Telethon Institute of Genetics and Medicine (TIGEM), SanRaffaele Biomedical Science Park, via Olgettina 58, 20132 Milan,Italy and 2Department of BiomedicalScience and Biotechnology, University of Brescia, via Valsabbina19, 25123 Brescia, Italy
Received on February 2, 1999. revisedon April 22, 1999; accepted on May 13, 1999.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In mammals, several sialidase enzymes which differ in subcellularlocalization, substrate preferences, and pH optimum have been described.Mammalian sialidases have been involved in several cellular processessuch us the catabolism of glycoconjugates, regulation of cell proliferation,clearance of plasma protein, and the developmental modeling of myelin. Detailedmolecular characterization of these mammalian proteins has beenhampered by both their low cellular content and their instabilityduring the purification procedures. Only three mammalian sialidaseshave been cloned so far: the cytosolic sialidase from rat skeletalmuscle (16Miyagi et al., 1993);the soluble sialidase secreted in the culture medium by Chinesehamster ovary (CHO) cells (8
Ferrari etal., 1994); and recently, the human lysosomal sialidase(4
Bonten et al., 1996; 15
Milner et al., 1997; 20
Pshezhetsky et al., 1997),responsible for the lysosomal storage disorder sialidosis (29
Thomas and Beaudet, 1995). All these mammalianproteins share an F(Y)RIP motif in the first part of the primarystructure, followed by Asp boxes, as previously described for themicrobic enzymes. In addition, a multiple alignment of the aminoacid sequences of these mammalian enzymes with some members of thebacterial sialidase family revealed that most of the amino acidresidues which form the catalytic site of the bacterial proteinsare highly conserved in all of the mammalian counterparts. Therefore,it is likely that mammalian sialidases, and bacterial and viralneuraminidases share a similar fold topology.
In a previous paper we described the identification of NEU2 (GenBankaccession number Y16535), a novel human gene homologous to rodentsoluble sialidases (17Monti etal., 1999). Here we demonstrate that NEU2 encodesa functional sialidase with cytosolic localization. In addition,a prediction of the three-dimensional structure of the enzyme wascarried out using a protein homology modeling approach.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The significant levels of sequence identity with both bacterialand viral sialidases allow us to further speculate on the structuralorganization of the NEU2 protein. Figure 3Ashows the structural alignment generated by SwissPdbViewer betweenNEU2 and the S.typhimurium sialidase. The location ofsecondary structure elements extracted using the STRIDE softwareis also mapped on the alignment: the identity of this structure-basedalignment corresponds to 71.1%.
|
Expression of NEU2 encoded protein in COS7 cells
To demonstrate that NEU2 encodes a sialidase enzyme, the genomicsequence from the putative ATG initiation codon to the TGA stopcodon (including the 1.25 kb intron) was amplified by PCR and subclonedinto a pCDL expression vector. The recombinant vector was subsequentlyused to transfect COS7 cells. Total lysates from cells transfectedwith pCDL-NEU2 or pCDL alone were tested for sialidase activitywith 4MU-NANA as substrate. As shown in Figure 4A,the transfection with pCDL-NEU2 leads to a dramatic increase inthe sialidase activity measured at pH 5.6. In fact, the specificactivity of the enzyme measured at pH 5.6 is barely detectable in COS7cells transfected with the pCDL vector alone (average value of 0.17nmol/min/mg protein), whereas we measured an averageactivity of 51 nmol/min/mg protein in the caseof pCDL-NEU2 transfected cells. To investigate the subcellular localizationof NEU2 encoded sialidase, a simple fractionation of the total celllysate into soluble and particulate cell compartments was carriedout by ultracentrifugation. The results obtained clearly demonstratethe soluble nature of NEU2 (Figure 4B) becausemore than 80% of the activity measured in the total celllysate was detected in the supernatant obtained by ultracentrifugationat 200,000 x g. Inaddition, the low level of endogenous sialidase activity detectablein COS7 cells is due to a particulate enzyme, because the enzymaticactivity was measurable only in the pelleted material (not shown).The homogenization conditions do not influence these results.
