Molecular Basis of GM1 Gangliosidosis and Morquio Disease, Type B. Structure^Function Studies of Lysosomal L-Galactosidase and T

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Review Molecular basis of GM1 gangliosidosis and Morquio disease, type B. Structure^function studies of lysosomal L-galactosidase and the non-lysosomal L-galactosidase-like protein

John W. Callahan *

Structural Biology and Biochemistry, The Research Institute and Genetic-Metabolic Laboratory, Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, and the Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, ON M5G 1X8, Canada

Received 14 January 1999; received in revised form 13 April 1999; accepted 13 April 1999

Abstract

GM1 gangliosidosis and Morquio B disease are distinct disorders both clinically and biochemically yet they arise from the same L-galactosidase enzyme deficiency. On the other hand, galactosialidosis and sialidosis share common clinical and biochemical features, yet they arise from two separate enzyme deficiencies, namely, protective protein/cathepsin A and neuraminidase, respectively. However distinct, in practice these disorders overlap both clinically and biochemically so that easy discrimination between them is sometimes difficult. The principle reason for this may be found in the fact that these three enzymes form a unique complex in lysosomes that is required for their stability and posttranslational processing. In this review, I focus mainly on the primary and secondary L-galactosidase deficiency states and offer some hypotheses to account for differences between GM1 gangliosidosis and Morquio B disease. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: GM1 gangliosidosis; Morquio disease; Galactosialidosis; Sialidosis; L-Galactosidase; Cathepsin A; Neuraminidase

Contents

1. Introduction ...... 86

2. Primary L-galactosidase de¢ciency diseases ...... 86 2.1. The GM1-gangliosidoses ...... 86 2.2. Morquio disease, type B ...... 87

3. Secondary L-galactosidase de¢ciencies ...... 87 3.1. Galactosialidosis ...... 87 3.2. Sialidosis, a primary neuraminidase de¢ciency ...... 89

4. Storage substances ...... 89 4.1. GM1-gangliosidosis and Morquio disease, type B ...... 89 4.2. Galactosialidosis and sialidosis ...... 89

* Fax: +1-416-813-8700; E-mail: [email protected]

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5. L-Galactosidase and elastin binding protein arise from the Gal gene by alternate splicing . 90 5.1. Biosynthesis and processing of Gal ...... 90 5.2. Gal: substrates, the role of saposin B, and its active site ...... 91 5.3. S-Gal, a component of the elastin binding (EBP) receptor? ...... 93

6. Neuraminidase ...... 94

7. Protective protein/cathepsin A ...... 95

8. The `complexes' formed by Gal and S-Gal with protective protein/cathepsin A and neuraminidase ...... 95 8.1. The lysosomal complex ...... 95 8.2. The elastin binding receptor ...... 97

9. Genetic heterogeneity in GM1 gangliosidosis and Morquio disease, type B ...... 97

10. Pathophysiological bases of GM1 gangliosidosis and Morquio, type B: a hypothesis . . . . . 100

Acknowledgements ...... 101

References ...... 101

1. Introduction Gal works, the role of S-Gal, and the nature and role of the complexes formed with PPCA and Neurami- De¢ciency in lysosomal L-galactosidase activity is nidase. For completeness, a brief summary of the the primary defect in patients with GM1-gangliosi- current knowledge on galactosialidosis and sialidosis, dosis, and in Morquio disease, type B. Clinical het- the primary neuraminidase de¢ciency will be in- erogeneity is a common feature of lysosomal dis- cluded. eases, and GM1 gangliosidosis and Morquio disease, type B represent the extremes in the spec- trum of clinical phenotypes arising from mutations 2. Primary L-galactosidase de¢ciency diseases in the L-galactosidase gene. Two related proteins arise by alternate splicing of the primary L-galacto- 2.1. The GM1-gangliosidoses sidase transcript, the most abundant one being the lysosomal L-galactosidase (Gal). The other, called All patients with GM1-gangliosidosis have a the L-galactosidase-like protein (S-Gal), does not dis- marked de¢ciency (0^5% of normal) of lysosomal play L-galactosidase activity and its speci¢c biologi- Gal activity [1,2]. In 1959, Norman and associates cal action, tissue speci¢city and its role in the etiol- reported an infant having a cherry red spot in the ogy of these diseases is not well de¢ned. Gal activity fundus of the eye, with facial features reminiscent of is also decreased to very low levels in galactosialido- Hurler disease, but with no skeletal X-ray abnormal- sis, a disorder resulting from de¢ciency in cathepsin ity, with an enlarged liver, but no splenomegaly [3]. A/protective protein (PPCA), one important function The nerve cell bodies of the neurons were ballooned of which is to form a complex with Gal and a second and foam cells with highly vacuolated cytoplasm was enzyme, Neuraminidase, whereupon these enzymes noted in all visceral organs examined (liver, spleen, are stabilized, or `protected' from inappropriate ly- lymph gland, thymus, bone marrow, lung, pancreas, sosomal proteolysis but allowed to undergo the post- intestine, kidney, adrenal medulla, and thyroid). translational processing required for them to become Chemical analysis documented a 3-fold increase in mature functional enzymes. In this review, I attempt ganglioside sialic acid, but no further chemical char- to de¢ne the pathophysiological basis for GM1-gan- acterization was reported. Landing and co-workers gliosidosis and Morquio disease by examining how [4] described a series of eight clinically-similar pa-

