. Sialidase II was purified further by Mono Q-FPLC. Overall purification was 450- and 2,150-fold, for sialidases I and II, respectively. Purified sialidases I and II were maximally active at near pH 5.0 and exhibited M = 70,000 by gel filtration. Sialidase I hydrolyzed gangliosides but scarcely other substrates including 4-methylumbelliferyl-NeuAc (4MU-NeuAc). Sialidase II hydrolyzed oligosaccharides, glycoproteins, and 4MU-NeuAc although gangliosides appeared to be preferential substrates. Sialidase II cleaved GM2 much faster than sialidase I. An antibody raised in rabbits against sialidase I reacted with only sialidase I and an antibody against sialidase II reacted with only sialidase II. A subcellular distribution study suggested sialidase I in the synaptosomal membrane and sialidase II in the synaptosomal and lysosomal membranes, and this was verified by using the above antibodies.