Human Molecular Genetics, 2000, Vol. 9, No. 9 1283-1290
© 2000 Oxford University Press
Biochemical and structural analysis of missense mutations in N-acetylgalactosamine-6-sulfate sulfatase causing mucopolysaccharidosis IVA phenotypes
1Department of Pediatrics, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan, 2Research Center for Structural Biology Institute for Protein Research, Osaka University, Osaka, Japan, 3Department of Biochemistry and Molecular Biology, St Louis University, St Louis, MO, USA, 4Gene Therapy Programme, Dibit Tiget St Raffaele Hospital, Milano, Italy, 5Department of Biochemistry, University of Milano, Milano, Italy and 6Department of Human Welfare, Faculty of Human Welfare, Chubu Gakuin University, Seki, Japan
Received 14 January 2000; Revised and Accepted 27 March 2000.
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ABSTRACT |
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Mucopolysaccharidosis IVA (MPS IVA; OMIM#253000), a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), has variable clinical phenotypes. To date we have identified 65 missense mutations in the GALNS gene from MPS IVA patients, but the correlation between genotype and phenotype has remained unclear. We studied 17 missense mutations using biochemical approaches and 32 missense mutations, using structural analyses. Fifteen missense mutations and two newly engineered active site mutations (C79S, C79T) were characterized by transient expression analysis. Mutant proteins, except for C79S and C79T, were destabilized and detected as insoluble precursor forms while the C79S and C79T mutants were of a soluble mature size. Mutants found in the severe phenotype had no activity. Mutants found in the mild phenotype had a considerable residual activity (1.3–13.3% of wild-type GALNS activity). Sulfatases, including GALNS, are members of a highly conserved gene family sharing an extensive sequence homology. Thus, a tertiary structural model of human GALNS was constructed from the X-ray crystal structure of N-acetylgalactosamine-4-sulfatase and arylsulfatase A, using homology modeling, and 32 missense mutations were investigated. Consequently, we propose that there are at least three different reasons for the severe phenotype: (i) destruction of the hydrophobic core or modification of the packing; (ii) removal of a salt bridge to destabilize the entire conformation; (iii) modification of the active site. In contrast, mild mutations were mostly located on the surface of the GALNS protein. These studies shed further light on the genotype–phenotype correlation of MPS IVA and structure–function relationship in the sulfatase family.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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A deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS; EC3164) leads to lysosomal accumulation of undegraded glycosaminoglycans, keratan sulfate and chondroitin-6-sulfate and mucopolysaccharidosis IVA (MPS IVA, Morquio disease type A; OMIM#253000) occurs (1). The condition commonly manifests as short trunk dwarfism, coxa valga, odontoid hypoplasia, corneal clouding and hepatosplenomegaly (2), and if untreated, most of these patients die from cervical myelopathy or valvular heart failure by the third decade of life. The mild phenotype is characterized by mild bone involvement and some such patients survive to 50–60 years of age with a relatively good quality of life (3–9).
GALNS enzyme protein has been purified from human placenta as an oligomer with a molecular mass of 120 kDa, consisting of 40 and 15 kDa polypeptides linked by a disulfide bond (10). The full-length cDNA and genomic sequences for human GALNS have been isolated and characterized (11,12). The GALNS gene is located on chromosome 16q24 (13–15) and encodes 522 amino acids, including a signal peptide of 26 residues. To date we have identified over 90 mutations (including submicroscopic, double gene and small deletions, insertions and point mutations) in the GALNS gene from MPS IVA patients (16–32). Most GALNS mutations identified have been point mutations and small deletions. Point mutations accounted for 65 of the missense mutations. Transient expression analysis of some mutant cDNAs revealed low residual GALNS activities in the cells of patients, but the correlation between genotype and clinical phenotype has remained unclear.
Sulfatases, including GALNS are members of a highly conserved gene family sharing extensive sequence homology (33) and a unique post-translational modification (34). This modification generates a 2-amino-3-oxopropanoic acid residue. In eukaryotes, 2-amino-3-oxopropanoic acid formation occurs in the endoplasmic reticulum through oxidation of a conserved cysteine residue. In multiple sulfatase deficiency, a rare lysosomal storage disorder, all sulfatases are catalytically inactive (35) because they are defective in this post-translational modification.
