Journal of Molecular Biology
The Structure of Human GALNS Reveals the Molecular Basis for Mucopolysaccharidosis IV A
Graphical Abstract
Highlights
► We have determined the crystal structure of human GALNS. ► The structure reveals the molecular basis of MPS IV A (Morquio A) disease. ► The structure shows posttranslational modification of an active‐site cysteine. ► The structure reveals that MPS IV A (Morquio A) is a protein-folding disease. ► Human GALNS is a promising target for rational drug design.
Introduction
Lysosomal enzymes are responsible for the catabolism of biomolecules. Deficiencies in lysosomal enzymes result in accumulation of undegraded substrates in tissues, leading eventually to lysosomal storage diseases. The human lysosomal galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.C. 3.1.6.4) removes sulfate groups from a terminal N-acetylgalactosamine-6-sulfate (or galactose-6-sulfate) in mucopolysaccharides such as keratan sulfate and chondroitin-6-sulfate (Fig. 1a and b). Defects in GALNS lead to accumulation of substrates, resulting in the development of the lysosomal storage disease mucopolysaccharidosis IV A (also known as MPS IV A and Morquio A disease).2
The autosomal recessive MPS IV A affects approximately 1 in 200,000 live births and is characterized by a wide variety of symptoms, including severe skeletal abnormalities, hearing loss, corneal clouding, heart valve disease, and other impairments.3 The disease presents two phenotypes depending on its severity: a mild form, which generally allows the patient a full life span, and a severe form, which often results in death before the second decade of life.3 However, the correlation between genotype and phenotype is not fully understood. At present, 157 different mutations have been identified in the GALNS gene in patients with MPS IV A, 120 of which are missense mutations leading to a change of a single side‐chain residue in the protein.4 While there is no currently approved treatment for MPS IV A, enzyme replacement therapy, where patients are injected weekly with recombinant enzyme, is in phase III clinical trials.5
Despite the intense clinical interest in GALNS and the large amounts of purified protein available for decades, to date, there has been no structure of the protein reported. GALNS was first purified from human placenta in 19796 and later was isolated from liver cells7 and fibroblasts.[8], [9] The recombinant enzyme used in clinical trials is purified from Chinese hamster ovary cells.10 Because of the importance of GALNS and its relationship to disease, those interested in the structure have resorted to homology modeling11 using the best available paralogs, the 36% and the 28% identical human arylsulfatase A12 (ASA) and arylsulfatase B13 (ASB) structures.
In general, sulfatases catalyze the hydrolysis of sulfate ester bonds from a diverse range of substrates. In vitro, most sulfatases can hydrolyze synthetic substrates such as 4-methylumbelliferyl sulfate (4-MU-S), which shares only a sulfate group with native substrates. Therefore, determining native substrate specificity for sulfatases is nontrivial.
Sulfatases require a modified amino acid nucleophile for catalytic function. The polypeptide encodes a cysteine (or occasionally serine), which is then enzymatically converted to a formylglycine aldehyde by formylglycine-generating enzyme (FGE).[14], [15], [16], [17] The ubiquitous FGE recognizes a specific sequence motif (typically CXPXR),[18], [19] and oxidative desulfurization of the side chain leads to an aldehyde.[20], [21] Hydration of the aldehyde leads to a dihydroxyalanine (DHA) nucleophile with geminal hydroxy groups.[22], [23], [24]
To elucidate the molecular basis for MPS IV A, we determined the structure of human GALNS to 2.2 Å resolution. Using stably transfected insect cells, we expressed human GALNS glycoprotein and demonstrated enzyme activity comparable to endogenous enzyme. Using X-ray crystallography and mass spectrometry, we identified FGE modification of the GALNS nucleophile in our insect cell expression system. To extend our structural knowledge about GALNS to the effects seen in patients with MPS IV A, we mapped the disease-causing mutations onto the structure. The mutations fall into three categories: disruption of the active site, perturbation of overall fold, and surface exposure, suggesting potential treatments depending on a patient's genotype. Overall, these results will improve the understanding of the molecular defects in MPS IV A and will provide insight into lysosomal storage diseases and other protein‐folding diseases.
Section snippets
Protein expression
We purified GALNS from Trichoplusia ni (Tn5) insect cells, using both baculovirus-infected and stable cell line approaches. The protein sequence contained the native signal sequence for secretion and a C-terminal hexahistidine tag for purification. Baculovirus-infected cells yielded about 0.2 mg of purified GALNS per liter of culture while stably transfected cell lines yielded approximately 0.5 mg/L. Monoclonal selection of the stable cell lines increased expression to 0.7 mg/L culture.
Overall description of the structure
The
Discussion
Many nearly identical carbohydrate-containing substrates appear in the lysosome. For example, glucosides and galactosides differ by a single chiral center (at the 4‐position of the sugar ring), α- and β-sugars differ by a single chiral configuration at the anomeric center, and so on. In response, lysosomal glycosidases have evolved exquisite specificity for their substrates, as they are able to distinguish among the many similar substrates differing by a single chiral center. In contrast,
Molecular biology
Human GALNS cDNA (National Center for Biotechnology Information Sequence ID: NM_000512.4) was purchased from Open Biosystems. The open reading frame, including the 26-residue native signal sequence (for secretion into the media) and a C-terminal hexahistidine affinity tag, was amplified by PCR using Hot Start Phusion polymerase (NEB). For transfection into insect cells, the PCR product was gel purified, incubated with Taq polymerase to add 3′ A-overhangs, and cloned into the pIB/V5-His-TOPO TA
Acknowledgements
This work was funded by National Institutes of Health grant R01 DK76877 to S.C.G., by National Institutes of Health grant T32 GM008515 to Y.R.-C., and by a Howard Hughes Medical Institute summer research internship to E.K.S. We gratefully acknowledge Igor Kaltashov for assistance with mass spectrometry and Matthew C. Metcalf and Elih M. Velázquez-Delgado for assistance with X-ray experiments. We thank Jean Jankonic, Marc Allaire, and Vivian Stojanoff at the National Synchrotron Light Source X6A
References (66)
- et al.
