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.     

Cloning, chromosomal mapping, and characteristic 5'-UTR sequence of murine cytosolic sialidase

We have totally sequenced a cytosolic sialidase [EC 3.2.1.18] by RT-PCR from the murine thymus (murine thymic sialidase, MTS) which has a 1844-base length (encoding 385 amino acids including two sialidase motifs) and is the longest cytosolic sialidase ever reported. MTS has high and relatively low homologies with those of mammalian cytosolic sialidases from the mouse brain (99%), rat (91%), and human skeletal muscle (75%), and those of the mouse lysosomal (47%) and membrane-bound (51%) sialidases, respectively. Chromosomal mapping, being the first report of mouse cytosolic sialidase gene, showed that the MTS gene is localized to the distal part of mouse chromosome 1D and to rat chromosome 9q36. RT-PCR with the site-specific primers revealed that the coding region was expressed in all organs tested, but expressions including the 5'-UTR were barely detectable except for in the upper-thymic fraction. Also, soluble sialidase activity in the thymus was the highest of these organs. There were mRNA instability signals and AT-rich regions in 143 bp of MTS 5'-end.     

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 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.     

Molecular cloning and functional expression of rat leukotriene A4 hydrolase using the polymerase chain reaction.

We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.     

The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins

Summary An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.     

Hamster D-amino-acid oxidase cDNA: rodents lack the 27th amino acid residue in D-amino-acid oxidase

Summary.  The nucleotide sequence of cDNA that encodes hamster d-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,590 nucleotides and a poly(A) tail. It had an open reading frame for a protein consisting of 346 amino acid residues. The number of the amino acid residues is the same as that of the rat DAO. However, the hamster DAO has one residue more than mouse DAO and one residue less than human, pig, rabbit, and guinea pig DAOs. Amino acid sequence of the hamster DAO was highly similar to those of mouse and rat DAOs: 89% and 88% of the amino acid residues were identical between the hamster and mouse DAOs and between the hamster and rat DAOs, respectively. The homology was slightly less between the hamster DAO and the human (81%), pig (78%), rabbit (78%), or guinea pig DAO (82%). It has been proposed that the mouse and rat DAOs lack an amino acid residue corresponding to the 25th residue of the DAOs of other mammals. However, a detailed comparison of the amino acid sequences as well as the underlying nucleotide sequences by inclusion of the hamster ones revealed that the rodent DAOs does not lack the 25th, but the 27th residue. Received January 16, 2002 Accepted June 20, 2002 Published online November 14, 2002 Authors' address: Dr. Ryuichi Konno, Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan, Fax: +81-282-86-5616     

Molecular cloning of a new human G protein. Evidence for two Gi alpha-like protein families

The amino acid sequence of a novel G protein alpha subunit (Gx alpha) has been deduced from the nucleotide sequence of a human cDNA clone isolated from a differentiated HL-60 cDNA library. The cDNA encodes a polypeptide of 354 amino acids (Mr 40,519) which is closely related to Gi alpha proteins. The amino acid sequence homology between Gx alpha and human myeloid Gi alpha is 86% with 15 nonconservative substitutions. Gx alpha also shares 86% homology with both rat brain and mouse macrophage Gi alpha but is more homologous (94%) to bovine brain Gi alpha with only 5 nonconservative amino acid differences. G proteins previously termed Gi alpha may fall into at least two distinct groups, with one including human myeloid Gi alpha, rat brain Gi alpha and mouse macrophage Gi alpha; and other Gx alpha and bovine brain Gi alpha. One group probably contains true Gi and the other a new class of G protein whose function remains to be determined.     

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.     

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.     

Cloning, sequencing and expression of the sialidase gene from Actinomyces viscosus DSM 43798.

Chromosomal DNA from Actinomyces viscosus was digested with restriction endonucleases and the fragments ligated with pUC-vectors were used to transform Escherichia coli cells. Clones bearing the required sialidase gene were detected by spraying the colonies with the fluorogenic sialidase substrate MU-Neu5Ac. The identity of the cloned sialidase was confirmed after the 5700-fold enrichment and comparison with the purified enzyme of A. viscosus. Both sialidases were identical with regard to molecular mass, substrate specificity tested with sialyllactoses, and the inhibition of their activity by heterologous antisialidase antibodies. The sequenced insert (EMBL accession number X62276) revealed a mol% G + C of 68.2, typical for A. viscosus. An open reading frame of 2739 bp follows a sequence with dyad symmetry and an AG-rich region, and codes for 913 amino acids representing a molecular mass of 113 kDa. The conserved amino acid sequence [Ser-X-Asp-X-Gly-X-Thr-Trp] typical for bacterial sialidases was found at five positions in the predicted amino acid sequence. The gene of this enzyme is expressed by E. coli, despite the low relatedness of both species.     

Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells.

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.     

Sialidase Activity in Nuclear Membranes of Rat Brain

Abstract: A highly purified nuclear membrane preparation was obtained from adult rat brain and examined for sialidase activity using GM3, GD1a, GD1b, or N -acetylneuramin lactitol as the substrate. The nuclear membranes contained an appreciable level of sialidase activity; the specific activities toward GM3 and N -acetylneuramin lactitol were 20.5 and 23.8% of the activities in the total brain homogenate, respectively. The sialidase activity in nuclear membranes showed substrate specificity distinct from other membrane-bound sialidases localized in lysosomal membranes, synaptosomal plasma membranes, or myelin membranes. These results strongly suggest the existence of a sialidase activity associated with the nuclear membranes from rat brain.     

Hamster estrogen receptor cDNA: cloning and mRNA expression

Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5'-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).     

Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition

Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade beta-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr(334), conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu(111) and the catalytic aspartate Asp(46) are disordered in the apo form but upon binding of DANA become ordered to adopt two short alpha-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses.     

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.