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Insights into Krabbe disease from structures of galactocerebrosidase.

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  • 1. Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, United Kingdom.
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    • Deane JE 1
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Proceedings of the National Academy of Sciences of the United States of America, 29 Aug 2011, 108(37):15169-15173
https://doi.org/10.1073/pnas.1105639108 PMID: 21876145 PMCID: PMC3174575

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A comment on this article appears in "Sneak peak at galactocerebrosidase, Krabbe disease's lysosomal hydrolase." Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15017-8.

Abstract 

Krabbe disease is a devastating neurodegenerative disease characterized by widespread demyelination that is caused by defects in the enzyme galactocerebrosidase (GALC). Disease-causing mutations have been identified throughout the GALC gene. However, a molecular understanding of the effect of these mutations has been hampered by the lack of structural data for this enzyme. Here we present the crystal structures of GALC and the GALC-product complex, revealing a novel domain architecture with a previously uncharacterized lectin domain not observed in other hydrolases. All three domains of GALC contribute residues to the substrate-binding pocket, and disease-causing mutations are widely distributed throughout the protein. Our structures provide an essential insight into the diverse effects of pathogenic mutations on GALC function in human Krabbe variants and a compelling explanation for the severity of many mutations associated with fatal infantile disease. The localization of disease-associated mutations in the structure of GALC will facilitate identification of those patients that would be responsive to pharmacological chaperone therapies. Furthermore, our structure provides the atomic framework for the design of such drugs.

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Proc Natl Acad Sci U S A. 2011 Sep 13; 108(37): 15169-15173.
Published online 2011 Aug 29. https://doi.org/10.1073/pnas.1105639108
PMCID: PMC3174575
PMID: 21876145
From the Cover

Insights into Krabbe disease from structures of galactocerebrosidase

Janet E. Deane,a,1 Stephen C. Graham,b Nee Na Kim,c Penelope E. Stein,c Rosamund McNair,c M. Begoña Cachón-González,c Timothy M. Cox,c and Randy J. Reada,1
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Abstract

Krabbe disease is a devastating neurodegenerative disease characterized by widespread demyelination that is caused by defects in the enzyme galactocerebrosidase (GALC). Disease-causing mutations have been identified throughout the GALC gene. However, a molecular understanding of the effect of these mutations has been hampered by the lack of structural data for this enzyme. Here we present the crystal structures of GALC and the GALC-product complex, revealing a novel domain architecture with a previously uncharacterized lectin domain not observed in other hydrolases. All three domains of GALC contribute residues to the substrate-binding pocket, and disease-causing mutations are widely distributed throughout the protein. Our structures provide an essential insight into the diverse effects of pathogenic mutations on GALC function in human Krabbe variants and a compelling explanation for the severity of many mutations associated with fatal infantile disease. The localization of disease-associated mutations in the structure of GALC will facilitate identification of those patients that would be responsive to pharmacological chaperone therapies. Furthermore, our structure provides the atomic framework for the design of such drugs.

Keywords: glycoside hydrolase family 59, glycosyl hydrolase

Krabbe disease, also known as globoid-cell leukodystrophy, is an autosomal recessive neurodegenerative disorder caused by a deficiency of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.1.46) (1). Defects in GALC that result in reduction of enzyme activity lead to the accumulation of the cytotoxic metabolite psychosine, resulting in demyelination due to the apoptosis of myelin-forming cells (2, 3). Most patients with Krabbe disease suffer from rapidly progressive leukoencephalopathy with onset before 6 mo of age and death occurring before the age of 2 y (4). Rare attenuated variants of Krabbe disease include late-infantile, juvenile, and adult presentations with disease severity and progression being highly variable. The prevalence of Krabbe disease is approximately 1 in 100,000 births, but there is wide variation among countries. Two genetically homogenous communities have an extremely high prevalence of infantile Krabbe disease with about 1 in 100–150 live births (5).

Sequence characterization of the human GALC gene and the discovery of the naturally occurring Twitcher mouse model of the disease have contributed to the molecular understanding of Krabbe disease (6–9). GALC is produced and glycosylated in the endoplasmic reticulum (ER)-Golgi complex after which it is trafficked, via the mannose-6-phosphate (M6P) pathway, to the lysosome (10). GALC is essential for normal catabolism of galactosphingolipids, including the principal lipid component of myelin, galactocerebroside (10). Sphingolipid degradation requires the combined action of water-soluble hydrolases and nonenzymatic sphingolipid activator proteins known as saposins (11, 12).

