Glycobiology, 1999, Vol. 9, No. 12 1313-1321
© 1999 Oxford University Press
Expression of a novel human sialidaseencoded by the NEU2 gene
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.
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Sialidases (E.C.3.2.1.18) belong to a group of glycohydrolyticenzymes, widely distributed in nature, which remove sialic acidresidues from glycoproteins and glycolipids. All of the sialidaseso far characterized at the molecular level share an Asp block,repeated three to five times in the primary structure, and an F/YRIPsequence motif which is part of the active site. Using a sequencehomology-based approach, we previously identified a human gene,named NEU2, mapping to chromosome 2q37. NEU2 encoded protein isa polypeptide of 380 amino acids with two Asp block consensusesand the YRIP sequence in the amino terminal part of the primarystructure. Here we demonstrate that NEU2 encodes a functional sialidase.NEU2 was expressed in COS7 cells, giving rise to a dramatic increasein the sialidase activity measured in cell extracts with the artificial substrate4-MU-NANA. Using a rabbit polyclonal antiserum, on Western blotsa protein band with a molecular weight of about 42 kDa was detectable,and its cytosolic localization was demonstrated with cell fractionation experiments.These results were confirmed using immunohistochemical techniques.NEU2 expression in E.coli cells allowed purificationof the recombinant protein. As already observed in the enzyme expressedin COS7 cells, NEU2 pH optimum corresponds to 5.6 and the polypeptideshowed a Km for 4-MU-NANA of 0.07 mM. In addition, basedon the detectable similarities between the NEU2 amino acid sequenceand bacterial sialidases, a prediction of the three-dimensionalstructure of the enzyme was carried out using a protein homologymodeling approach.
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Introduction |
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Neuraminidases or sialidases (E.C.3.2.1.18) are a family of glycohydrolyticenzymes that remove N-acetyl neuraminic acid residues from varioussubstrates such as glycolipids and glycoproteins. Viral and bacterialneuraminidases have been well characterized, and the three-dimensionalstructure of several of these enzymes has been determined (6
Crennell et al., 1993, 1994; 12Janakiraman et al., 1994).All of the microbic enzymes characterized so far share the F(Y)RIPdomain in the amino terminal portion of the protein, followed bya series of so-called "Asp boxes" (S-X-D-X-G-X-X-T/W),which are repeated three to five times depending on the protein(21Roggentin et al., 1989),but have an overall amino acid sequence identity of about 35%.Despite this low level of amino acid similarity, the overall foldof the molecules and organization of the amino acids involved inthe catalysis are remarkably similar.
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 (8Ferrari etal., 1994); and recently, the human lysosomal sialidase(4Bonten et al., 1996; 15Milner et al., 1997; 20Pshezhetsky et al., 1997),responsible for the lysosomal storage disorder sialidosis (29Thomas 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.
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The derived amino acid sequence of NEU2 and alignment similaritiesto bacterial and mammalian sialidases
The NEU2 gene encodes a protein of 380 amino acids (Figure 1A), just one amino acid (Pro 267) more thanthe two rodent enzymes. The calculated molecular weight of the NEU2 encodedprotein is 42.23 kDa and the isoelectric point is 6.82. The polypeptidehas one potential N-linked glycosylation site (Asn-Val-Thr) at Asn120, exactly in the same position reported for the CHO sialidase,instead of the three present in both rat cytosolic and human lysosomalsialidase. The Kyte–Doolittle hydrophobicity plot (Figure 1B) suggests that NEU2 is a soluble protein:no hydrophobic loops consistent with transmembrane domain are detectablein its primary structure. Database searches performed using the2.0 version of BLAST (2
Altschul etal., 1997) revealed that NEU2 shares significant sequenceidentities with mammalian, viral, and bacterial sialidases (seeTable ). As expected, the highest similarityis detectable with the hamster (73.7%) and the rat (72.4%) cytosolicsialidases. A lower but still significant level of sequence similaritywas found with the G9 human lysosomal sialidase (42.5%)and the two bacterial enzymes, Salmonella typhimurium (11Hoyer et al., 1992) and Micromonospora viridifaciens (23Sakuradaet al., 1992), along the entire lengthof the protein.
