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Human Molecular Genetics Pages 115-121

Cloning of the cDNA and gene encoding mouse lysosomal sialidase and correction of sialidase deficiency in human sialidosis and mouse SM/J fibroblasts
Introduction
Results
   Cloning of sialidase cDNA
   Structure of the mouse lysosomal sialidase gene
   Intracellular localization
   Tissue distribution of sialidase mRNA
   Transient expression of cDNA
Discussion
Materials And Methods
   Cell lines
   Antibodies
   Cloning of mouse sialidase cDNA and gene
   Subcloning into expression vectors
   Northern blot analysis
   Expression in mammalian cells
   Immunocytochemical localization of lysosomal sialidase
   Statistical analysis
Acknowledgements
References

Cloning of the cDNA and gene encoding mouse lysosomal sialidase and correction of sialidase deficiency in human sialidosis and mouse SM/J fibroblasts

Cloning of the cDNA and gene encoding mouse lysosomal sialidase and correction of sialidase deficiency in human sialidosis and mouse SM/J fibroblasts Suleiman A. Igdoura1, Christopher Gafuik1,2, Carmen Mertineit1,5, Farzad Saberi1, Alex V. Pshezhetsky6, Michel Potier6, Jacquetta M. Trasler1,3,4,5 and Roy A. Gravel1,2,3,4,*

1Montreal Children's Hospital Research Institute and Departments of 2Biology, 3Human Genetics, 4Pediatrics and 5Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada and 6Département de Pédiatrie, Hôpital Sainte-Justine, Université de Montréal, Montreal, Quebec, Canada

Received September 5, 1997 Revised and Accepted October 19, 1997

DDBJ/EMBL/GenBank accession no. U93702

Lysosomal sialidase occurs in a multienzyme complex that also contains [beta]-galactosidase and cathepsin A. We previously cloned the human lysosomal sialidase cDNA and characterized mutations in human sialidosis patients. Here, we report the cloning and expression of the mouse lysosomal sialidase cDNA and gene. The 1.77 kb cDNA encodes an open reading frame of 408 amino acids which shows high homology to the human lysosomal sialidase (80%), the rat cytosolic sialidase (65%) and viral and bacterial sialidases (50-55%). The sialidase gene is ~4 kb long and contains six exons. The five introns range in size from 96 to 1200 bp. Northern blot analysis revealed high expression of multiple sialidase transcripts in kidney and epididymis, moderate levels in brain and spinal cord, and low levels in adrenal, heart, liver, lung and spleen. Transient expression of the cDNA clone in sialidase-deficient SM/J mouse fibroblasts and human sialidosis fibroblasts restored normal levels of sialidase activities in both cell types. Immunocytochemically expressed sialidase co-localized with a lysosomal marker, LAMP2, confirming its lysosomal nature. Since sialidase activity requires its association with [beta]-galactosidase and cathepsin A, the expression of mouse sialidase within human sialidosis cells underlines the structural similarity between mouse and human enzymes and suggests that the mechanism for complex formation and function is highly conserved.

INTRODUCTION

Sialidases cleave terminal [alpha]2 -> 3 and [alpha]2 -> 6 sialyl linkages of oligosaccharides and glycoproteins, a process associated with numerous important biological reactions such as antigenic expression and recognition of cell surface receptors (1 ,2 ). Generally, sialidases have been classified based on their subcellular distribution and substrate specificity into three types: cytosolic, lysosomal and plasma membrane (3 ,4 ). Although the rat and Chinese hamster cytosolic sialidases had been cloned previously (5 ,6 ), it is only recently that the human lysosomal sialidase cDNA has been identified successfully (7 -10 ). The identity of the cDNA was confirmed through mutation analysis of sialidosis patients and expression of sialidase activity in deficient human fibroblasts.

The human lysosomal sialidase is a glycoprotein which exists in two isoforms of 44 and 48 kDa (8 ) and is only active as a part of a high molecular weight lysosomal multienzyme complex that also contains [beta]-galactosidase and cathepsin A (11 ). The human lysosomal sialidase gene was mapped to chromosome 6p21 within the human major histocompatibility complex (MHC) (10 ). Mapping of the mouse sialidase gene was possible because the SM/J strain mouse carries a defective sialidase allele and as a result has a tissue-specific deficiency in sialidase activity (12 ). The mouse sialidase gene was mapped near the H-2D end of the mouse MHC on chromosome 17, a region which is syntenic to the human MHC region on chromosome 6 (13 ), suggesting that these may be analogous genes.