More than 70 mutations in the GALC gene have been associated with severe clinical phenotypes and these mutations are distributed throughout the sequence of GALC. The mutational profile of patients is highly heterogeneous, often occurring in compound heterozygote patterns, such that the course of the disease cannot easily be predicted from the genotype at the GALC locus (13–15). As with many other lysosomal storage diseases, Krabbe patients with similar or identical genotypes can have varied clinical presentations and course of their disease (4).

Here we present the structure of mouse GALC (83% identity with human GALC, Fig. S1) alone and in complex with d-galactose. These structures reveal the presence of a unique domain arrangement, identify the nature of substrate binding and catalysis, and provide insight into the differing roles of mutations occurring in human Krabbe disease variants.

Results

Crystal Structure of GALC.

The crystal structure of GALC refined to 2.1-Å resolution contains one molecule per asymmetric unit comprising residues 25 to 668 (Fig. 1 and Table S1); GALC is the archetype of the carbohydrate-active enzymes (CAZy) glycoside hydrolase family 59 (GH59). The overall fold comprises three domains: a central triosephosphate isomerase (TIM) barrel, a β-sandwich domain, and a lectin domain (Fig. 1). The first 24 residues of GALC encode the signal peptide for targeting to the ER that in our construct is replaced with the sequence required for secretion by HEK293 cells and is cleaved during secretion (as occurs with the wild-type signal peptide in vivo). The structure of GALC reveals that residues 25–40 contribute two β-strands to the β-sandwich domain before forming the (β/α)8 TIM barrel (residues 41–337) that lies at the center of the structure. Residues 338–452 then form the remainder of the β-sandwich domain. Residues 453–471 lie across one face of the structure stretching from the β-sandwich to the lectin domain (residues 472–668). The interfaces formed between each of the domains of GALC are very large: 22% (1,770 Å2) and 15% (1,480 Å2) of the solvent accessible surface areas of the β-sandwich domain and the lectin domain, respectively, are buried in their interfaces with the TIM barrel. Thus, the domains of GALC are not likely to move relative to each other.

An external file that holds a picture, illustration, etc. Object name is pnas.1105639108fig1.jpg

Structure of GALC shown in two orthogonal views. Ribbon diagram of GALC colored by domain: β-sandwich (red), TIM barrel (blue), linker (orange), and lectin domain (green). The disulfide bond (yellow spheres), calcium ion (gray sphere), and glycosylation moieties (violet sticks) are shown. The N and C termini are marked by labeled circles (Top).

The central TIM barrel is composed of eight parallel β-strands surrounded by α-helices. The connecting loops on the C-terminal side of the barrel (proximal to the lectin domain) are generally longer than those on the opposite side of the barrel. The β-sandwich domain comprises two twisted β-sheets with a similar topology to that seen in other glycosyl hydrolases except for a very long loop that wraps over the top of the TIM barrel. A cysteine residue in this loop, C378, forms a disulfide bridge with C271 in the TIM barrel. A calcium ion is bound in the lectin domain of GALC in a pentagonal bipyramidal configuration (Fig. S2). The lectin domain of GALC possesses a similar fold and calcium-binding site to that of β-glucanase (PDB ID code 3d6e, rmsd = 2.38 Å over 155 Cα atoms). However, the lectin domain of GALC does not possess the catalytic residues present in the β-glucanase family. The lectin domain of GALC also possesses structural similarity to galectin proteins (PDB ID code 2yv8, rmsd = 2.45 Å over 129 Cα atoms) and other carbohydrate-binding lectins. Residues N284, N363, N387, and N542 displayed electron density for glycosylation moieties, and these have been modeled in the GALC structure (Fig. 1). No ordered electron density was observed for glycosylation at additional predicted sites in the lectin domain (N585 and N629).

Substrate Specificity.

GALC catalyses the hydrolysis of the galactosyl moiety from glycosphingolipids such as galactocerebroside and psychosine (Fig. 2A). To better understand the substrate binding of GALC, we solved the structure of the enzyme in complex with d-galactose to 2.4-Å resolution (Fig. 2B and Table S1). The overall fold of GALC is unchanged upon galactose binding (rmsd = 0.17 Å over 641 Cα atoms), the core of the binding pocket being formed by the long loops on the C-terminal face of the TIM barrel as has been observed for other enzymes containing TIM barrels. However, in our structure we see that loops from both the β-sandwich and lectin domains also contribute to the substrate-binding pocket (Fig. 2C). The position and orientation of galactose in the active site is unambiguous as shown by the characteristic shape of the difference density calculated before inclusion of galactose in the model (Fig. 2D). The interactions and atomic distances between atoms of galactose and GALC identify E258 as the active site nucleophile and E182 as the proton donor (Fig. 2E). The average distance between the carboxyl oxygens of these catalytic residues is 5.0 Å, consistent with the retaining mechanism of enzymatic glycosidic bond hydrolysis in which the product retains the same stereochemistry as the substrate (16).