|
These data were confirmed by Western blot analysis of COS 7cells transfected with pCDL-NEU2 or HA-NEU2 (Figure 4D). The anti-NEU2 antiserum recognized a bandof about 42 kDa, as expected from the calculated molecular weightof NEU2. Moreover, the band was lightly enriched in the supernatantand barely detectable in particulate fraction (lanes 24, anti-NEU2).A superimposable pattern was obtained with anti-HA monoclonal antibody,with a recognized band of about 46 kDa, corresponding to the predictedmolecular weight of the HA-NEU2 chimera (lanes 24, anti-HA).An additional light band of about 32 kDa was detectable in the fractionsobtained by ultracentrifugation using the latter antibody reagent,a signal probably related to proteolytic degradation of HA-NEU2. This32 kDa band appears enriched in the particulate fraction (lane 4,anti-HA), suggesting a trapping effect and/or low solubilityof the polypeptide. Moreover, with longer posttransfection timesa larger amount of the 32 kDa band was detectable, together withthe appearance of a novel 30 kDa band, both showing roughly thesame signal intensity of the 46 kDa main product (not shown). ByWestern blot analysis, both the antibodies detected additional proteinbands of about 7275 kDa. The presence of these bands incells transfected with the expression vector alone (lanes 1) indicatesthat they are unrelated to NEU2.
No evidence of glycosylation of the polypeptide was detectable,as demonstrated by transfecting cells in the presence of tunicamycinor by N-glycosidase F treatment of the cell extracts (not shown).Moreover, no protein was detected in the cultured media, even after6x concentration of the media by ultrafiltration,indicating that NEU2 is not secreted from COS7 cells (not shown).
Immunofluorescence localization of NEU2 in COS7cells
Immunofluorescence staining was carried out in COS7 cells transfectedwith HA-NEU2 and pCDL-NEU2. Transient expression of NEU2 up to 72h posttransfection yielded an extensive fluorescence labeling, astaining pattern associated with soluble protein (Figure 5). These results were obtained by detectingthe chimera HA-NEU2 and the wild type protein (Figure 5, panels A,B and C,D, respectively), and byusing a different procedure of cell fixation and permeabilization(Figure 5C,D). In some cells overexpressingHA-NEU2, an extensive nuclear staining was detectable (Figure 5B). This could be due to the high expressionlevel of the polypeptide. No crystal-like structures described inthe case of lysosomal sialidase overexpression (4Bontenet al., 1996; 15
Milneret al., 1997) were recognized by our antibodies,even in cells with very high production of NEU2 (Figure 5C).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary structure analysis revealed that NEU2 has significanthomology with bacterial and mammalian components of the sialidasefamily. Moreover, the secondary structure prediction of NEU2 indicatesa high ß-sheet content as already reportedfor the microbic enzymes purified from S.typhimurium and V.cholerae. Thus, we decided to predict the three dimensionalstructure of the polypeptide by homology modeling, using as templatethe solved structure of S.typhimurium sialidase.It should be noted that protein models based on homology modelingare fully reliable when the known protein structure shares at least40% sequence identity with the unknown protein structure(3Blundell, 1991). Although inour case the sequence identity corresponds to 28.4%, thehigh structural similarities between viral and bacterial sialidases crystallizedso far give a strong rationale support to this prediction. The modelobtained shows the same fold topology of the S.typhimurium enzymeand allows the conservation in topologically equivalent positionsof both the two conserved Asp box and the nine amino acid residuesinvolved in the active site. The role of the Asp box structuralmotifs is still unknown, although their presence on the surfaceof the sialidase enzymes may suggest a potential role as functionaland/or recognition site. In addition, a recent study demonstratesthat the recombinant hamster enzyme and the S.typhimurium sialidaseshare the same stereoselectivity of catalysis (13
Kaoet al., 1997). Since a strong correlationof this catalytic behavior with active site architecture has beendemonstrated (10
Gebler et al.,1992), these data strongly suggest that microbial andmammalian sialidases have similar active site topology even thoughthe proteins do not share high amino acid sequence similarities.These features further support the accuracy of the NEU2 three-dimensional structuremodel.