Here we report the identification and characterization of the mouse lysosomal sialidase cDNA and gene. Transient expression of the cDNA in sialidase-deficient SM/J mouse fibroblasts and in human sialidosis fibroblasts restored sialidase activity. Expression in normal cells was dependent on co-transfection with cathepsin A cDNA.

RESULTS

Cloning of sialidase cDNA

The complete cDNA is 1.77 kb with an open reading frame of 1227 bp encoding a 409 amino acid polypeptide (Fig. 1 a). The protein contains a putative 40 amino acid signal peptide, a `FRIP' sequence, four `aspartic boxes' and four potential glycosylation sites. The predicted molecular weight of the protein is 44.6 kDa. With the exception of the signal peptide, the mouse sequence shows high homology with the human lysosomal sialidase sequence (>85%) (Fig. 1 b), with the rat cytosolic sialidase (65%) and with viral and bacterial sialidases (50-55%).

Structure of the mouse lysosomal sialidase gene


Figure 1. (a) Nucleotide sequence and deduced amino acid sequence of the mouse lysosomal sialidase. The putative signal peptide is underlined. The four potential glycosylation sites are marked with asterisks and the four conserved aspartic boxes are boxed. Arrows indicate positions of intron-exon boundaries. (b) Amino acid sequence alignment of the mouse and human sialidases. While the putative signal peptides show little similarity, the rest of the sequences share extensive homology, including identical locations for the aspartic boxes and three of the four potential glycosylation sites. Note that the fourth glycosylation site is substituted by a glycine in the human sialidase sequence. (c) Schematic showing the genomic organization of the mouse lysosomal sialidase gene. The introns vary in size from 96 to 1200 bp. The intron-exon junctions are conserved between mouse and human sialidase genes.

Four genomic DNA fragments containing five introns were amplified by PCR from mouse (C57BL/6) genomic DNA (Fig. 1 c). The mouse sialidase gene is ~4 kb long from the translation initiation codon (ATG) to the termination signal (in exon 6), and is structurally similar to the human gene. There is no distinguishable polyadenylation signal. The introns range in size from 96 bp to 1.2 kb (Table 1 ). All the exon-intron boundary sequences were consistent with the established splice consensus sequences (14 ).

Intracellular localization

In order to determine if the expressed sialidase is targeted to the lysosomes, SM/J lung fibroblasts (Fig. 4 A-C) and sialidosis fibroblasts (Fig. 4 D-F) transfected with a pCMV-sialidase vector were double immunolabeled for sialidase and for LAMP2 (lysosomal marker) (15 ). In sialidosis cells, only the two successfully transfected cells in Figure 4 E show reactivity to the anti-sialidase antibody, confirming the specificity of the antibody. In SM/J and sialidosis cells transfected with sialidase cDNA, anti-sialidase immunofluorescence was observed in perinuclear punctate structures that co-localized with anti-LAMP2 immunofluorescence, confirming a lysosomal location for the expressed mouse sialidase (Fig. 4 A-F).

Tissue distribution of sialidase mRNA

To evaluate the levels of sialidase expression in different mouse tissues, total RNA was analyzed by Northern blot using the mouse sialidase cDNA as a probe. The highest levels of expression were found in the kidney and epididymis, with moderate levels in the brain and spinal cord, and low levels in adrenal, liver, lung, spleen and heart (Fig. 2 ). Transcript size varies from 1.8 to 2.6 kb, with only the epididymis showing predominantly faster migrating mRNA transcripts. Reprobing the same blot with an 18S oligonucleotide probe confirmed that loading was similar in all lanes, with the exception of the kidney and epididymis samples where only 7.5 µg/lane were used.

Table 1 . Intron-exon boundaries in the mouse lysosomal sialidase gene
Primer pairs Intron Exon 5'-splice
donor		 Size
(bp)		 Exon 3'-splice
acceptor
5'-GAGCCAGGGCAGAGGATGACTTCAGC I AGCCTGgtgagcctt 420 tctgcgcagGTGCAG
5'-GTCGTCCTTACTCCAAACAACATGG II ACCAGGgtaacaagc 430 ttcttctagGTAGCA
5'-CCGGAATCTCTCTGTGGATATTGG III ATTCAGgtttcaccc 1200 tcttaacagAAACAG
5'-CAAAGGGAATGCCGCTCACTCC
5'-CCCAAACACGATCACGATTTCAACC IV TGCCAGgtcaggagt 96 cccacgcagCCCTAC
5'-GGTTCAGGCCTTTCTCGTACAG V AGTTCCgtgagtgcc 101 gctctctagGAGTGA