An external file that holds a picture, illustration, etc. Object name is pnas.1105639108fig2.jpg

Substrate-binding site of GALC. (A) Schematic diagram of the GALC substrate galactocerebroside. (B) Ribbon diagram of GALC illustrating bound galactose (pink sticks) with domains colored as in Fig. 1. (C) Surface representation of GALC zoomed in on the galactose-binding site, colored and oriented as in B. (D) Unbiased difference (FO-FC) electron density (green mesh) corresponding to galactose bound to GALC. (E) Active site of GALC illustrating the relevant atomic distances (purple dashed lines) between the bound galactose (pink sticks) and the proposed catalytic residues: the nucleophile (E258, blue sticks) and the proton donor (E182, blue sticks). (F) Substrate specificity for galactose- (pink sticks) rather than glucose- (yellow sticks) containing glycolipids is conferred by W291 and T93 (blue sticks).

In addition to the catalytic glutamates E182 and E258, several residues contribute to the formation of the substrate-binding pocket and form hydrogen bonds with the bound galactose molecule (Fig. S3). The position and orientation of galactose in the GALC active site differs from that seen in other galactose-containing hydrolase structures, such as acid α-galactosidase (Fig. S4), but is similar to the position of glucose analogues in glucocerebrosidase (GlcCerase, also known as acid β-glucosidase), the hydrolase responsible for cleaving glucose-containing sphingolipids (17, 18). Our structure reveals how GALC specifically recognizes galactose-containing lipids. Galactose and glucose differ only in the position of a hydroxyl group at one carbon (C4, Fig. 2F). In the galactose-bound structure of GALC, this hydroxyl forms a hydrogen bond with T93: a residue with unusual backbone dihedral angles ([var phi] = 139°, ψ = -48°) that are maintained in the absence of galactose, indicating this residue confers specificity rather than undergoing an induced fit. Glucose is not compatible with the substrate-binding pocket as the position of its hydroxyl group would clash with residue W291. The conformation of W291 is stabilized by a hydrogen bond with the backbone carbonyl of G47. Interestingly, other lysosomal glycosyl hydrolases, including GlcCerase and β-glucuronidase, conserve a tryptophan residue at this position but in a different conformation that is incompatible with the structure of GALC (Fig. S5). The binding of galactose is further stabilized by hydrogen bonding to R380 at the tip of the long loop that stretches from the β-sandwich domain. This loop is secured in position by the disulfide bond formed between C271 in the TIM barrel and C378 in the loop. The position of the side chain of R380 alters slightly upon galactose binding to accommodate the ligand.

Discussion

Substrate Binding.

The galactose-bound structure of GALC reveals that the active site pocket accommodates only the galactose moiety and does not have space for the lipid tails that are present in the natural substrates (Fig. 2 A and C). Analysis of the loops surrounding the active site suggests that GALC is unlikely to undergo a conformational change similar to that described for GlcCerase in order to accommodate substrate (Fig. S6) (18, 19). The scissile bond points out toward the surface of the protein but in order to gain access to the membrane-embedded substrate, glycolipid hydrolases require saposins (saps) (20). GALC-mediated degradation of galactolipids requires the activity of sapA (21–23) and possibly also sapC (21, 22, 24). Two mechanisms of action for saposins have been postulated. The first suggests saposins extract the lipids from bilayers to form water-soluble lipid–protein complexes that present the substrate to the appropriate enzyme (11). The second involves the binding of enzyme at the bilayer surface where saposin molecules facilitate the access to substrate by distorting the bilayer (12, 25). During GALC activation it is possible that both sapA and sapC will be involved via these different mechanisms. Indeed, it has been shown for GlcCerase that both sapA and sapC can activate the enzyme and that they mediate their activating effects by binding to distinct sites on the enzyme (26). It has been noted previously that saposins possess a highly negatively charged surface (27) and that in the case of sapC this needs to be partially neutralized to trigger membrane binding (28). Our structure reveals that the surface charge of GALC at pH 4.8, equivalent to the acidified lysosome, is +10 e- with a very large, highly positively charged patch surrounding the substrate-binding site (Fig. S7). As this patch is near to the substrate-binding pocket and possesses a surface charge that would be compatible with sapC or negatively charged lipid binding, it is likely to be the site of their association. Interestingly, human GlcCerase, which also binds sapC, possesses a similar surface charge distribution but without conserving the residues that confer this charge (Fig. S7).

