| Human Molecular Genetics | Pages |
A point mutation in the neu-1 locus causes the neuraminidase defect in the SM/J mouse
Introduction
Results
Isolation of the murine neuraminidase cDNA and expression pattern of neu-1 in mouse tissues
Phenotypic characterization of SM/J mice and identification of the mutation in the neu-1 gene
Expression of L209I mutant neuraminidase in deficient fibroblasts
Biosynthesis of the L209I mutant in COS-1 cells and its association with the PPCA precursor
Discussion
Materials And Methods
Isolation of the mouse cDNA
RNA isolation and Northern blot analysis
Mutation analysis and mutagenesis
Cell culture, electroporation of fibroblasts and transfection of COS-1 cells
Lysosomal/mitochondrial extract
Enzyme activities and urine analysis
Immunoprecipitation
Acknowledgements
References
 |
A point mutation in the neu-1 locus causes the neuraminidase defect in the SM/J mouse
Lysosomal neuraminidase (sialidase) occurs in a high molecular weight complex with the glycosidase [beta]-galactosidase and the serine carboxypeptidase protective protein/cathepsin A (PPCA). Association of the enzyme with PPCA is crucial for its correct targeting and lysosomal activation. In man two genetically distinct storage disorders are associated with either a primary or a secondary deficiency of lysosomal neuraminidase: sialidosis and galactosialidosis. In the mouse the naturally occurring inbred strain SM/J presents with a number of phenotypic abnormalities that have been attributed to reduced neuraminidase activity. SM/J mice were originally characterized by their altered sialylation of several lysosomal glycoproteins. This defect was linked to a single gene, neu-1, on chromosome 17, which was mapped by linkage analysis to the H-2 locus. In addition, these mice have an altered immune response that has also been coupled to a deficiency of the Neu-1 neuraminidase. Here we report the identification in SM/J mice of a single amino acid substitution (L209I) in the Neu-1 protein which is responsible for the partial deficiency of lysosomal neuraminidase. We propose that the reduced activity is caused by the enzyme's altered affinity for its substrate, rather than a change in substrate specificity or turnover rate. The mutant enzyme is correctly compartmentalized in lysosomes and maintains the ability to associate with its activating protein, PPCA. We propose that it is this mutation that is responsible for the SM/J phenotype.
INTRODUCTION
Neuraminidases (sialidases) constitute a large and important family of hydrolytic enzymes that cleave the terminal sialic acid residues from a variety of sialoglycoconjugates [for a review see (10)]. This event influences many cellular processes, including cell-cell interaction/adhesion, protection from pathogens and antigen recognition (10-14). Some family members share certain characteristic features, including the F(Y)RIP domain in the N-terminal region of the protein, where the arginine is part of the active site, and two to five evenly spaced Asp boxes (S/T-X-D-X-G-X-T-W/F), which are located C-terminal of the F(Y)RIP sequence (15,16). The three-dimensional structure of bacterial and viral sialidases has shown that these enzymes have a common catalytic core of [sim]40 kDa with a characteristic six-bladed [beta]-propeller fold (17,18). Human lysosomal N-acetyl-[alpha]-neuraminidase is deficient or defective in two distinct metabolic storage disorders: sialidosis, which is caused by structural lesions in the neuraminidase gene; and galactosialidosis, in which neuraminidase deficiency is secondary to a primary defect in the serine carboxypeptidase protective protein/cathepsin A (PPCA) (1,4). Recently we and others cloned the human neuraminidase cDNA and identified a number of independent mutations in the neuraminidase gene that we associated with different clinical variants of sialidosis (3,19,20). The neuraminidase locus maps to the HLA region on chromosome 6p21 (3,21,22).