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A multiple alignment of the amino acid sequences of these mammalianand bacterial sialidase enzymes is shown in Figure 2. The sequence motif F/YRIP, whichis highly conserved in all of the sialidase enzymes described sofar and occurs near the N-terminus of these polypeptides, is alsofound in NEU2 (amino acids 20–23). Moreover, NEU2 containstwo Asp blocks, both in agreement with the consensus sequence (STDHGRTW,amino acids 129–136; SHDHGRTW, amino acids 199–206).These characteristic motifs correspond to the second and third Aspblocks observed in G9 human sialidase and the bacterial enzyme from S.typhimurium LT2, being the first (amino acid58–64) and the fourth (amino acid 247–254) Asp blockspoorly conserved in NEU2, as well as in the two rodent enzymes.NEU2 differs from G9 protein in the N-terminal region. In fact,neither a cleavage site nor a transmembrane segment is detectablein the NEU2 amino acid sequence. In addition, as already reportedfor G9 protein, 9 out of 12 of the amino acid residues which formthe catalytic site of S.typhimurium enzyme areconserved in NEU2 sialidase. The differences concern Trp-121, Trp-128,and Leu-175 residues which in the S.typhimurium sialidase,together with the conserved Met-99, form a hydrophobic pocket whichaccommodates the N-acetyl group of sialic acid (6
Crennellet al., 1993). These amino acids are replacedin NEU2 and the two rodent enzymes by Phe-102 and Phe-157. Theseamino acid residues are also different in the other sialidases,suggesting their involvement in variations of substrate specificityin these enzymes.
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NEU2 secondary and tertiary structure prediction
The three-dimensional structure of the 42 kDa sialidase from S.typhimurium LT2 has been reported. It comprisessix ß-sheets, each composed of fourantiparallel ß-strands, arranged aroundan axis passing through the active site (6
Crennellet al., 1993). This folding closely resemblesthe crystal structure of the influenza virus neuraminidase (5Burmeister et al., 1992).A similar structure has been described previously (7Crennellet al., 1994) for the catalytic subunitof the larger enzyme purified from V.cholerae (9Galen et al., 1992). 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%.
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The refined three-dimensional structure of the NEU2 model, obtainedaccording to a protein homology modeling approach (22
Rostand Sander, 1996), is reported in Figure 3B. The predicted structure of NEU2 appearsto fold into 24 ß-strands,2 
-helices, and 26 connecting segments,and closely resembles the general molecular folding found in microbicand viral enzymes. Moreover, in the NEU2 model the residues Arg21, Arg41,Asp46, Met85, Glu218, Arg237, Arg304, Tyr334, and Glu355 that arepredicted to form the active site are organized in a shallow crevicein the same fashion as the S.typhimurium sialidase.Ramachandran plot analyses (data not shown) show that in the NEU2model 51 out of 380 residues are in disallowed regions (13.4%),whereas in the S.typhimurium sialidase 25 out of381 are in disallowed regions (6.6%).
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.
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The rate of hydrolysis of 4-MU-NANA to 4-MU by the soluble fractionof pCDL-NEU2 transfected cells was measured over the pH range 3.6–6.6in citrate/phosphate buffer (Figure 4C).The enzyme exhibited considerable activity over a broad pH range,from 4.2 to 6.6, with a plateau at pH 5.6–6.0. The samepH curve was obtained with the recombinant enzyme purified by E.coli cellextracts (not shown).
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 2–4, 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 2–4, 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 72–75 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; 15Milneret al., 1997) were recognized by our antibodies,even in cells with very high production of NEU2 (Figure 5C).
