Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH.

The Fe(2+)/α-ketoglutarate (αKG)-dependent oxygenases use molecular oxygen to conduct a wide variety of reactions with important biological implications, such as DNA base excision repair, histone demethylation, and the cellular hypoxia response. These enzymes follow a sequential mechanism in which O2 binds and reacts after the primary substrate binds, making those structural factors that promote productive O2 binding central to their chemistry. A large challenge in this field is to identify strategies that engender productive turnover. Factor inhibiting HIF (FIH) is a Fe(2+)/αKG-dependent oxygenase that forms part of the O2 sensing machinery in human cells by hydroxylating the C-terminal transactivation domain (CTAD) found within the HIF-1α protein. The structure of FIH was determined with the O2 analogue NO bound to Fe, offering the first direct insight into the gas binding geometry in this enzyme. Through a combination of density functional theory calculations, {FeNO}(7) electron paramagnetic resonance spectroscopy, and ultraviolet-visible absorption spectroscopy, we demonstrate that CTAD binding stimulates O2 reactivity by altering the orientation of the bound gas molecule. Although unliganded FIH binds NO with moderate affinity, the bound gas can adopt either of two orientations with similar stability; upon CTAD binding, NO adopts a single preferred orientation that is appropriate for supporting oxidative decarboxylation. Combined with other studies of related enzymes, our data suggest that substrate-induced reorientation of bound O2 is the mechanism utilized by the αKG oxygenases to tightly couple O2 activation to substrate hydroxylation.

Variations in the GLA gene correlate with globotriaosylceramide and globotriaosylsphingosine analog levels in urine and plasma.

Recent data have shown that lyso-Gb3, the deacylated derivative of globotriaosylceramide (Gb3), is possibly involved in the pathogenesis of Fabry disease (FD) and might be a clinically useful biomarker of its metabolic load. To test this hypothesis, we assayed Gb3 and lyso-Gb3 and related analogs in plasma and/or urine samples of 12 clinically well-characterized subjects carrying several different GLA variant alleles associated with a wide range of residual α-galactosidase A activities. Urinary Gb3 was measured by HPLC-MS/MS; plasma and urinary lyso-Gb3 and related analogs were measured by UPLC-MS/MS. Individual profiles of Gb3 and lyso-Gb3 and related analogs closely correlated with the phenotypic data for each subject, discerning the classical FD patient from the two patients carrying cardiac variants as well as those from all the others without FD. The lyso-Gb3 analog at m/z 836 was found at increased levels only in patients manifesting clinically severe heart disease, irrespective of the pathogenicity of the GLA variant they carried. This finding suggests that this lyso-Gb3 analog might be an earlier biomarker of progressive heart disease, non-specific of the FD cardiomyopathy. The possibility that urinary Gb3 is a specific marker of kidney involvement in FD deserves further study.

Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.

Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the NH group of the apical imidazole ligand from His1 and the Glu17 carboxylate from a neighboring subunit in the hexameric enzyme. This interaction is supported by another intrasubunit H-bond between Glu17 and Arg47. In this study, four mutant NiSOD proteins were produced to experimentally evaluate the roles of this H-bonding network and compare the results with prior predictions from density functional theory calculations. The X-ray crystal structure of H1A-NiSOD, which lacks the apical ligand entirely, reveals that in the absence of the Glu17-His1 H-bond, the active site is disordered. Characterization of this variant using X-ray absorption spectroscopy (XAS) shows that Ni(II) is bound in the expected N2S2 planar coordination site. Despite these structural perturbations, the H1A-NiSOD variant retains 4% of wild-type (WT) NiSOD activity. Three other mutations were designed to preserve the apical imidazole ligand but perturb the H-bonding network: R47A-NiSOD, which lacks the intramolecular H-bonding interaction; E17R/R47A-NiSOD, which retains the intramolecular H-bond but lacks the intermolecular Glu17-His1 H-bond; and E17A/R47A-NiSOD, which lacks both H-bonding interactions. These variants were characterized by a combination of techniques, including XAS to probe the nickel site structure, kinetic studies employing pulse-radiolytic production of superoxide, and electron paramagnetic resonance to assess the Ni redox activity. The results indicate that in addition to the roles in redox tuning suggested on the basis of previous computational studies, the Glu17-His1 H-bond plays an important structural role in the proper folding of the "Ni-hook" motif that is a critical feature of the active site.

