Journal of Molecular Biology
Volume 423, Issue 5, 9 November 2012, Pages 736-751
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The Structure of Human GALNS Reveals the Molecular Basis for Mucopolysaccharidosis IV A

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Abstract

Lysosomal enzymes catalyze the breakdown of macromolecules in the cell. In humans, loss of activity of a lysosomal enzyme leads to an inherited metabolic defect known as a lysosomal storage disorder. The human lysosomal enzyme galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.C. 3.1.6.4) is deficient in patients with the lysosomal storage disease mucopolysaccharidosis IV A (also known as MPS IV A and Morquio A). Here, we report the three-dimensional structure of human GALNS, determined by X-ray crystallography at 2.2 Å resolution. The structure reveals a catalytic gem diol nucleophile derived from modification of a cysteine side chain. The active site of GALNS is a large, positively charged trench suitable for binding polyanionic substrates such as keratan sulfate and chondroitin-6-sulfate. Enzymatic assays on the insect‐cell-expressed human GALNS indicate activity against synthetic substrates and inhibition by both substrate and product. Mapping 120 MPS IV A missense mutations onto the structure reveals that a majority of mutations affect the hydrophobic core of the structure, indicating that most MPS IV A cases result from misfolding of GALNS. Comparison of the structure of GALNS to paralogous sulfatases shows a wide variety of active‐site geometries in the family but strict conservation of the catalytic machinery. Overall, the structure and the known mutations establish the molecular basis for MPS IV A and for the larger MPS family of diseases.

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

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