Expression of the NEU2 in COS7 cells and E.coli demonstratesthat this new human gene encodes a sialidase. Moreover, the molecularweight of the protein detected by specific antiserum in COS7 cells,and the sialidase activity measured in E.coli cellextracts showed that the transfected NEU2 gene was spliced as predictedfrom the genomic sequence (17Monti etal., 1999).
A comparison of the specific activity of the purified recombinantNEU2 with the rodent counterparts is possible only by consideringthe values reported for the originally purified enzymes, since nodata on the corresponding recombinant proteins are available. NEU2measured specific activity toward 4-MU-NANA (3.12 µmol/minmg) is very similar to the reported values in the case of the hamster(10.1 µmol/min mg) and rat liver(5.1 µmol/min mg) enzymes.The observed Km value of 0.07 mM is about 5.7 and 9.6times lower than the reported value for hamster and rat protein,respectively. In addition, the pH optimum at pH 5.6, very similarto the values of the hamster (5.9) and rat (5.5) soluble sialidase,together with the high amino acid sequence similarities of theseproteins, strongly suggest a similar kinetic behavior for the threepolypeptides. No glycosylation is detectable in either hamster orhuman sialidase, but NEU2 is not released in the culture media,at least in COS7 cells, whereas the hamster enzyme was originallypurified from culture media of Chinese hamster ovary cells (32Warner et al., 1993).
NEU2 appears to be a soluble protein, as demonstrated by bothsubcellular fractionation and Western blot analysis. A simple fractionationinto soluble and membrane associated proteins was carried out andthe fractions assayed for sialidase activity. The enzymatic activitywas detectable mainly in the soluble material, and these resultswere confirmed by the amount of antigen detected by specific antibodiesin the two subcellular fractions. Moreover, immunofluorescence localizationof both the wild type protein or the HA-NEU2 chimera, using differentcell-fixation and permeabilization procedures to avoid artifacts,indicates a cytosolic localization of the enzyme.
The localization in adult tissue of the rat skeletal muscle cytosolicsialidase was carried out by immunohistochemical techniques (1Akita et al., 1997). Theenzyme seems to be widely distributed inside the muscle fiber, butis also detectable in axons, Schwann cells, and cells of endomysiumand blood vessels. These observations clearly indicate that this cytosolicsialidase is also present in cells other than skeletal muscle fibers.The cytosolic localization of a sialidase enzyme is rather puzzling,and still unknown is its physiological role and the nature of itsnatural substrate(s).
The nucleotide sequence of the putative NEU2 gene promoter providessome insight into the possible biological role of this protein (17Monti et al., 1999). Infact, as already reported for the promoter of the rat cytosolicsialidase (26
Sato and Miyagi, 1995),there are four E-box sequences (-CAXXTG-), which are involved inthe developmental and tissue-specific regulation of muscle genetranscription (18
Olson and Klein, 1994),and a classical TATA box. These promoter features strongly suggestthat the protein is expressed in a tissue specific manner. In addition,during rat myoblast differentiation the cytosolic sialidase contentincreases, and the myotube formation can be stopped by suppressingthe cytosolic sialidase mRNA transcription (27
Satoand Miyagi, 1996). These results suggest an involvementof cytosolic sialidase proteins in myoblast differentiation.
An intriguing paper by Tokuyama et al. describes the suppressionof pulmonary metastasis in murine melanoma cells by transfectionwith the rat cytosolic sialidase cDNA (30Tokuyamaet al., 1997). Surprisingly, this changein melanoma cell behavior appears to be related to a variation ofintracellular glycolipid levels. In fact, a decrease in gangliosideGM3 and an increase in lactosylceramide were the major changes detected.These results suggest an involvement of this soluble type of sialidasein the mechanism(s) that regulate tumor cell invasiveness and motility.
The study of NEU2 may contribute to a better understanding ofthe physiological role of soluble sialidases, providing anotherpiece to the sialic acid biology puzzle.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Computer sequence analysis
Amino acid sequences were compared to the non redundant sequencedatabases present at the NCBI (National Center for BiotechnologyInformation) using the BLAST network service (2Altschulet al., 1997). Pairwise and multiple aminoacid sequence alignments were performed using the Bestfit and PileUpprograms, respectively (Genetics Computer Group, Wisconsin Package,Version 9, Madison, WI). Secondary structure prediction was carriedout with the PeptideStructure program of GCG package.