The crystal structures of N-acetylgalactosamine-4-sulfatase (4S) (36) and arylsulfatase A (ASA) (37) were reported and structures of the sulfatase family were seen to share important features such as the active site.
To determine the contribution of each mutation to the clinical phenotype of MPS IVA, we characterized 15 missense mutations and two newly engineered active site mutations, using transient expression analysis. In addition, we discussed the effects of 32 missense mutations, based on a GALNS tertiary structural model constructed from the X-ray crystal structure of 4S and ASA.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Characterization of mutant GALNS proteins by transient expression assay
To determine the individual effect of mutations on GALNS, mutant vectors were constructed by site-directed mutagenesis. The wild-type and 11 mutant expression pCAGGS vectors were defined in previous work, as described in Materials and Methods. Six mutant vectors (P77R, G155R, S162F, R259Q, C79S and C79T) were constructed. Using the same conditions, the wild-type and 17 mutant proteins were analyzed, for comparative purposes.
GALNS activity in the cell supernatant, the cell pellet and the cell culture medium was determined. Total GALNS activity of cell supernatants from the mutants was all reduced relative to the wild-type GALNS (Table 1). The P77R, I113F, P125L, P151L, G155R, S162F, G301C and R386C mutants, which were found in patients with the severe phenotype, had a very low activity at the resolution limit of the GALNS assay, or no activity. The R94G, V138A mutants found in the intermediate patients were similar. While the F97V, N204K, R259Q and T312S mutants had residual activity (1.3–13.3% of wild-type GALNS activity), all were present in the mild patients. Although the R94C mutant was found in a mild patient who was a compound heterozygote with F97V/R94C, this mutant had no activity. F97V activity might be expressed dominantly, relative to R94C (29). In addition C79S and C79T, engineered as the predictive active site mutations, had no activity. Thus, significant residual activity was found in mutants from the mild patients and this finding served as a guide to predict the biochemical phenotype of the mutants.
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Cell pellets from the wild type and all of the mutants had no activity (data not shown). In the culture medium, GALNS activity was not detected for any mutant except for the wild type (data not shown).
GALNS protein was analyzed by western blotting (Fig. 1A and B). The 55 kDa precursor and 40 kDa mature polypeptides were observed in the cell supernatant from the wild type and C79S, C79T of the active site mutants. However, in the 15 mutants, except for C79S and C79T, the 40 kDa mature polypeptide was not detected and the 55 kDa polypeptide was faintly visible or below the level of detection. We reported that GALNS mature protein had two subunits (40 and 15 kDa), each of which had a carbohydrate chain (10). In the present study we used an anti-GALNS specific oligopeptide (amino acid position 249–271) antibody, as described in Materials and Methods, so that the antibody reacted to the 40 kDa subunit but not to the 15 kDa subunit. The N204K mutant was predicted to destroy the N-glycosylation site and lose one of the carbohydrate chains (16). As expected the 52–53 kDa polypeptide was detected in the N204K mutant instead of the 55 kDa polypeptide. The decreased 2–3 kDa corresponds to one carbohydrate chain. On the other hand a 55 kDa polypeptide (52–53 kDa for N204K mutant) was detected in cell pellets from the wild type and all the mutants studied. The presence of 55 kDa (or 52–53 kDa) polypeptides in cell pellets may be due to accumulation and/or aggregation, under conditions of high levels of GALNS overexpression. The expression of very high levels of lysosomal enzymes may lead to poor processing and this situation may give rise to saturation of the mannose-6-phosphate targeting pathway, following the accumulation and/or aggregation of the enzyme protein in cell compartments (38). In addition we suggest that the mutant proteins, except for the active site mutations C79S and C79T, destabilized the entire conformation and aggregated in cell compartments.