Transient expression of cDNA

Transfection with mouse sialidase cDNA restored sialidase enzymatic activities in SM/J-deficient fibroblasts and in human sialidosis fibroblasts to normal levels (Fig. 3 a). Co-transfection of cathepsin A cDNA with the sialidase cDNA in deficient cells increased sialidase activity further to 10-fold in SM/J mouse cells and 40-fold in human sialidosis cells. However, expressing mouse sialidase cDNA in normal human and wild-type mouse fibroblasts did not alter levels of sialidase activity. Only when human cathepsin A cDNA was co-transfected with sialidase cDNA did sialidase activity increase by 2-fold in both cell types. In all transfections, levels of a control lysosomal enzyme, [beta]-galactosidase, remained unchanged (Fig. 3 b). The pH curve for expressed sialidase revealed optimum activity at pH 4.5 which is characteristic of lysosomal enzymes.

DISCUSSION


Figure 2. Northern blot analysis of lysosomal sialidase mRNA in various mouse tissues. The multiple transcripts reflect heterogeneity possibly due to alternative splicing. Total RNA from various tissues (10 µg/lane) was electrophoresed through a 1.5% formaldehyde-agarose gel and transferred to a nylon membrane. The membrane was hybridized with 32P-labeled mouse sialidase cDNA. A, adrenal; S, spleen; Sc, spinal cord; C, cerebellum; Bs, brain stem; Bc, brain cortex; H, heart; K, kidney; Li, liver; Lu, lung; Eh, epididymal head; Et, epididymal tail.


Figure 3. Sialidase (a) or [beta]-galactosidase (b) activities in transiently transfected normal human fibroblasts (Norm), human sialidosis fibroblasts (sialidosis), mouse C57BL/6 lung fibroblasts (wild-type) and mouse SM/J lung fibroblasts (SM/J). Cells were either transfected with mock vector, mouse sialidase cDNA or a combination of mouse sialidase cDNA and human cathepsin A (hCatA) cDNA. *Significant differences (P<0.01) from mock-treated sialidase activity of cells. The data represent three separate experiments. Error bars represent standard deviation. Note the synergistic effects of cathepsin A and sialidase cDNAs on elevating levels of sialidase expression.


Figure 4. Expressed sialidase co-localizes with LAMP2 within distinct lysosomal compartments. SM/J mouse cells (A-C) and sialidosis human fibroblasts (D-F) transfected with mouse sialidase cDNA were incubated for 72 h then fixed and immunolabeled for sialidase (B and E) and LAMP2 (A and D) proteins. Note that the lysosomal marker LAMP2 co-localizes with expressed sialidase (C and F), confirming the lysosomal nature of the expressed sialidase enzyme. (Bars: C = 6 µm and F = 13 µm).

In this study, we report the cloning, sequencing and expression of the mouse lysosomal sialidase cDNA. The alignment of the amino acid sequences demonstrated that the mouse cDNA sequence shares significant homology with the human lysosomal, rodent cytosolic and bacterial sialidases. This homology includes the highly conserved FRIP domain and aspartic boxes (16 ). In addition, the residues which bind the carboxylate group of the sialic acid substrate are conserved between bacterial (Arg37, 246 and 309), human (Arg78, 280 and 347) and mouse (Arg72, 274 and 341) sialidases (10 ,17 ). The three-dimensional structure of the mouse lysosomal sialidase appears therefore to resemble that of bacterial sialidases which consist of six four-stranded antiparallel [beta]-sheets arranged as a propeller (17 ). The highly conserved sequences shared among the various sialidases support a common phylogenetic origin for mammalian and bacterial sialidases.

The structure of the mouse lysosomal sialidase gene is similar to the human gene, with exon-intron junctions at identical sites (9 ). However, intron sizes varied considerably between the mouse and human genes, in particular intron III which is 1.2 kb in mouse and 0.425 kb in human. Furthermore, while Northern blot analysis of human tissues revealed a single transcript of 1.9 kb in all tissues (8 ), mouse tissues show substantial heterogeneity in sialidase mRNA transcripts, pointing to the possibility of alternative splicing.