Tertiary structure prediction
A first model of the three-dimensional structure of NEU2 was predictedthrough the program Modeller 4 (24Sali andBlundell, 1993), using as a template the sialidase from Salmonella typhimurium LT2 (Protein Data Bank accessionnumber 2SIM). Modeller 4 copied the atomic coordinates from 2SIMto NEU2 and minimized the obtained structure. The model-default.top scriptwas employed. A refined model was obtained through the program SwissPdbViewer3.0 (19
Peitsch, 1996), generatinga sequence alignment from a structural superposition between NEU2and 2SIM atomic coordinates. This sequence alignment was used asan input file for Modeller 4 to produce a refined model. Locationof secondary structure elements for both NEU2 and 2SIM structureswas performed by the STRIDE program available at http://www.embl-heidelberg.de/argos/stride/down_stride.html.It extracts from the atomic coordinates of a protein the positionof secondary structure elements and maps them on the amino acidsequence. The structural quality of the NEU2 model was evaluatedcomparing the Ramachandran plots, obtained through SwissPdbViewer3.0, for both NEU2 and 2SIM structures. Amino acids taking partin the active site were predicted from the multiple sequence alignmentof sialidases and mapped on the NEU2 structure. Three-dimensionalstructures of model and template were visualized using the Rasmol2.6 program available at http://www.umass.edu/microbio/rasmol/.
Expression of NEU2 encoded protein in COS7 cells
The genomic region of about 2.4 kb containing the entire NEU2ORF, including the 1.25 kb intron, was amplified by PCR using clonedPfu polymerase (Stratagene), a sense primer with a BglII site (Neu2-Nt:5'-CGTAGATCTATGGCGTCCCTTCCTGTCCTG-3'), an antisense primer with an EcoRI site(Neu2-Ct: 5'-CATGAATCCTCACTGAGGCAGGTACTG-3'), and HG7 genomic clone as template.This clone was isolated from a human genomic library using as aprobe a portion of the hamster cDNA encoding CHO soluble sialidase, andcontains the two predicted exons and a single intron of about 1.25kb (17Monti et al., 1999).The PCR product was cloned in different expression vectors. HA-NEU2was constructed by cloning the amplified insert 5' in-framewith the hemoagglutinin (HA) epitope into plasmid pCDNAI (Invitrogen).The amplified insert was also cloned into an RSV promoted vector(28
Takebe et al., 1988),yielding the pCDL-NEU2 construct. COS7 cells were grown in petridishes (100 mm diameter) using Dulbeccos modified Eaglesmedium (DMEM) with 10% fetal calf serum. Transfectionswere performed overnight using 8 µgof plasmid DNA and LipofectAMINE reagent, according to the manufacturers guidelines(Life Technologies). Cells were harvested by scraping, washed inPBS, and resuspended in the same buffer containing 1 mM EDTA, 1 µg/ml pepstatin A, 10 µg/ml apoprotinin,and 10 µg/ml leupeptin. Totalcell extracts were prepared 24 h after transfection by sonicationor using a glass potter. The supernatant obtained after a centrifugationat 800 x g for 10 minrepresented the crude cell extract and was subsequently centrifugedat 200,000 x g for15 min on an Optima TL 100 ultracentrifuge (Beckman). Aliquots ofthe original crude cell extract, 200,000 x g supernatant and pellet were used for proteinassay (Coomassie Protein Assay Reagent, Pierce), enzymatic activity,and Western blot analysis.
Preparation of anti-NEU2 antiserum and Westernblot analysis
To generate anti-NEU2 serum, two rabbits were immunized withan E.coli expressed and purified NEU2 protein,following the standard immunization protocol. Immunoreactivity ofthe obtained anti-NEU2 antiserum was determined by ELISA; the serumwas used without further purification. Protein samples correspondingto 20 µg of each cell fraction wereelectrophoresed on SDS-10% (w/v) polyacrylamidegels (14Laemmli, 1970) and subsequentlytransferred onto nitrocellulose extra blotting membrane (Sartorius)by electroblotting. The membranes were incubated for 30 min in TBS,0.1% (v/v) Tween 20 (TTBS) containing 10% (w/v)dried milk (Blocking Buffer: BB). The primary antibody, either anti-NEU2rabbit antiserum or anti-HA monoclonal antibody (Boehringer), wasadded at appropriate dilution in BB and blots were incubated for1 h. After washing in TTBS, the membranes were incubated for 1 h withthe appropriate horseradish peroxidase-conjugated IgG (Amersham)diluted in BB. After final washing in TTBS, visualization of theantibody binding was carried out with ECL (Amersham) according tothe manufacturers instructions. All incubations were carriedout at room temperature under constant shaking.