Disease-Associated Mutations of GALC.

Mutations have been identified in the GALC gene that affect gene splicing and mRNA stability or cause deletions, frameshifts, and missense mutations (5, 13–15, 29–34). A large proportion of the missense mutations in GALC that cause Krabbe disease are likely to result in protein mistargeting or premature degradation (33). Mistargeting could occur at several stages in the GALC processing pathway, including failure to transit from the ER to Golgi due to misfolding, and blockage of trafficking to the lysosome due to altered binding to the M6P receptor. In addition, if mutations affect the binding of GALC to essential activating factors in the lysosome (including saposins) then, despite being correctly processed and localized, GALC may not be able to access substrate for efficient glycosphingolipid cleavage.

The structure of GALC provides detailed insight into the role of many disease-associated mutations. Nearly 70% of the missense mutations that result in Krabbe disease involve the modification of residues that are buried within the structure of the enzyme (Table S2). These mutations will lead to instability or misfolding of GALC resulting in the premature degradation of the enzyme. Examples of such mutations are not restricted to the core TIM barrel domain but can be found in each domain of GALC. Mutations that are likely to result in severe misfolding include E114K and S257F in the TIM barrel, L364R and W410G in the β-sandwich domain, and G537R and L629R in the lectin domain (Fig. 3 and Table S2). Previous work has shown that GALC possessing this last mutation, L629R, does indeed accumulate in the ER and is not secreted by cells or detected in the lysosome (33). Misfolding or destabilizing mutations of GALC do not necessarily result in an enzyme that is catalytically inactive. Several disease-associated mutations that are completely buried in the structure have been shown to retain residual enzyme activity (Table S2) (14, 15, 33, 35, 36). In those cases where the mutated form of GALC is trapped in the ER but still capable of cleaving substrate the use of pharmacological chaperone therapy (PCT) would be clinically relevant. PCT involves the use of small molecules that stabilize the enzyme structure allowing it to escape the ER, thus correcting the trafficking defect. Pharmacological chaperones have been identified for similar lysosomal storage diseases such as Fabry, Gaucher, and Pompe diseases and are now being translated into clinical applications (37).

An external file that holds a picture, illustration, etc. Object name is pnas.1105639108fig3.jpg

Disease-associated mutations of GALC. A selection of residues that are mutated in Krabbe disease are shown as sticks (beige) on the structure of GALC. For each residue a relevant view is provided (Insets) illustrating the surrounding region of the structure that would be perturbed by the mutation. Because of the insertion of a residue in the human sequence at position 507, residues 528 and 629 correspond to 527 and 628, respectively, in the mouse structure.

Within the ER-Golgi complex GALC undergoes modification by the addition of glycans to specific asparagine residues. This N-linked glycosylation is essential for the correct trafficking of GALC including binding to the M6P receptor (36). A common mutation found in Krabbe disease patients of Arab ancestry is D528N (5), which has been shown to introduce a new glycosylation site into GALC (33). The mutant GALC protein is hyperglycosylated, not efficiently taken up by cells and not present in the lysosome (33). D528 lies on a loop of the lectin domain with the side chain buried in the interface with the TIM barrel (Fig. 3). Mutation to asparagine will not only alter the glycosylation state of GALC but will destabilize this part of the structure.

Some disease-causing mutations may not affect the residual enzyme activity, processing, or localization of GALC, but may interfere with binding to activating factors. Consistent with this hypothesis, the disease-associated mutation E215K has been shown to have a very mild effect on GALC enzymatic activity (29), and our structure reveals that it is exposed on the surface of the TIM barrel (Fig. 3). This mutation confers an opposite charge on the same face as the substrate-binding pocket suggesting that the mechanism of disease for this mutation will involve the perturbation of a binding face for an activating factor, such as a saposin, that is essential for efficient in vivo glycolipid metabolism. Similarly, residue P302 that is found on the surface of GALC very close to the substrate-binding pocket is mutated to arginine in Krabbe disease, a mutation that would significantly alter the surface properties in this region (Fig. 3).