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Expression of NEU2 encoded protein in E.coli
To obtain a large amount of NEU2 we subcloned the coding regionof the gene in the pGEX-2T bacterial expression vector as describedin Materials and methods. The polypeptide was expressedin transformed E.coli cells as glutathione S-transferasefusion protein. As expected, a new protein band with an apparentmolecular mass of about 65 kDa appeared on SDS–PAGE ofthe transformed cell extracts, indicating expression of the fusionprotein (Figure 6, lane 3). The sialidaseactivity toward 4MU-NANA substrate was detectable only in lysateof bacterial cells harboring pGEX-2T-NEU2, while cells transformedwith pGEX-2T alone did not show any sialidase activity (see Table
). The observed specific activity was roughly fourtimes higher than the reported value for the recombinant rat cytosolicenzyme expressed in E.coli with the same vector (16Miyagi et al., 1993), andabout half of the value detectable in crude homogenate of insectcells expressing the soluble enzyme from Chinese hamster ovary cells(8Ferrari et al., 1994).Batch purification of glutathione S-transferase-NEU2 fusion protein,followed by thrombin cleavage, leads to selective enrichment ofa protein band with a molecular mass of about 43 kDa, and a roughlycomparable amount of uncleaved fusion protein (Figure 6, lanes 4 and 5). The purified preparationshowed sialidase activity at pH 5.6 of 3.12 µmol/min/mg proteinand a Km value toward 4MU-NANA of 0.07 mM. Purificationwith glutathione-Sepharose-4B led to a 376x increase inthe enzymatic specific activity.
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Discussion |
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We report the characterization of a second human sialidase encodedby the NEU2 gene, mapping to chromosome 2q37 (17
Montiet al., 1999). Briefly, the gene was isolatedby screening a human genomic library using as probe a portion ofthe hamster sialidase cDNA. Using the same probe, we were not ableto isolate cDNA clones from a large set of cDNA libraries. In fact,expression studies in adult tissues demonstrate that the NEU2 geneis only poorly transcribed in skeletal muscle. From these data,it is not surprising that no ESTs encoding NEU2 could be found amongmore than 1,200,000 human and 400,000 mouse partial cDNA sequences.
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 (13Kaoet al., 1997). Since a strong correlationof this catalytic behavior with active site architecture has beendemonstrated (10Gebler 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 (26Sato and Miyagi, 1995),there are four E-box sequences (-CAXXTG-), which are involved inthe developmental and tissue-specific regulation of muscle genetranscription (18Olson 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 (27Satoand 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.
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Materials and methods |
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In general, standard molecular biology techniques were carried outas described by Sambrook et al. (25
Sambrooket al., 1989). DNA restriction and modifyingenzymes were from Boehringer unless otherwise indicated.
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 (19Peitsch, 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(28Takebe et al., 1988),yielding the pCDL-NEU2 construct. COS7 cells were grown in petridishes (100 mm diameter) using Dulbecco’s modified Eagle’smedium (DMEM) with 10% fetal calf serum. Transfectionswere performed overnight using 8 µgof plasmid DNA and LipofectAMINE reagent, according to the manufacturer’s 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 manufacturer’s 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 BamHI–EcoRI 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 manufacturer’sinstructions (Pharmacia Biotech, GST Gene Fusion System, 18–1115–70).Samples of E.coli crude lysates (20 µg)and affinity purified NEU2 (0.5–1 µg)were subjected to SDS–PAGE 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 (31Venerando 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 5–10min. 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.
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We thank the Tigem Informatics Core, Maria Teresa Bassi, NicaBorgese, Germana Meroni, and Bruno Venerando for helpful discussion;Luigi Croci for technical assistance in sialidase assay; ClaudioGattuso for graphic assistance; and Melissa Smith for help in preparationof the manuscript. This work was partly supported by 60% MURSTfunds to E.M. The financial support of the Italian Telethon Foundationis gratefully acknowledged.
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a To whom correspondence should be addressed.E-mail: monti{at}tigem.it
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References |
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