Immunofluorescence staining of NEU2 in COS7 cells
For immunofluorescence, COS7 were grown in 8-wellchamber slide culture chambers (Nunc). Transfections were carried outas previously described, using 0.2 µgDNA for each well. Indirect immunostaining of HA-NEU2 and NEU2 wasperformed on 4% (w/v) paraformaldehyde in PBSor methanol/acetone 1:1 (v/v) fixed cells. Cellsfixed with the former reagent were permeabilized with Triton X-100or saponin 0.2% (w/v) in PBS, blocked with porcineserum, and incubated with anti-HA monoclonal antibody (4 µg/ml)or anti-NEU2 rabbit antiserum (1:500 dilution) in PBS plus porcineserum alone or containing the permeabilizing reagents. Stainingwas obtained after incubation with fluorescein 5-isothiocyanatedconjugated isotype-specific antibodies (1:100 dilution) (DAKO).Fluorescence microscopy was carried out using an Axioplan microscope(Zeiss).
Expression of NEU2 encoded protein in E.coli
The entire coding region of NEU2 was obtained by PCR amplificationusing the isolated genomic clone HG7 as template. The DNA sequencefrom the Met to the Gln in position 67 (exon 1) was amplified usingNeu2-Nt (see above) with a 5'-BamHI restrictionsite and a 5'-phosphorylated antisenseprimer Neu2-67R (5'-CTGAACCTGGTGGGTGGGTGC-3'). The DNA sequence from the Trp inposition 68 to the stop codon (exon 2) was amplified using Neu2-68F(5'-TGGCAAGCTCAGGAGGTGGTG-3') and Neu2-Ct (see above) with a 5'-EcoRI restriction site. The two PCRproducts, obtained using cloned pfu polymerase (Stratagene), wereligated using T4 ligase and the resulting fragment of about 1.15kb was subcloned into BamHIEcoRI sites of pGEX-2T expressionvector (Pharmacia Biotech). The recombinant plasmid, pGEX-2T-NEU2,was completely sequenced using both vector- and gene-specific primers. E.coli DH5 alpha cells were transformed with therecombinant plasmid pGEX-2T-NEU2 or the expression vector pGEX-2Talone, and the transformed cells grown in LB-ampicillin medium at37°C to mid-log phase before additionof isopropyl-ß-D-thiogalacto-pyranoside(IPTG) to 0.1 mM. After overnight growth at room temperature, cellswere pelleted and suspended in 1/50 volume of phosphate-bufferedsaline (PBS). Cells lysed by sonication and supernatants were assayedfor sialidase activity. Purification of glutathione-S-transferase-NEU2fusion protein was carried out according to the manufacturersinstructions (Pharmacia Biotech, GST Gene Fusion System, 18111570).Samples of E.coli crude lysates (20 µg)and affinity purified NEU2 (0.51 µg)were subjected to SDSPAGE under denaturing conditions (14Laemmli, 1970).
Sialidase assay
The enzymatic activity of NEU2 in total cell lysates and in cellularsubfractions toward the artificial substrate 2'-(4-methylumbelliferyl)-D-N-acetylneuraminicacid (4MU-NANA) (Sigma) was determined by fluorimetric assay (31
Venerando et al., 1994).Reactions were set up in duplicate using up to 50 µg oftotal protein in 0.1 M Na citrate/phosphate buffer pH 5.6,in the presence of 400 µg bovine serumalbumin, with 0.2 mM 4MU-NANA in a final volume of 100 µl,and incubated at 37°C for 510min. Reactions were stopped by addition of 1 ml of 0.2 M glycine/NaOHpH 10.2. Fluorescence emission was measured on a Jasco FP-770 fluorometerwith excitation at 365 nm and emission at 445 nm, using 4-methylumbelliferone(4-MU) to obtain a calibration curve.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 Altschul,S.F.,Madden,T.L., Schäffer,A.A., Zhang,J., Zhang,Z., Miller,W.and Lipman,D.J. (1997) Gapped BLAST and PSI-BLAST:a new generation of protein database search programs. NucleicAcids Res., 25, 33893402.