BBADIS 61880 10-9-99 J.W. Callahan / Biochimica et Biophysica Acta 1455 (1999) 85^103 87 tients, in 1964, and from the ninth patient of the 2.2. Morquio disease, type B series, O'Brien et al. isolated the storage substance and showed it was GM1 ganglioside [5]. The ¢rst Classical Morquio disease is a rare autosomal re- indication of variable clinical expression was recog- cessive disorder characterized by massive spondylo- nized by Derry and co-workers, in 1968 [6], who epiphyseal dysplasia (short stature, sternal protru- reported a French-Canadian boy who was clinically sion, odontoid hypoplasia, severe genua valga), well and had displayed appropriate milestones up to normal intelligence and storage of keratan sulfate the age of 11 months, noticeably lost these over the (see Section 3 for structural properties). Two distinct next few months, and after a relentless downhill neu- forms are recognized, the most common of which, rodegenerative course, died at 4 years of age. Three type A, results from a de¢ciency of N-acetylgalactos- distinct phenotypic variants are now recognized ^ the amine-6-sulfate sulfatase. The ¢rst indication of clin- infantile form of Norman and Landing is now called ical heterogeneity in Morquio disease was a case of a type 1, the late infantile/juvenile form is called type 2 14-year-old girl of Italian parents who was described and a later onset adult, chronic form is type 3. Type with a 4-year course of increasing limitation of mo- 1 patients, who usually die before the age of 3 years, tion, coxa vara, £attening of the femoral heads, and display the coarse facial features, hepatosplenomeg- narrowing of the intervertebral spaces, £attening of aly and the skeletal dysmorphology reminiscent of the vertebral disks, and progressive dysplastic Hurler's disease, suggesting that if these patients changes [12]. Residual L-galactosidase activities in were to live longer their skeletal dysplasia would ¢broblasts were between 1.3 and 7.0% of the normal likely be worse than that seen in Morquio disease. with synthetic, and natural substrates. Although uri- Consistent with the extent of visceral organ involve- nary levels of keratan sulfate excretion were not ment, type 1 patients accumulate in these tissues and measured, Arbisser et al. [13] documented this in a ultimately excrete in their urine 30 or more L-linked second case in 1977, and con¢rmed the enzyme de¢- galactose-terminal oligosaccharides which arise from ciency. Later van Gemund et al. described three cases the lysosomal digestion of a variety of glycoproteins. in a single family [14], clearly establishing it as a On the other hand, patients with GM1 gangliosido- distinct variant and thereby enlarged the range of sis, type 2 are characterized primarily as neuronal clinical entities attributable to primary L-galactosi- lipidoses and while they do not display the skeletal dase de¢ciencies. Patients with Morquio disease usu- changes associated with type 1 they nonetheless ex- ally show normal intelligence in spite of neuropatho- crete elevated amounts of galactose-terminal oligo- logical evidence of abnormalities [15] but neither saccharides [6,7]. Neuropathological examination of keratan sulfate nor GM1-ganglioside appear to be long-lived, enzymatically-proven type 2 patients has stored in the brain [15]. Giugliani et al. [16] reported documented accumulation of ceroid lipofuscin mate- a brother and sister who displayed mental regression rial in addition to GM1 ganglioside in the brain [8]. and the skeletal abnormalities of Morquio B Adult or chronic (type 3) GM1 gangliosidosis was with keratan sulfaturia and oligosacchariduria, ¢rst reported in 1977 by Suzuki et al. [9] who de- thus obscuring the lines of clinical demarcation be- scribed two Japanese patients (30 and 34 years of tween GM1 gangliosidosis and Morquio disease, age) with dysarthria, gait disturbance, dystonia and type B. visual impairment. Many more cases of type 3 have been described in the Japanese and in other ethnic groups, with clinical signs of both gray matter and 3. Secondary L-galactosidase de¢ciencies white matter involvement [10] (for a recent review, see [11]) although there have been no complete de- 3.1. Galactosialidosis scriptions documenting accumulation of GM1 ganglioside in the brain of the type 3 form. A summary Galactosialidosis as the name implies arises from of some of the major clinical and biochemical fea- the simultaneous de¢ciencies of L-galactosidase and tures of the primary and secondary L-galactosidase neuraminidase (also called sialidase). These de¢cien- de¢ciencies is given in Table 1. cies are the secondary consequences of primary de-