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A tertiary structure model for GALNS protein
The X-ray crystal structures of the human 4S (36) and human ASA (37) were reported. These sulfatases share 24 and 29% identity with GALNS, respectively (Fig. 2A). Using their structures as the templates, we constructed a tertiary structural model of the human GALNS, by homology modeling.
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The model structure of GALNS has a monomeric form with two domains; the larger, N-terminal and the smaller, C-terminal (Fig. 2B). The main structural feature of the larger domain is a ß-pleated sheet with 10 strands sandwiched between
-helices. All the connections between the strands have the same orientation, and eight -helices have comparable lengths and positions in 4S and ASA. The smaller, C-terminal domain consists of a four-stranded antiparallel ß-sheet with an orthogonal -helix as seen in 4S and ASA. The overall topology of the GALNS structure model was practically identical for 4S and ASA proteins.
The active site in GALNS
Eukaryotic sulfatases contain a unique post-translational modification in their active site, a formylglycine residue generated from a cysteine (34), and the active site is located by the presence of a cluster of conserved residues surrounding this cysteine residue (36). To confirm the catalytic site of GALNS protein, we generated two engineered mutations on Cys79 (C79S and C79T) and demonstrated the expressed proteins. Both mutations destroyed enzyme activity (Table 1). In addition, the structural model of GALNS protein revealed that Cys79 changed to formylglycine lies at the bottom of a cavity. This active site cavity was lined mainly with charged amino acids. These charged groups were in loops (Asp288, Asn289) (Asp39, Asp54) (His236) (Lys140, His142) (Lys310) and the -helix (Arg83), which were highly conserved in 4S and ASA. The active site of the sulfatase family also contained a metal ion. In 4S and ASA the active site carries Ca2+ or Mg2+, respectively (36,37). It is suggested that the sulfatase family requires the presence of a metal ion to stabilize formation of the sulfate ester at the active site. Thus we confirmed which ions are required for GALNS protein. When GALNS protein was incubated with EGTA, the enzyme activity was inhibited and significant enzyme activity was recovered by adding Ca2+ but not Mg2+ ion (data not shown). GALNS protein may require a divalent metal ion at the active site.
Deduced effects of mutations on the tertiary structural model of the human GALNS protein
This model afforded the opportunity to analyze potential structural consequences of missense mutations in GALNS protein. To probe effects of various amino acid substitutions on catalytic activity or the stability of GALNS protein, we located the mutations on the GALNS model and characterized the structural effects in four groups (Table 2, Fig. 3A and B).
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Destruction of the hydrophobic core or modification of the packing.
G47R, P77R, G96V, I113F, P125L, P151L, G155R, F156C, S162F, W230G, S287L, G301C, M343R, F346L, A351V (found in the severe phenotype patients) and V138A, F97V, T312S (found in intermediate or mild patients) are found in the hydrophobic core.
P77R, M343R: Pro77, Met 343 prevent internalization in hydrophobic patch and the presence of the positive charged side chain of Arg is expected to be destabilizing.
I113F, S162F, S287L, A351V: the more bulky side chains of these substitution residues appear to break the packing.
F156C, W230G, F346L, V138A, F97V, T312S: residues of these substitutions form the cavity in the internal part of the protein and lead to modification of the packing.
P125L, P151L: Pro125 is located on the N-cap of the -helix and Pro151 is a conserved Pro. These substitutions lead to the modification of the packing.
G47R, G96V, G155R, G301C: Gly47, Gly96, Gly155 and Gly301 are in the -helix or loops and are highly conserved in the sulfatase family. The torsion angles of these Gly residues may be necessary in the folding pathway.
Removal of a salt bridge to destabilize the entire conformation.
R90W, R376Q, D344E (found in the severe phenotype patients), R94G, R253W (found in the intermediate phenotype patients) remove the hydrogen bond to destabilize the entire conformation. Arg90, Arg376, Asp344, Arg94 and Arg253 are directly involved in salt bridges with Glu121, Asp379, Arg380, Asp364 and Glu298, respectively. As these residues are conserved in 4S and ASA, they may stabilize the internal-domain interactions in the protein.
Modification of the active site geometry to reduce or lose the enzymatic activity.