3 Blundell,T. (1991)Comparative analysis of protein three-dimensional structures andan approach to the inverse folding problem. Ciba Found. Symp., 161, 2836.[ISI][Medline]
4 Bonten,E.,van der Spoel,A., Fornerod,M., Grosveld,G. and dAzzo,A.(1996) Characterization of human lysosomal neuraminidasedefines the molecular basis of the metabolic storage disorder sialidosis. GenesDev., 10, 31563169.[Abstract]
5 Burmeister,W.P.,Ruigrok,R.W. and Cusack,S. (1992) The 2.2 Å resolution crystalstructure of influenza B neuraminidase and its complex with sialic acid. EMBOJ., 11, 4956.[Abstract]
6 Crennell,S.J.,Garman,E.F., Laver,W.G., Vimr,E.R. and Taylor,G.L. (1993) Crystalstructure of a bacterial sialidase (from Salmonellatyphimurium LT2) shows the same fold as an influenza virusneuraminidase. Proc. Natl Acad. Sci. USA, 90, 98529856.[Abstract]
7 Crennell,S.,Garman,E., Laver,G., Vimr,E. and Taylor,G. (1994) Crystalstructure of Vibrio cholerae neuraminidase revealsdual lectin-like domains in addition to the catalytic domain. Structure, 2, 535544.[ISI][Medline]
8 Ferrari,J.,Harris,R. and Warner,T.G. (1994) Cloning and expressionof a soluble sialidase from Chinese hamster ovary cells: sequencealignment similarities to bacterial sialidases. Glycobiology, 4, 367373.
9 Galen,J.E.,Ketley,J.M., Fasano,A., Richardson,S.H., Wasserman,S.S. and Kaper,J.B.(1992) Role of Vibrio cholerae neuraminidase in the function of cholera toxin. Infect.Immun., 60, 406415.[Abstract]
10 Gebler,J.,Gilkes,N.R., Claeyssens,M., Wilson,D.B., Beguin,P., Wakarchuk,W.W.,Kilburn,D.G., Miller,R.C.,Jr., Warren,R.A. and Withers,S.G. (1992)Stereoselective hydrolysis catalyzed by related ß-1,4-glucanases and ß-1,4-xylanases. J. Biol. Chem., 267, 1255912561.
11 Hoyer,L.,Hamilton,A., Steenbergen,S. and Vimr,E. (1992) Cloning,sequencing and distribution of the Salmonella typhimurium LT2sialidase gene, nanH, provides evidence for interspecies gene transfer. Mol.Microbiol., 6, 873884.[ISI][Medline]
12 Janakiraman,M.N.,White,C.L., Laver,W.G., Air,G.M. and Luo,M. (1994) Structureof influenza virus neuraminidase B/Lee/40 complexedwith sialic acid and a dehydro analog at 1.8-Å resolution:implications for the catalytic mechanism. Biochemistry, 33, 81728179.[ISI][Medline]
13 Kao,Y.H.,Lerner,L. and Warner,T.G. (1997) Stereoselectivityof the Chinese hamster ovary cell sialidase: sialoside hydrolysiswith overall retention of configuration. Glycobiology, 7, 559563.[Abstract]
14 Laemmli,U.K. (1970)Cleavage of structural proteins during the assembly of the headof bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
15 Milner,C.,Smith,S., Carrillo,M., Taylor,G., Hollinshead,M. and Campbell,R. (1997)Identification of a sialidase encoded in the human major histocompatibilitycomplex. J. Biol. Chem., 272, 45494558.
16 Miyagi,T.,Konno,K., Emori,Y., Kawasaki,H., Suzuki,K., Yasui,A. and Tsuik,S.(1993) Molecular cloning and expression of cDNA encodingrat skeletal muscle cytosolic sialidase. J. Biol. Chem., 268, 2643526440.