In the mouse a partial deficiency of a neuraminidase was first identified in the naturally occurring strain SM/J (23). These inbred mice had already been selected in the early 1940s for their relative small body size following cross matings with seven different inbred strains. Later, biochemical analysis demonstrated abnormal sialylation of at least four lysosomal glycoproteins that showed an altered migration pattern on starch gel electrophoresis. This defect was corrected by treatment with bacterial sialidase [reviewed in (5)]. This hypersialylation was attributed to a reduction in activity of a liver-specific sialidase (5,24,25), although some reports suggested that other organs were also affected (23,26). The responsible gene was designated neu-1 and mapped, by linkage analysis, to the histocompatibility locus on chromosome 17, in the region between H-2D and H-2E[alpha] (6,7), which is syntenic to the human HLA locus on chromosome 6p21. Besides the abnormal sialylation of lysosomal glycoproteins, SM/J mice also have an impaired immune response, which is thought to result from the altered processing of sialic acids pres-ent on cell surface molecules of subpopulations of T cells (8,9,27-31). An important step in the development of an immune response is differentiation of activated naive T cells into either IFN-[gamma]-producing (Th1) or IL-4-producing (Th2) cells [for a review see (32)]. Although SM/J mice can stimulate a Th1- mediated immune response, they cannot stimulate the conversion of naive T cells into IL-4-producing Th2 lymphocytes. This altered response has been attributed to reduced activity of Neu-1 neuraminidase, which is thought to result in: (i) improper desialylation of surface antigens on Th2-committed cells; (ii) reduction in early IL-4 production; and (iii) absence of IgG1 and IgE production by B cells after in vivo immunization of SM/J mice with pertussis toxin (31). T cell Neu-1 neuraminidase has also been implicated in conversion of vitamin D3 binding protein into macrophage activating factor (27). Together these data suggest an important role for Neu-1 neuraminidase in processing of selected sialoglycoconjugates at either the plasma membrane or within intracellular compartments.
In this paper we describe identification of a single amino acid substitution, L209I, in the neu 1-encoded lysosomal neuraminidase of SM/J mice. Analysis of the biochemical properties of this mutant enzyme demonstrates that its reduced neuraminidase activity is indeed caused by the presence of this mutation and not by improper compartmentalization of the protein, altered turnover or a lack of association with PPCA.
RESULTS
Isolation of the murine neuraminidase cDNA and expression pattern of neu-1 in mouse tissues
Two murine neuraminidase cDNAs (1.8 and 2.4 kb) were isolated using the human cDNA as probe. Both contained the same open reading frame, but the 2.4 kb clone lacked the first two codons and had an extended 3[prime]-untranslated region (UTR). The deduced amino acid sequence of the mouse protein is 91% similar to its human counterpart: the N-terminus begins with a conventional 39 amino acid signal sequence (33) and includes a FRIP domain as well as three conserved and two degenerated Asp boxes. The protein has four potential N-linked glycosylation sites; the first three are found at identical positions in the human neuraminidase, whereas the fourth, which is close to the C-terminus, is only present in the mouse sequence (Fig. 1). Northern blot analysis of multiple tissues, using probes spanning the cDNA (Fig. 1) demonstrated two major and two minor transcripts, which vary in length from 1.8 to 4.0 kb (Fig. 1). The 3[prime]-UTR probe, unique for the 2.4 kb cDNA, recognized only the 2.4 and 4.0 kb transcripts, indicating that the four mRNAs use alternative 3[prime]-UTRs. The hybridization results suggest that all four transcripts contain the same protein encoding sequence. The 1.8 and 2.4 kb mRNAs were the most abundant and displayed a differential pattern of expression which closely correlated with expression of PPCA, which forms a complex with the neuraminidase protein (34,35). The murine gene coding for the isolated cDNAs contains six coding exons (Table 1). The gene spanned a small region of 4 kb and was mapped, using the 1.8 kb cDNA insert as probe, to the H-2 region of chromosome 17 (data not shown).