The alpha-galactosidase A p.Arg118Cys variant does not cause a Fabry disease phenotype: data from individual patients and family studies.

Lysosomal α-galactosidase A (α-Gal) is the enzyme deficient in Fabry disease (FD), an X-linked glycosphingolipidosis caused by pathogenic mutations affecting the GLA gene. The early-onset, multi-systemic FD classical phenotype is associated with absent or severe enzyme deficiency, as measured by in vitro assays, but patients with higher levels of residual α-Gal activity may have later-onset, more organ-restricted clinical presentations. A change in the codon 118 of the wild-type α-Gal sequence, replacing basic arginine by a potentially sulfhydryl-binding cysteine residue - GLA p.(Arg118Cys) -, has been recurrently described in large FD screening studies of high-risk patients. Although the Cys118 allele is associated with high residual α-Gal activity in vitro, it has been classified as a pathogenic mutation, mainly on the basis of theoretical arguments about the chemistry of the cysteine residue. However its pathogenicity has never been convincingly demonstrated by pathology criteria. We reviewed the clinical, biochemical and histopathology data obtained from 22 individuals of Portuguese and Spanish ancestry carrying the Cys118 allele, including 3 homozygous females. Cases were identified either on the differential diagnosis of possible FD manifestations and on case-finding studies (n=11; 4 males), or on unbiased cascade screening of probands' close relatives (n=11; 3 males). Overall, those data strongly suggest that the GLA p.(Arg118Cys) variant does not segregate with FD clinical phenotypes in a Mendelian fashion, but might be a modulator of the multifactorial risk of cerebrovascular disease. The Cys118 allelic frequency in healthy Portuguese adults (n=696) has been estimated as 0.001, therefore not qualifying for "rare" condition.

Unlocking a caged lysosomal protein from a polymeric nanogel with a pH trigger.

A polymeric nanogel has been used to sequester and turn off a lysosomal protein, acid α-glucosidase (GAA). The nanogel contains a ß-thiopropionate cross-linker, which endows the nanogel with pH-sensitivity. While encapsulation of the enzyme fully turns off its activity, approximately 75% of the activity is recovered upon reducing the pH to 5.0. The recovered activity is ascribed to pH-induced degradation of the ß-thiopropionate cross-linker causing the swelling of the nanogel and ultimately causing the release of the enzyme. We envision that strategies for sequestering protein molecules and releasing them at lysosomal pH might open up new directions for therapeutic treatment of lysosomal storage diseases.

The structure of human GALNS reveals the molecular basis for mucopolysaccharidosis IV A.

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 2.2Å 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.

The molecular basis of pharmacological chaperoning in human α-galactosidase.

Fabry disease patients show a deficiency in the activity of the lysosomal enzyme α-galactosidase (α-GAL or α-Gal A). One proposed treatment for Fabry disease is pharmacological chaperone therapy, where a small molecule stabilizes the α-GAL protein, leading to increased enzymatic activity. Using enzyme kinetics, tryptophan fluorescence, circular dichroism, and proteolysis assays, we show that the pharmacological chaperones 1-deoxygalactonojirimycin (DGJ) and galactose stabilize the human α-GAL glycoprotein. Crystal structures of complexes of α-GAL and chaperones explain the molecular basis for the higher potency of DGJ over galactose. Using site-directed mutagenesis, we show the higher potency of DGJ results from an ionic interaction with D170. We propose that protonation of D170 in acidic conditions leads to weaker binding of DGJ. The results establish a biochemical basis for pharmacological chaperone therapy applicable to other protein misfolding diseases.

Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.

The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.22) and alpha-N-acetylgalactosaminidase (alpha-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of alpha- GAL and alpha-NAGAL. The engineered alpha-GAL (which we call alpha-GAL(SA)) retains the antigenicity of alpha-GAL but has acquired the enzymatic specificity of alpha-NAGAL. Conversely, the engineered alpha-NAGAL (which we call alpha-NAGAL(EL)) retains the antigenicity of alpha-NAGAL but has acquired the enzymatic specificity of the alpha-GAL enzyme. Comparison of the crystal structures of the designed enzyme alpha-GAL(SA) to the wild-type enzymes shows that active sites of alpha-GAL(SA) and alpha-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.