Perhaps the most unexpected observation from the structure of GALC is the contribution from the β-sandwich domain of a long loop that forms an integral part of the substrate-binding site. R380 at the tip of this loop directly binds the galactose molecule in the active site (Fig. 3). The importance of this residue for GALC activity is confirmed by its mutation to tryptophan or leucine leading to severe infantile Krabbe disease (13, 38).

Role of the Lectin Domain.

A striking feature of the GALC structure is the presence of the lectin domain sitting over one face of the TIM barrel. Although several lysosomal enzymes possess TIM barrel and β-sandwich domains similar to that seen in GALC, the presence of a lectin domain is unique. This domain contributes directly to the formation of the substrate-binding cleft (Fig. 2C) and the presence of several disease-causing mutations in this domain highlights its importance in the normal processing and activity of GALC (Fig. 3 and Table S2). It is likely that the lectin domain will be involved in processes that are not found in related enzymes lacking this domain (18). For example, the lectin domain is unlikely to be involved in saposin binding as GlcCerase, which does not possess the lectin domain, retains both the binding of sapA and sapC as activators (26, 39) and the positively charged patch on the TIM barrel near the active site (Fig. S7). The lectin fold is almost universally a carbohydrate recognition domain (40), suggesting that this region of GALC may play a role via carbohydrate binding. This activity may involve binding of glycosylated proteins during the processing and trafficking of GALC. In support of this, one family of lectins known as galectins, to which the GALC lectin domain possesses structural homology, has identified roles in protein trafficking and sorting into specific vesicle populations (41). It has been noted previously that GALC may be taken up by neighboring cells via M6P-independent mechanisms (13). In cultured skin fibroblasts the uptake of GALC by untransduced cells is inhibited less than 50% by M6P, indicating that the uptake is not solely via the M6P receptor-mediated pathway. It is possible that the lectin domain of GALC is responsible for conferring this additional uptake mechanism. However, we note that other lysosomal enzymes also undergo M6P-independent trafficking via mechanisms that do not require the presence of a lectin domain (42).

Cleavage of GALC.

Many studies of GALC have focused on the processing of GALC into 50- and 30-kDa fragments following uptake into the lysosome (6, 10, 43, 44). The cleavage site lies within a loop of the β-sandwich domain, far from the active site. Following cleavage it is extremely unlikely that the two fragments would dissociate given the very large buried surface area between them. We therefore conclude that this cleavage event has no relevance for enzyme activity, consistent with the previous identification of activity of the 80-kDa “precursor” (33). Thus, it is incorrect targeting, with the side effect of failure to encounter processing enzymes, rather than lack of cleavage per se that is likely to be the critical deficit in mutant forms of GALC that are not cleaved.

The structure of GALC has provided essential insights into the role of disease-associated mutations in the pathogenesis of Krabbe disease. GALC is now seen to possess a unique domain arrangement, including a previously uncharacterized lectin domain and a substrate-binding site that involves each of the three domains of the protein. This structure provides a rich framework for further understanding of Krabbe disease and the development of potential therapeutics.

Materials and Methods

His-tagged mouse GALC was produced recombinantly in HEK293T cells and purified from the supernatant using cobalt-affinity chromatography. The protein was crystallized at 4 mg/mL using sitting-drop vapor diffusion with microseeding (45) against a reservoir of 0.2 M sodium acetate, 0.1 M sodium cacodylate, 34% PEG 8000. Diffraction data were recorded at Diamond Light Source beamline I03. The structure was solved by MIRAS using AutoSol (46) with data from Mercury and Platinum derivatives. For the product complex crystals were soaked with 20 mM d-galactose for 45 min before data collection. The structure was built using ARP/wARP (47) and COOT (48) and refined using phenix.refine (49). Detailed methods, data collection, and refinement statistics are provided in SI Materials and Methods.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank the staff of beamline I03 at the Diamond Light Source. J.E.D. is funded by a Wellcome Trust grant (to R.J.R.). S.C.G. is an 1851 Research Fellow. N.N.K. was supported by a grant from the Jean Shanks Foundation and the Cambridge Overseas Trust. M.B.C.-G. was funded by the Hunter’s Hope Foundation and the Biomedical Research Centre of the National Institute of Health Research.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3zr5 and 3zr6).

See Commentary on page 15017.

This article contains supporting information online at www.pnas.org/lookup/suppl/10.1073/pnas.1105639108/-/DCSupplemental.

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