Figure
Phenotypic characterization of SM/J mice and identification of the mutation in the neu-1 gene
All four neuraminidase transcripts displayed similar patterns of expression in kidney, brain, liver and spleen RNA preparations from SM/J mice (Fig. 1). In addition, a single polypeptide was immunoprecipitated with anti-human neuraminidase antibodies (anti-neur) from radiolabeled lysates of SM/J fibroblasts; this immunoprecipitated protein was comparable in size with the normal murine protein (data not shown). Although we found no overt changes at the RNA or protein level, we did find that neuraminidase activity of SM/J Neu-1 differed from that of wild-type Neu-1. Using sodium 4-methyl-umbelliferyl-[alpha]-D-N-acetylneuraminate (4-MU-NANA) as substrate, SM/J Neu-1 activity was significantly reduced in lysosomal/mitochondrial extracts derived from several SM/J tissues and this partial deficiency was clearly not restricted to any one tissue (Fig. 2). SM/J neuraminidase activity in kidney and liver extracts was also lower than that of control values when assayed with either [alpha]-2,3- and [alpha]-2,6-sialyllactose ([alpha]-2,3- and [alpha]-2,6-NANA-lactose) as substrate, thus demonstrating that the defective enzyme did not show altered specificity for either of the two linkages (Fig. 2). However, using fibroblast extracts we could demonstrate that SM/J neuraminidase assayed with substrate concentrations ranging from 0.1 to 1.5 mM 4-MU-NANA had an [sim]3-fold lower Vmax than the wild-type enzyme (Fig. 2). This suggests that the L209I substitution influences the kinetic properties of the mutant enzyme. Furthermore, the mutant mice displayed an abnormal pattern of urinary oligosaccharides (Fig. 2), indicative of oligosacchariduria, a condition commonly observed in galactosialidosis mice (2). Histological analysis of the SM/J mice showed evidence of storage products in specific cells, such as the Purkinje cells of the cerebellum and the glomerular epithelium, which appeared to accumulate over time (data not shown). Because these parameters are commonly used in biochemical diagnosis of sialidosis and galactosialidosis patients (1,4), it is clear that SM/J mice share similar phenotypic abnormalities with these two human diseases.
Figure
To identify the underlying genetic lesion responsible for these abnormalities we searched for a mutation(s) in the neuraminidase gene. Using RT-PCR on brain and liver RNA derived from SM/J mice of different ages and from different litters we amplified four overlapping fragments that span the entire neuraminidase cDNA (Fig. 3). Three mouse strains, BALB/c, 129/Sv and FVB, were used as controls. Sequence comparison identified seven nucleotide changes in the SM/J cDNA; four involved the wobble base of amino acid codons Lys93, Arg202, Thr295 and Ala316, two were present in the 3[prime]-UTR and one was a C[rarr]A transversion at nt 625 within exon IV of the gene. This transversion resulted in the amino acid change Leu209 to Ile (L209I). Because exon IV is present in all four neuraminidase transcripts (Fig. 1), we inferred that this point mutation must be present in all of the mRNAs and in the corresponding protein.
Figure 
Table 1
| Exons | cDNA position | Introns | Intron size | ||||
| Number | Size | 5[prime] splice site | 3[prime] splice site | ||||
| I | 171 | -30 to 141 | AGCCTGgtgagc | ...... | gcgcagGTGCAG | 365 | (1) |
| II | 190 | 142 to 331 | ACCAGGgtaaca | ...... | ttctagGTAGCA | 453 | (2) |
| III | 266 | 332 to 597 | ATTCAGgtttca | ...... | taacagAAACAG | 1200 | (3) |
| IV | 183 | 598 to 780 | TGCCAGgtcagg | ...... | acgcagCCCTAC | 97 | (4) |
| V | 221 | 781 to 1001 | AGTTCCgtgagt | ...... | tcctagGAGTGA | 99 | (5) |
| VI | 1365 | 1002 to 2366 | |||||
To assess the impact of the L209I change on biochemical properties of the normal enzyme we engineered this mutation into the normal 1.8 kb murine cDNA. The resulting mutant clone (Mo-smj) was completely sequenced to confirm correct introduction of the mutation. This mutant cDNA was transiently expressed in two human sialidosis type II fibroblasts. These cells were chosen because, unlike SM/J fibroblasts, they totally lack neuraminidase activity (3). Mo-smj cDNA only partially corrected the deficient fibroblasts, generating neuraminidase activity of between 40 and 65% of that of the wild-type murine (Mo-neur) enzyme. Given the strict dependence of neuraminidase on PPCA for full enzymatic activity (1,2), we also tested the effect of both mouse and human PPCA on SM/J neuraminidase. Co-transfection of the Mo-smj and Mo-neur cDNAs with either the mouse or human PPCA cDNA (Mo-ppca and