Catalytic mechanism of human alpha-galactosidase.

The enzyme alpha-galactosidase (alpha-GAL, also known as alpha-GAL A; E.C. 3.2.1.22) is responsible for the breakdown of alpha-galactosides in the lysosome. Defects in human alpha-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of alpha-galactosylated substrates in the tissues. alpha-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human alpha-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-alpha-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a (1)S(3) skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on alpha-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones.

Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin.

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.

Structure-function relationships in alpha-galactosidase A.

UNLABELLED: With recent interest in the molecular mechanisms responsible for Fabry disease, the number of known mutations in the GLA gene which encodes alpha-galactosidase A has expanded considerably. Combining a large database of Fabry disease-causing mutations with the recently determined crystal structure of human alpha-galactosidase A allows for a new understanding of the atomic defects in the protein responsible for Fabry disease. We have conducted a systematic survey of the known Fabry disease-causing mutations and analyzed the mutations in the context of the alpha-galactosidase A structure. We have applied quantitative methods for identifying the plausible effect of each mutation on the alpha-galactosidase A protein. We present the analysis of 331 different defects in the GLA gene leading to non-native proteins in patients with Fabry disease. These mutations include 278 missense mutations, 49 nonsense mutations, and four single amino acid deletions. CONCLUSION: Over half of the residues in the protein have been found to have changes in patients with Fabry disease. Most of these genetic mutations lead to disruption of the hydrophobic core of the protein, thus Fabry disease is primarily a disease of protein-folding. Further understanding of alpha-galactosidase A, one of the best studied members of the lysosomal storage disease family, will lead to increased understanding of other lysosomal storage diseases and other protein-folding diseases.

The 1.51-Angstrom structure of the poxvirus L1 protein, a target of potent neutralizing antibodies.

Although eradicated from nature more than two decades ago, the threat of smallpox has reemerged because of concerns over its use as a biological weapon. We present the structure of the poxvirus L1 protein, a molecule that is conserved throughout the poxvirus family and is nearly identical in vaccinia virus and in variola virus, which causes smallpox. L1 is a myristoylated envelope protein that is a potent target for neutralizing antibodies and an important component of current experimental vaccines. The L1 structure reveals a hydrophobic cavity located adjacent to its N terminus. The cavity would be capable of shielding the myristate moiety, which is essential for virion assembly. The structure of L1 is a step in the elucidation of molecular mechanisms common to all poxviruses that may stimulate the design of safer vaccines and new antipoxvirus drugs.

Pediatric Fabry disease.

BACKGROUND: Fabry disease is an underdiagnosed, treatable, X-linked, multisystem disorder. OBJECTIVES: To test the hypothesis that quality of life and sweating are decreased among pediatric patients with Fabry disease, compared with control subjects, and to provide quantitative natural history data and novel clinical end points for therapeutic trials. DESIGN: Prospective, cross-sectional, observational study. SETTING: Referral to the National Institutes of Health. PARTICIPANTS: Twenty-five male children with Fabry disease (mean age: 12.3 +/- 3.5 years) and 21 age-matched control subjects. MAIN OUTCOME MEASURES: Quality of life (measured with the Child Health Questionnaire) and sweating (assessed with the quantitative sudomotor axon reflex test). RESULTS: Quality of life scores for pediatric patients <10 years of age with Fabry disease, compared with published normative values, were 55 +/- 17 vs 83 +/- 19 for bodily pain and 62 +/- 19 vs 80 +/- 13 for mental health. Bodily pain scores for patients > or =10 years of age were 54 +/- 22 vs 74 +/- 23. Sweat volume in the Fabry disease group was 0.41 +/- 0.46 microL/mm2, compared with 0.65 +/- 0.44 microL/mm2 in the control group. Renal function, urinary protein excretion, and cardiac function and structure were normal for the majority of patients. The 3 patients with residual alpha-galactosidase A activity > or =1.5% of normal values were free of cornea verticillata and had normal serum and urinary globotriaosylceramide levels. All other children had glycolipid levels comparable to those of adult patients with Fabry disease. Acroparesthesia and cardiac abnormalities were generally present before anhidrosis and proteinuria. Mapping of the missense mutations on the crystallographic structure of alpha-galactosidase A revealed that the mutations were partially surface-exposed and distal to the active site among individuals with residual enzyme activity. Mutations associated with left ventricular hypertrophy (defined as left ventricular mass index of >51 g/m2.7) were localized near the catalytic site of the enzyme. CONCLUSIONS: Despite the absence of major organ dysfunction, Fabry disease demonstrates significant morbidity already in childhood. We have identified important, potentially correctable or preventable, outcome measures for future therapeutic trials. Prevention of complications involving major organs should be the goal for long-term specific therapy.

Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.

Understanding the mechanisms underlying the poor immunogenicity of human self/tumor antigens is challenging because of experimental limitations in humans. Here, we developed a human-mouse chimeric model that allows us to investigate the roles of the frequency and self-reactivity of antigen-specific T cells in determination of the immunogenicity of an epitope (amino acids 209-217) derived from a human melanoma antigen, gp100. In these transgenic mice, CD8+ T cells express the variable regions of a human T cell receptor (hTCR) specific for an HLA-A*0201-restricted gp100(209-217). Immunization of hTCR-transgenic mice with gp100(209-217) peptide elicited minimal T cell responses, even in mice in which the epitope was knocked out. Conversely, a modified epitope, gp100(209-217(2M)), was significantly more immunogenic. Both biological and physical assays revealed a fast rate of dissociation of the native peptide from the HLA-A*0201 molecule and a considerably slower rate of dissociation of the modified peptide. In vivo, the time allowed for dissociation of peptide-MHC complexes on APCs prior to their exposure to T cells significantly affected the induction of immune responses. These findings indicate that the poor immunogenicity of some self/tumor antigens is due to the instability of the peptide-MHC complex rather than to the continual deletion or tolerization of self-reactive T cells.

The molecular defect leading to Fabry disease: structure of human alpha-galactosidase.

Fabry disease is an X-linked lysosomal storage disease afflicting 1 in 40,000 males with chronic pain, vascular degeneration, cardiac impairment, and other symptoms. Deficiency in the lysosomal enzyme alpha-galactosidase (alpha-GAL) causes an accumulation of its substrate, which ultimately leads to Fabry disease symptoms. Here, we present the structure of the human alpha-GAL glycoprotein determined by X-ray crystallography. The structure is a homodimer with each monomer containing a (beta/alpha)8 domain with the active site and an antiparallel beta domain. N-linked carbohydrate appears at six sites in the glycoprotein dimer, revealing the basis for lysosomal transport via the mannose-6-phosphate receptor. To understand how the enzyme cleaves galactose from glycoproteins and glycolipids, we also determined the structure of the complex of alpha-GAL with its catalytic product. The catalytic mechanism of the enzyme is revealed by the location of two aspartic acid residues (D170 and D231), which act as a nucleophile and an acid/base, respectively. As a point mutation in alpha-GAL can lead to Fabry disease, we have catalogued and plotted the locations of 245 missense and nonsense mutations in the three-dimensional structure. The structure of human alpha-GAL brings Fabry disease into the realm of molecular diseases, where insights into the structural basis of the disease phenotypes might help guide the clinical treatment of patients.

Structural basis of Fabry disease.

Fabry disease is a lysosomal storage disease caused by deficiency in the enzyme alpha-galactosidase (alpha-GAL). To understand the molecular defects responsible for Fabry disease, we have collected more than 190 reported point and stop mutations and mapped them onto a model of human alpha-GAL based on the X-ray structure of the closely related enzyme alpha-N-acetylgalactosaminidase (alpha-NAGAL). The locations of the human alpha-GAL point mutations reveal two major classes of Fabry disease protein defects: active site mutations and folding mutations. Active site mutations reduce enzymatic activity by perturbing the active site without necessarily affecting the overall alpha-GAL structure. Folding mutations reduce the stability of alpha-GAL by disrupting its hydrophobic core. Examining the frequency of mutation around each alpha-GAL residue identifies the active site as a hotspot for mutations leading to Fabry disease. This study furthers our understanding of the structural basis for mutations leading to Fabry disease, from which new avenues for the treatment of lysosomal storage diseases may be developed.