The low endogenous levels of sialidase activity in liver of SM/J mice has been known for many years (12 ). In this report, we have established a cell line of SM/J lung fibroblasts with a sialidase-deficient phenotype. The very low endogenous levels of lysosomal sialidase in these SM/J cells made them suitable for expression of the mouse sialidase clone and for the subsequent detection of elevated sialidase enzymatic activity and sialidase immunolocalization. The expression of the mouse sialidase clone with and without human cathepsin A cDNA in mouse SM/J and human sialidosis cells confirms that the mouse lysosomal sialidase can form a catabolically active complex with human cathepsin A and [beta]-galactosidase proteins. These interspecies interactions among the various components demonstrate a conserved mechanism for complex assembly and activation. Furthermore, the elevation of sialidase activity in normal human fibroblasts by co-transfection of mouse sialidase and human cathepsin A cDNAs together, but not by sialidase alone, illustrates the role of cathepsin A in the activation of sialidase. This is consistent with previous findings that the presence of cathepsin A is essential for the expression of sialidase activity (18 ).

The lysosomal sialidase has been shown biochemically to cleave sialic acid from oligosaccharides and glycoproteins (1 ) and from GM3 gangliosides (19 ). We (20 ) and others (21 ,22 ) have described mouse models for Tay-Sachs (Hexa -/-) and Sandhoff (Hexb -/-) diseases which implicated lysosomal sialidase in the degradation of GM2 to GA2, allowing the Hexa -/- mice to escape disease symptoms. This sialidase-mediated sparing effect does not occur in human Tay-Sachs patients, although the human brain expresses lysosomal sialidase. This may implicate a substrate specificity difference between mouse and human lysosomal sialidases or it may reflect a difference in the level of the enzyme in the CNS. Further studies will be required to differentiate between these alternatives.

Overall, the molecular characterization of the mouse lysosomal sialidase has strengthened our understanding of the conserved nature of mammalian sialidases and may shed light on other roles for sialidase revealed through disease.

MATERIALS AND METHODS

Cell lines

Normal and sialidosis human fibroblasts were obtained from the Repository for Mutant Human Cell Strains, Montreal Children's Hospital, Montreal (sialidosis cell strain code WG0544). Primary cultures of SM/J and C57BL/6 lung fibroblast were established as described before (23 ). All cell types were maintained in modified Eagle's medium (MEM) supplemented with 10% fetal calf serum (FCS) and antibiotics.

Antibodies

Mouse and rat monoclonal antibodies against human and mouse LAMP2 respectively were obtained from the Developmental Studies Hybridoma Bank in Baltimore, MD (15 ). Tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rat IgG were purchased from Sigma (St Louis). Rabbit polyclonal antibodies against recombinant human sialidase were prepared as follows. A human sialidase cDNA fragment of 755 bp was obtained by BstEII-TaqI restriction, treated with a Klenow fragment DNA polymerase, subcloned into a pGEX-2T vector (Pharmacia) and expressed in Escherichia coli to produce a SIAL-glutathione transferase (GST) fusion protein. The fusion protein was purified as described by the manufacturer (Pharmacia). Purified fusion protein was used to immunize rabbits as described before (24 ). The IgG fraction from the obtained antiserum was purified further using a recombinant GST coupled to CNBr-Sepharose to absorb the anti-GST-specific antibodies. The specificty of the antibody was tested by Western blot analysis (at dilution of 1:2000, data not shown) and by immunofluorescent microscopy (at 1:200, Fig. 4 E).

Cloning of mouse sialidase cDNA and gene

Homology searches in the dbEST database (National Center for Biotechnology Information) were performed using the Online BLAST program in order to identify clones with high homology to the human sialidase sequence. The complete mouse lysosomal sialidase cDNA was found in I.M.A.G.E.Consortium cDNA clone AA107584 and was obtained from Genome Systems, Inc. (St Louis).

DNA was amplified from 1 µg of genomic DNA using polymerase chain reaction (PCR) procedures as described elsewhere (25 ). Based on previously published human sialidase gene structure (9 ), primers were designed to amplify the introns of the mouse sialidase gene by PCR. The primer pairs used to amplify the introns are given in Table 1 . PCR-amplified genomic fragments were separated on agarose gels and extracted from the gel using a GeneClean kit (Bio/Can Scientific, La Jolla, CA). PCR products were subcloned into a pCR2.1 vector using the TA-cloning Kit (Invitrogen) and sequenced manually or at the DNA Core facility of the Canadian Genetic Disease Network, Ottawa, Ontario.

Subcloning into expression vectors

The mouse sialidase cDNA (1.77 kb) flanked with XhoI and SalI restriction sites was subcloned into the SalI site of a pCMV-Sport2 vector (Gibco). The human cathepsin A cDNA was a gift from Y. Suzuki (The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). The cDNA was excised from a pGEM vector using EcoRI and subcloned into the EcoRI site of the pCMV-Sport2 vector. The orientation of the cDNA was confirmed in both vectors with NcoI restriction analysis.