Hu-ppca) resulted in a clear increase in neuraminidase activity for both the normal and mutant protein (Fig. 3). The induced SM/J activity, however, remained lower than that of the wild-type. The L209I mutation was also introduced into the human neuraminidase cDNA (Hu-smj). Expression of this mutant clone alone or in combination with human or mouse PPCA again resulted in reduced neuraminidase activity (Fig. 3), unequivocally demonstrating that the L209I substitution is responsible for the enzyme defect. Immunofluorescence analysis of singly and doubly transfected cells showed that the presence of the mutation in either the mouse or the human neuraminidase molecule does not alter the subcellular distribution of the enzyme, which maintained a typical punctate lysosomal staining (Fig. 3). The lysosomal localization of the mutant enzyme was confirmed using Percoll density gradients with transfected COS-1 cells (data not shown). Co-expression of either mouse or human PPCA clearly enhanced the lysosomal signal, further indicating that PPCA has a stabilizing effect on the mutant protein. The increase in SM/J neuraminidase activity in cells co-expressing mutant Neu-1 neuraminidase and PPCA suggested that interaction between the two proteins was not affected by the L209I mutation. We tested this assumption by overexpressing the Smj-neu1 and the PPCA cDNAs in COS-1 cells and then immunoprecipitating radiolabeled proteins with anti-neur or anti-PPCA antibodies (Fig. 4). Although the anti-neur antibodies recognized the murine protein with lower affinity than they did the human protein, more SM/J protein than wild-type mouse protein was immunoprecipitated from equally transfected cells (Fig. 4, lanes 2 and 3). Nevertheless, neuraminidase activity in singly transfected cells was again 50% of that of control values (data not shown). In co-transfected cells both the mouse and human PPCA precursors were co-precipitated with anti-neur antibodies, together with the SM/J polypeptide (Fig. 4, lanes 8 and 11). The mutant protein, the wild-type mouse protein and human neuraminidase all co-precipitated the PPCA precursor equally efficiently (lanes 7, 9, 10 and 12). Therefore, the L209I substitution did not interfere with association between the mutant protein and either mouse or human PPCA, excluding the possibility that the SM/J mutation affects complex formation. Sequential immunoprecipitation of all samples with anti-PPCA antibodies explained the difference in the ability of the mouse and human PPCA to enhance neuraminidase activity (Fig. 3). The murine PPCA precursor in overexpressing cells was not as well processed to the mature two chain form as the human PPCA precursor (Fig. 4, lanes 7-9 and 10-12). This reduced level of processing could have led to a smaller pool of mature PPCA available for `protection' of the lysosomal neuraminidase.
Figure
Turnover of the SM/J neuraminidase was apparently not influenced by the L209I substitution, as determined by pulse-chase labeling of transfected COS-1 cells (Fig. 4). Both the mutant and wild-type neuraminidase appeared to be stabilized upon co-expression of mouse PPCA, since immunoprecipitable material could still be detected at the 3-6 h time points (Fig. 4, middle panels). The stabilizing effect was less apparent, but still recognizable, when human PPCA was co-expressed with mutant or wild-type neuraminidase (Fig. 4, lower panels). These results clearly correlate with the observed increase in enzyme activity in cells co-expressing mutant or wild-type neuraminidase with PPCA. Overall the results we present here provide strong evidence that the subtle L209I substitution is responsible for the altered neuraminidase activity in SM/J mice. Leu209 in the murine enzyme falls in an amino acid stretch that is highly conserved among the different sialidases (10,36). This residue coincides with Leu221 and Leu199 of the Micromonospora viridifaciens and Salmonella typhimurium sialidases respectively, which are located in the three-dimensional structure of these enzymes in the vicinity of the active site (17,18). It is therefore conceivable that this amino acid substitution in the SM/J neuraminidase could affect substrate recognition, rate of substrate cleavage or release of the product, as evidenced by the altered Vmax of the mutant protein. Although SM/J mice present with some of the biochemical abnormalities that are associated with the human lysosomal disorder sialidosis, the relatively high residual neuraminidase activity prevents the occurrence in young mice of excessive storage in their tissues. Older mice, on the other hand, eventually develop visible