Structure of a human lysosomal sulfatase
Structure
(1997) - et al.
The multiple sulfatase deficiency gene encodes an essential and limiting factor for the activity of sulfatases
Cell
(2003) - et al.
Multiple sulfatase deficiency is caused by mutations in the gene encoding the human Cα-formylglycine generating enzyme
Cell
(2003) - et al.
Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann–Pick C1 disease—lysosomal storage disorders caused by defects of non-lysosomal proteins
Biochim. Biophys. Acta
(2009) - et al.
Sulfotransferases, sulfatases and formylglycine-generating enzymes: a sulfation fascination
Curr. Opin. Chem. Biol.
(2008) - et al.
1.3 Å structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family
Structure
(2001) - et al.
Characterization and pharmacokinetic study of recombinant human N-acetylgalactosamine-6-sulfate sulfatase
Mol. Genet. Metab.
(2007) - et al.
Structure of human estrone sulfatase suggests functional roles of membrane association
J. Biol. Chem.
(2003) - et al.
Lectin–carbohydrate interactions: different folds, common recognition principles
Trends Biochem. Sci.
(1997) - et al.
A fluorimetric enzyme assay for the diagnoisis of Morquio disease type A (MPS IV A)
Clin. Chim. Acta
(1990)
Three-dimensional structures of sulfatases
Methods Enzymol.
Mucopolysaccharidosis type IV: N-acetylgalactosamine-6-sulfatase mutations in Tunisian patients
Mol. Genet. Metab.
GALNS gene expression profiling in Morquio A patients' fibroblasts
Clin. Chim. Acta
Lysosomal multienzyme complex: biochemistry, genetics, and molecular pathophysiology
Prog. Nucleic Acid Res. Mol. Biol.
Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of β-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate
J. Biol. Chem.
Enhancement of drug delivery: enzyme-replacement therapy for murine Morquio A syndrome
Mol. Ther.
Processing of X-ray diffraction data collected in oscillation mode
Crystal structure of a covalent intermediate of endogenous human arylsulfatase A
J. Inorg. Biochem.
POVScript+: a program for model and data visualization using persistence of vision ray-tracing
J. Appl. Crystallogr.
The mucopolysaccharidoses
Mucopolysaccharidosis type IVA (Morquio syndrome): a clinical review
J. Inherited Metab. Dis.
Mutation and polymorphism spectrum of the GALNS gene in mucopolysaccharidosis IVA (Morquio A)
Hum. Mutat.
Biomarker analysis of Morquio syndrome: identification of disease state and drug responsive markers
Orphanet J. Rare Dis.
Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta
Biochem. J.
Human liver N-acetylgalactosamine 6-sulphatase. Purification and characterization.
Biochem. J.
Different properties of residual N-acetylgalactosamine-6-sulfate sulfatase in fibroblasts from patients with mild and severe forms of Morquio disease type A
Pediatr. Res.
Impaired degradation of keratan sulphate by Morquio A fibroblasts
Biochem. J.
Enzyme replacement in a human model of mucopolysaccharidosis IVA in vitro and its biodistribution in the cartilage of wild type mice
PLoS One
Biochemical and structural analysis of missense mutations in N-acetylgalactosamine-6-sulfate sulfatase causing mucopolysaccharidosis IVA phenotypes
Hum. Mol. Genet.
Crystal structure of human arylsulfatase A: the aldehyde function and the metal ion at the active site suggest a novel mechanism for sulfate ester hydrolysis
Biochemistry
New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo
J. Am. Chem. Soc.
Site-specific chemical protein conjugation using genetically encoded aldehyde tags
Nat. Protoc.
A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme
Proc. Natl Acad. Sci. USA
Cited by (96)
Molecular Trojan Horses for treating lysosomal storage diseases
2023, Molecular Genetics and MetabolismBlood RNA alternative splicing events as diagnostic biomarkers for infectious disease
2023, Cell Reports MethodsRNA analysis of the GALNS transcript reveals novel pathogenic mechanisms associated with Morquio syndrome A
2022, Molecular Genetics and Metabolism ReportsClinical outcomes in elderly patients with Morquio a syndrome receiving enzyme replacement therapy - experience in a Colombian center
2020, Molecular Genetics and Metabolism ReportsCitation Excerpt :This molecule contains an oligosaccharide with a mannose 6-phosphate terminal which reaches the lysosome when taken up by the body, triggering the correct catabolism of chondroitin sulfate and keratan sulphate, thus avoiding their accumulation in the body as well as the complications of the disease. It is administered intravenously on a weekly basis at a dose of 2 mg/kg/week. [4,5] This molecule has been available since 2014, when approved by the European Medicine Agencies (EMA) and the Food and Drug Administration (FDA), for the treatment of mucopolysaccharidosis IVA; [20,23,24] in Colombia it was approved on March 2015. [30]
Structural analysis of the sulfatase AmAS from Akkermansia muciniphila
2021, Acta Crystallographica Section D: Structural Biology