H166Q, G168R (found in the severe phenotype patients): His166, Gly168 are located on the active site cavity.
Location on surface of the protein.
P179L, R386C (found in severe phenotype patients), D60N, D171A, N204K, R259Q, S295F (found in mild phenotype patients) are found on the surface of GALNS protein.
P179L: Pro179 is part of the ß-hairpin, which may be a possible recognition element for targeting to the lysosome (36), and is on the surface. When Pro179 is mutated with Leu, it appears to aggregate on this position.
R386C: Arg386 is found on the molecular surface, and forms a hydrogen bond with the side chain of Asn76.
D60N: Asp60 is highly conserved in mammalian sulfatases and is in the -helix. This Asp residue probably forms hydrogen bonds with the backbone.
D171A: Asp171 is in a loop and is engaged in hydrogen bonding distance from Lys173. Ala cannot form hydrogen bonds with a Lys residue.
R259Q: Arg259 is part of the -helix. This residue is engaged in hydrogen bonds with Glu55
N204K: Asn204 has the carbohydrate chain on the surface. The substitution to Lys destroys the N-glycosylation site.
S295F: Ser 295 is located on the threshold of the active site. The more bulky side chain of the Phe residue may interfere with enzyme reactions.
Phenotype–genotype correlation
Characterization of missense mutations, using a structural GALNS model and individual transient expression proved to be useful for structure–function studies of GALNS protein and for phenotype–genotype correlation in MPS IVA patients
GALNS activities in the expressed cells with the mutant vectors were all markedly reduced, relative to the wild type. While mutants found in the severe clinical phenotype had no activity, mutants found in the mild phenotype had low residual activity (1.3–13.3% of wild-type GALNS activity) (Table 1). This low residual activity may lead to a significant reaction in lysosomal particles and result in pronounced differences in clinical severity.
To determine how these mutations might be affecting catalytic activity or stability of the enzyme protein, a tertiary structural model of the human GALNS was constructed and the location of individual mutations was defined (Table 2, Fig. 3A and B). Consequently, we suggest that there are at least three different reasons for the severe phenotype: (i) destruction of the hydrophobic core or modification of the packing; (ii) removal of a salt bridge to destabilize the entire conformation; (iii) modification of the active site. Of the 22 severe mutations analyzed in this study 20 could be explained by these three reasons (20/22, 91%). In contrast, the mild mutations were mostly located on the surface of the GALNS protein (5/7, 71%). In the case of mild mutations, F97V and T312S were located in the hydrophobic core, but residues of these substitutions form the cavity in the internal part of the protein and are far from the active site. Thus it is likely that the milder mutations, probably far from the active site such as the surface, would not crucially affect the enzyme protein. Pemberton et al. (39) constructed a molecular model for the A domains of human factor VIII, based on the crystal structure of human ceruloplasmin, and they discussed the possible effects on the factor VIII A domain model of each missense mutation from the hemophilia A database. Wacey et al. (40) reported the construction of a multi-domain molecular model of factor IXa. Using the model and factor IXa gene mutations from the hemophilia B database, they deduced that the substitution of a buried residue is more likely to result in severe haemophilia B than the substitution of a surface residue. Zurutuza et al. (41) attempted to correlate the nature of the tissue-non-specific alkaline phosphatase gene mutation and the phenotype of hypophosphatasia, using clinical data, site-directed mutagenesis and computer-assisted modeling. They found that moderate mutations were not at the active site; rather most of the severe missense mutations were located in crucial domains such as the active site (41).
Enzyme and gene replacement as methods for treating subjects with mucopolysaccharidoses are the subjects of research. Findings from studies of the relationship between genotypes and clinical phenotypes have considerable relevance and significance in the selection of the patients for therapy and for predicting the efficacy in response to therapy. Based on the tertiary structural model, our study revealed effects of mutations on GALNS structure and function, and added to understanding of correlation between genotype and phenotype of MPS IVA. A structural model of the corresponding protein may also be used to engineer drugs for enzyme replacement therapy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations of GALNS gene in MPS IVA patients
In previous work, we identified 65 missense mutations. In the present study we analyzed 32 missense mutations. Because it is difficult to correlate genotype and phenotype for each heteroallelic mutation, we mainly selected homoallelic mutations for analysis. Of the 32 mutations, 20 were homoallelic and 12 were heteroallelic.