cellular changes, especially in the CNS. Therefore this animal model may be regarded as a mild form of sialidosis. The residual SM/J activity varied slightly in different tissues. This could be attributed to the occurrence in some tissues, like brain, of neuraminidase `isoenzymes' thought to be localized in the lysosomal (37,38) or plasma membrane (38-42) and the cytosol (16,38,43-45). However, the existence of various lysosomal neuraminidases is questionable, since in PPCA-deficient mice no residual neuraminidase activity is detected at acidic pH (2). The same holds true for human sialidosis patients with structural mutations in the neuraminidase gene that result in complete loss of neuraminidase activity (3). Modification of sialic acid residues, which are present as terminal sugars on various types of sialoglycoconjugates, is essential for regulation of many cellular activities. The Neu-1 neuraminidase plays a key role in such modifications, for example in processing of cell surface molecules that are involved in modulating an immune response (9,27-31). T lymphocyte activation is normally accompanied by an increase in endogenous Neu-1 neuraminidase (30,31), which, in turn, results in hyposialylation of glycoproteins on the surface of activated T cells (9,46-49). These surface glycoproteins are required for T cell differentiation [for a review see (32)] and several of them are known to be internalized from the plasma membrane and subsequently re-exposed by a `recycling' process. MHC class I and class II molecules and the T cell receptor are examples of such molecules (50-53). Therefore, it may be that processing of the sialic acid residues on these and other glycoproteins present on the surface of specific T cells is mediated intracellularly by lysosomal neuraminidase. If this enzyme is part of the main mechanism for sialic acid processing in T cells then the altered Vmax value of SM/J neuraminidase would quite logically account for the abnormal sialylation of these molecules. Our data suggest that the mutant enzyme retains the capacity to recognize its substrate but that its rate of catalysis and/or release of product is impaired. The type of substrates that are cleaved by the enzyme may determine whether or not a certain cell type can compensate for a reduction in activity of mutant neuraminidase. Again, this is best exemplified in the T cell system, where the immune response in SM/J mice involves differentiation of naive T cells to Th1 but not to Th2 cells. Interestingly, reduced neuraminidase activity has also been detected in rat strain KGH (54). The responsible gene, neu-1, was mapped to the RT1 locus (55), which is syntenic to the mouse H-2 and human HLA loci. It is unclear whether this defect results in the same phenotypic alterations identified in SM/J mice. It will be instructive to identify the molecular basis of the defective neuraminidase activity in this rat strain and to compare it with that found in SM/J mice. A second gene, neu-2, has also been described in both mouse and rat (54,56). However, the encoded enzyme is localized in the cytoplasm and does not cleave the fluorimetric substrate (57). Furthermore, linkage analysis demonstrated that the neu-2 is not linked to the neu-1 locus (54). Once the three-dimensional structure of the lysosomal mammalian neuraminidase becomes available we could gain a better understanding of the impact of the L209I mutation on structure and function of the enzyme. Our findings on SM/J mice will hopefully facilitate further genetic and immunological studies on this animal model. A mouse BALB/c cDNA library was screened according to the manufacturer's instructions (Clontech). cDNA clones were sequenced with the Amersham thermocycler kit and subcloned into the mammalian expression vector pSCTOP (58). RNA was prepared from SM/J mouse tissues by the LiCl/urea method as previously described (59). Total RNA was separated on 1% agarose gels that contained 0.66 M formaldehyde in MOPS buffer, was then blotted onto Zetaprobe membranes (BioRad) and was finally hybridized under standard conditions (60). The multiple tissue northern blot was purchased from Clontech and handled according to the manufacturer's instructions. Total RNA preparations from different SM/J mice and FVB controls were subjected to RT-PCR (3,61). The following primers were used in the reactions: 5[prime]-CCCTAGGACACCGGGCCTTC-3[prime] (antisense, primer R); 5[prime]-CCTGGACAGGGATCGCCG-3[prime] and 5[prime]-GTAGAGGCCACCTGGCAG-3[prime] (fragment I); 5[prime]-CGGACCAGGGTAGCACGTGG-3[prime] and 5[prime]-GGGTGTCACAGGCGTCATAG-3[prime] (fragment II); 