17 Monti,E.,Preti,A., Rossi,E., Ballabio,A. and Borsani,G. (1999)Cloning and characterization of NEU2, a human gene homologous torodent soluble sialidases. Genomics, 57, 137143.[ISI][Medline]
18 Olson,E. andKlein,W. (1994) bHLH factors in muscle development:dead lines and commitments, what to leave in and what to leave out. GenesDev., 8, 18.[ISI][Medline]
19 Peitsch,M.C. (1996)ProMod and Swiss-Model: Internet-based tools for automated comparativeprotein modelling. Biochem. Soc. Trans., 24, 274279.[ISI][Medline]
20 Pshezhetsky,A.,Richard,C., Michaud,L., Igdoura,S., Wang,S., Elsliger,M., Qu,J.,Leclerc,D., Gravel,R., Dallaire,L. and Potier,M. (1997)Cloning, expression and chromosomal mapping of human lysosomal sialidaseand characterization of mutations in sialidosis. Nature Genet., 15, 316320.[ISI][Medline]
21 Roggentin,P.,Rothe,B., Kaper,J., Galen,J., Lawrisuk,L., Vimr,E. and Schauer,R.(1989) Conserved sequences in bacterial and viral sialidases. Glycoconj.J., 6, 349353.
22 Rost,B. andSander,C. (1996) Bridging the protein sequence-structuregap by structure predictions. Annu. Rev. Biophys Biomol. Struct., 25, 113136.[ISI][Medline]
23 Sakurada,K.,Ohta,T. and Hasegawa,M. (1992) Cloning, expressionand characterization of the Micromonospora viridifaciens neuraminidasegene in Streptomyces lividans. J.Bacteriol., 174, 68966903.[Abstract]
24 Sali,A. andBlundell,T. (1993) Comparative protein modelling bysatisfaction of spatial restraints. J. Mol. Biol., 234, 779815.[ISI][Medline]
25 Sambrook,J.,Fritsch,E.F. and Maniatis,T. (1989) MolecularCloning: A Laboratory Manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY.
26 Sato,K. andMiyagi,T. (1995) Genomic organization and the 5'-upstream sequence of the rat cytosolicsialidase gene. Glycobiology, 5, 511516.[Abstract]
27 Sato,K. andMiyagi,T. (1996) Involvement of an endogenous sialidasein skeletal muscle cell differentiation. Biochem. Biophys.Res. Commun., 221, 826830.[ISI][Medline]
28 Takebe,Y.,Seiki,M., Fujisawa,J., Hoy,P., Yokota,K., Arai,K., Yoshida,M. and Arai,N.(1988) SR alpha promoter: an efficient and versatilemammalian cDNA expression system composed of the simian virus 40early promoter and the R-U5 segment of human T-cell leukemia virustype 1 long terminal repeat. Mol. Cell. Biol., 8, 466472.[ISI][Medline]
29 Thomas,G.H. andBeaudet,A.L. (1995) In Scriver,C.R.,Beaudet,A.L., Sly,W.S. and Valle,D. (eds.), The Metabolicand Molecular Bases of Inherited Disease. McGraw-Hill,New York, pp. 25292561.
30 Tokuyama,S.,Moriya,S., Taniguchi,S., Yasui,A., Miyazaki,J., Orikasa,S. and Miyagi,T.(1997) Suppression of pulmonary metastasis in murineB16 melanoma cells by transfection of a sialidase cDNA. Int.J. Cancer, 73, 410415.[ISI][Medline]
31 Venerando,B.,Fiorilli,A., Di Francesco,L., Chiarini,A., Monti,E., Zizioli,D. andTettamanti,G. (1994) Cytosolic sialidase from pig brain:a protein complex containing catalytic and protectiveunits. Biochim. Biophys. Acta, 1208, 229237.[ISI][Medline]
32 Warner,T.G.,Chang,J., Ferrari,J., Harris,R., McNerney,T., Bennett,G., Burnier,J.and Sliwkowski,M.B. (1993) Isolation and propertiesof a soluble sialidase from the culture fluid of Chinese hamsterovary cells. Glycobiology, 3, 455463.[Abstract]