Construction of expression vectors
The wild-type GALNS cDNA (11) was subcloned into pUC13, and site-directed in vitro mutagenesis was performed as described (24), or we used a commercially available mutagenesis kit (Stratagene, La Jolla, CA) and followed the manufacturer’s instructions. Each of the mutant cDNAs was excised from the pUC13 vector with EcoRI and introduced into the eukaryotic expression vector pCAGGS (42).
Transient expression in GALNS deficient fibroblasts
Four micrograms of wild-type and mutant cDNA expression vectors were transfected using the liposome mediated method (Gene Transfer lyo, Wako, TX) into 3 x 105 GALNS-deficient fibroblasts, which were from a severe phenotype patient with a double gene deletion. The transfected cells were incubated for 110 h. The cultured adherent cells were harvested, kept at –80°C until use then resuspended and sonicated in extraction buffer (10 mM Tris–HCl, pH 7.0, 0.15 M NaCl, 50 µM phenylmethylsulfonyl fluoride) at 4°C. After centrifugation at 10 000 g for 10 min, the supernatants and pellets were used for enzyme assays of GALNS activity and for western blot analysis, as described below.
GALNS enzyme assay
Cell supernatants and cell pellets were assayed either directly or after dialysis in 10 mM Tris–HCl, pH 7.0. GALNS activity was assayed using the radiolabeled trisaccharide substrate GalNAc6S-GlcA-[3H]GalitolNAc6S, as described by Masue et al. (10).
Western blot analysis
For western blot analysis, 10–20 µg of protein from the supernatants and the pellets of expressing cells were separated in 10% SDS–polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Advantec, Tokyo, Japan) and then blocked for 1 h at room temperature in 3% bovine serum albumin/TBS (10 mM Tris–HCl, pH 8.0, 0.15 M NaCl). Membranes were washed for 1 x 10 min. in TBS-T (TBS containing 0.1% Tween 20) and were then incubated overnight at 4°C with human GALNS-specific oligopeptide (2HN-C-SQRGRYGDAVREIDDSIGKILEL-COOH) antiserum. Subsequently, membranes were washed for 3 x 15 min in TBS-T and were incubated with alkaline phosphatase-conjugated anti-rabbit IgG (H+L; Promega, Madison, WI) in a 1:7500 dilution in TBS for 2 h at room temperature. The final wash was 3 x 15 min in TBS-T and 1 x 10 min in alkaline phosphatase buffer (0.1 M Tris–HCl pH 9.5, 0.1 M NaCl, 5 mM MgCl2). Blots were developed with BCIP/NBT color substrate (Promega).
Construction of a tertiary model for GALNS protein
A tertiary model of the human GALNS protein was constructed by homology modeling using our original programs: a loop search method for the backbone structure (43), a dead-end elimination method for side-chain conformation (44) and conformation energy minimization for structure refinement (45), using the AMBER force field (46). Human 4S (36) and human ASA (37) were used as templates for construction of a model.
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ACKNOWLEDGEMENTS |
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We thank Dr M. Onozuka and M. Ohara for helpful comments and M. Yamada for technical assistance. This study was supported in part by a Grant-in Aid for Scientific Research 10670720, from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Ministry of Health and Welfare of Japan.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 58 265 1241; Fax: +81 58 265 9011; Email: sukegawa@cc.gifu-u.ac.jp
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REFERENCES |
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  S Tomatsu, T Nishioka, A M Montano, M A Gutierrez, O S Pena, K O Orii, W S Sly, S Yamaguchi, T Orii, E Paschke, et al. Mucopolysaccharidosis IVA: identification of mutations and methylation study in GALNS gene J. Med. Genet., July 1, 2004; 41(7): e98 - e98. [Full Text] [PDF]
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