5[prime]-GATGACCACGGTGCCTCC-3[prime] and 5[prime]-GGTGTACCGGTTCAGGCC-3[prime] (fragment III); 5[prime]-CCTGGCAGAAGGAGAGGG-3[prime] and 5[prime]-CTGTTCATCTCTCCAGGG-3[prime] (fragment IV). Amplified products were purified by phenol/chloroform extraction, on Centricon-100 columns (Amicon) and by ethanol precipitation. The purified products were directly sequenced with the fmol sequencing kit (Promega). The mutation was inserted into the wild-type cDNA by combining fragments II and III (Fig. 3) and using the StuI restriction sites at positions 377 and 1168 to substitute the StuI fragment for the wild-type fragment. Human skin fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics and 10% fetal bovine serum (FBS). COS-1 cells were maintained in DMEM supplemented with 5% FBS. Fibroblasts were electroporated according to the manufacturer's instructions (BioRad) with the following modifications. Cells were harvested by trypsinization and washed once in Iscove's medium. They were then counted and 25 µg plasmid DNA were electroporated into 1 × 106 cells suspended in 500 µl Iscove's medium using a BioRad Gene Pulser set at 0.32 kV and 500 µF. Electroporated cells were seeded in 6-well plates for 14-18 h before the medium was changed. They were harvested 72 h later. Immunofluorescence of electroporated fibroblasts was performed as described previously (3). COS-1 cells were transfected with Qiagen's Superfect according to the manufacturer's instructions and harvested 72 h post-transfection. Transfection efficiency was checked by immunofluorescence and the total amount of synthesized neuraminidase protein was estimated by western blot analysis of total cell lysates. Comparable transfection efficiencies were obtained among samples within each experiment and similar levels of neuraminidase protein were synthesized. Mice were sacrificed by cervical dislocation and their tissues immediately isolated and placed in ice-cold 10 mM HEPES, pH 7.4, 250 mM sucrose. After the tissues were washed several times in this buffer they were weighed and homogenized in a tight-fitting dounce (Kontes) in 4 vols HEPES-buffered sucrose. A lysosomal/mitochondrial extract was prepared according to the procedure described by Gieselmann (62). The resulting lysosomal/mitochondrial pellet was dissolved in HEPES-buffered sucrose and analyzed for enzyme activity. Lysosomal/mitochondrial extracts and cell lysate from either transfected COS-1 cells or electroporated fibroblasts were assayed for neuraminidase activity using the artificial substrate 4-MU-NANA according to Galjaard (63). Protein concentrations were determined using the BCA kit from Pierce Chemical Co. Neuraminidase activity also was assayed with [alpha]-2,3- and [alpha]-2,6- NANA-lactose as substrates, according to the procedure described previously (64,65). Urine samples were collected and analyzed using a FACE® Urinary Carbohydrate Analysis kit purchased from Glyko Inc. following the manufacturer's instructions. Transfected COS-1 cells were seeded in 6-well plates and labeled for 16 h with 50 µCi [3H]-4,5-leucine. Radiolabeled proteins were immunoprecipitated with anti-neur antibodies, as described previously (66). For the pulse-chase experiment transfected cells were labeled with 50 µCi [3H]-4,5-leucine for 1 h and then chased in fresh DMEM over different time periods (67). We are indebted to Dr Neal Copeland (Director of the Mammalian Genetics Laboratory at the Frederick Cancer Research and Development Center, Frederick, MD) for chromosomal localization of the mouse neuraminidase gene. We thank Maria del Pilar Martin for help with the enzyme analyses and maintenance of the SM/J strain, Thasia Leimig for culturing the SM/J fibroblasts, Andrew Hollenbach for help with the kinetic studies, Jimmy Toy for generation of the mutant human cDNA and Aarnoud van der Spoel for useful suggestions and discussions. We also thank Sue Vallance for editing the manuscript and Charlette Hill for secretarial assistance. These studies were supported by the American Lebanese Syrian Associated Charities (ALSAC) of St Jude Children's Research Hospital.Expression of L209I mutant neuraminidase in deficient fibroblasts
Biosynthesis of the L209I mutant in COS-1 cells and its association with the PPCA precursor
DISCUSSION
MATERIALS AND METHODS
Isolation of the mouse cDNA
RNA isolation and Northern blot analysis
Mutation analysis and mutagenesis
Cell culture, electroporation of fibroblasts and transfection of COS-1 cells
Lysosomal/mitochondrial extract
Enzyme activities and urine analysis
Immunoprecipitation
ACKNOWLEDGEMENTS
REFERENCES
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