Northern blot analysis

To evaluate the level of sialidase mRNA in mouse tissues, total RNA from adrenal, spleen, brain cortex, brain cerebellum, brain stem, spinal cord, heart, kidney, liver, lung and epididymis was analyzed by Northern blot analysis. Total RNA was isolated from different tissues using the acid guanidinium thiocyanate-phenol-chloroform method (26 ). The amount of RNA loaded per lane was 15 µg for all tissues except kidney and epididymis, for which 7.5 µg of RNA per lane was loaded. The RNA was denatured and subjected to electrophoresis in 1.5% agarose formaldehyde gels and transferred to Zetabind nylon membranes. Hybridization and washing conditions were as described previously (25 ). An end-labeled synthetic oligonucleotide recognizing 18S rRNA was used to assess equivalence of loading between lanes.

Expression in mammalian cells

For transient expression, cells (normal human fibroblasts, human sialidosis fibroblasts, wild-type mouse C57BL/6 fibroblasts and mouse SM/J fibroblasts) were transfected with the mouse sialidase cDNA with or without the human cathepsin A cDNA. Transfection was done using lipofectamine [5 µg of vector(s) and 20 µl of lipofectamine solution in 2 ml of Optimem solution for 75 mm flask of cells] as described by the manufacturer (Gibco). For control, a pCMV-Sport2 vector containing no insert was used as a mock vector. After transfection, cells were incubated for 24 h in MEM medium containing 15% FCS with no antibiotics. On the second day, the medium was replaced with medium containing 15% FCS and antibiotics. Transfection efficiency was variable among cell types and, therefore, statistical comparisons between cell types were not considered. After 72 h of transfection, the cells were washed once with phosphate-buffered saline (PBS) and then scraped in cold PBS. Cells were pelleted and then sonicated for 5 s in 100 µl of distilled water. Cell homogenates were assayed for lysosomal sialidase and [beta]-galactosidase activity using sodium 4-methylumbelliferyl-d-N-acetylneuraminate (Sigma) and 4-methylumbelliferyl-[beta]-d-galactoside (ICN) substrates respectively (27 ). The data collected represent three separate experiments. Analysis of pH effect on sialidase activity was performed after expression of the mouse sialidase in sialidosis human fibroblast for 72 h. Sialidase activity was measured at the following pHs: 1.5, 2.5, 3.7, 4.2, 5.1, 6.3 and 6.9.

Immunocytochemical localization of lysosomal sialidase

Transfected cells expressing mouse sialidase cDNA, grown on chambered slides, were prepared for immunocytochemistry as described before (28 ). Cells were mounted on slides with 50% glycerol in PBS containing 1,4-diazabicyclo-[2,2,2]-octane (DABCO, Sigma) antifading reagent. Double antibody labeling experiments were analyzed on a Zeiss LSM 410 inverted confocal microscope (Carl Zeiss Inc., Thornwood, NY) as described previously (28 ). The green and red images were overlaid and pseudo-colored using built-in LSM software. Images were printed on a Kodak XLS8300 printer.

Statistical analysis

Data were analyzed using Bartlett's test for homogeneity. One-way analysis of variance was used to test for differences within each cell type (29 ). For comparisons between treatments of each cell type, a modified least significant difference test was used and significance was set at P<0.01 (29 ).

ACKNOWLEDGEMENTS

We are grateful to D. Leclerc for his helpful discussions. S.A.I. is a recipient of The Montreal Children's Hospital Research Institute Postdoctoral Fellowship and C.G. is a recipient of the National Scientific and Engineering Research Council studentship. J.M.T. and A.P. are scholars of the Fonds de la Recherche en Santé du Québec. This project was supported by grants to R.A.G., A.P. and J.M.T. from the Medical Research Council of Canada.

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*To whom correspondence should be addressed. Tel: +1 514 934 4358; Fax: +1 514 934 4331; Email: mc84@musica.mcgill.ca
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J. Biol. Chem., March 10, 2000; 275(11): 8007 - 8015.
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T. Miyagi, T. Wada, A. Iwamatsu, K. Hata, Y. Yoshikawa, S. Tokuyama, and M. Sawada
Molecular Cloning and Characterization of a Plasma Membrane-associated Sialidase Specific for Gangliosides
J. Biol. Chem., February 19, 1999; 274(8): 5004 - 5011.
[Abstract] [Full Text] [PDF]

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