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Table 1 Di¡erential diagnosis of L-galactosidase and neuraminidase de¢ciencies Disorder Enzyme Onset Symptoms Diagnosis Genetics de¢ciency GM1 L-Galactosidase 1. Infantile Facial dysmorphism, organomegaly, 1. Oligosaccharides AR gangliosidosis (EC 3.2.1.23) (birth to 6 months) dysostosis multiplex, cherry-red spots, in urine Chr 3p21.3 developmental retardation 2. Juvenile Onset of symptoms delayed, 2. L-Galactosidase progression slower de¢ciency in leukocytes and ¢broblast cultures 3. Adult Ataxia, dysarthria, mild to moderate intellectual deterioration, skeletal changes Galacto- Primary: 1. Early infantile Hydrops fetalis, skeletal dysplasia, 1. Sialyloligosaccharides AR sialidosis cathepsin A (prebirth^6 months) developmental retardation in urine Chr 20q13.1 (EC 3.4.16.1) Secondary: 2. Late infantile Organomegaly, cherry-red spots, 2. Cathepsin A neuraminidase, (6^12 months) dysostosis multiplex, mild mental de¢ciency or L-galactosidase retardation 3. Juvenile Dysmorphism, corneal clouding, 3. Neuraminidase and skeletal dysplasia, cherry-red spots, L-galactosidase de¢ciency progressive neurologic deterioration, in ¢broblast cultures dementia Sialidosis Neuraminidase 1. Early infantile Hydrops fetalis, organomegaly, 1. Sialyloligosaccharides AR (sialidase) failure to thrive, death within in urine Chr 6p21.3 (EC 3.2.1.18) 12 months 2. Late infantile Coarse facial appearance, 2. Neuraminidase (6^12 months) organomegaly, motor retardation, de¢ciency in ¢broblast progressive neurologic cultures deterioration, renal involvement, death in early childhood 3. Juvenile Hurler-like phenotype, progressive neurologic deterioration 4. Adult Begins in adolescence, myoclonus, bilateral cherry-red spots, visual loss, normal intelligence AR, autosomal recessive. fects in another lysosomal enzyme, Protective pro- galy are consistent ¢ndings. Several recent cases of tein-cathepsin A (PPCA). In a review in 1994 [17] early infantile Galactosialidosis have been described we summarized the major clinical ¢ndings of 55 where nonimmune hydrops fetalis was diagnosed by cases, documented the major clinical features of the ultrasound between the 24th and 28th week of ges- infantile, late infantile, juvenile and adult cases of tation [19^22]. At birth, all infants presented with galactosialidosis and described the salient biochemi- massive edema, £attened coarse facial features, with cal features. A more recent review has also appeared no or with mild dysostosis multiplex. Vacuolated [18]. The majority of cases described to date are ex- lymphocytes were noted in all cases. Diagnosis was amples of the juvenile/adult variant where coarse fa- con¢rmed by the presence of sialyloligosaccharides in cial features, mental retardation, cherry-red spots, the urine, de¢cient L-galactosidase in peripheral myoclonus, and ataxia are the dominant ¢ndings. blood leukocytes (due to its instability, neuramini- In the relatively few early infantile cases that have dase is not usually measured reliably in leukocytes) been noted at birth or soon after birth, ascites, and de¢ciency of both L-galactosidase and neurami- coarse facies, bony deformities, and hepatosplenome- nidase in cultured skin ¢broblasts.

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3.2. Sialidosis, a primary neuraminidase de¢ciency from urine of types 1 and 2 GM1 gangliosidosis [11,29,30]. Storage of undersulfated forms of keratan Sialidosis is due to a primary de¢ciency in neur- sulfate, a galactose-containing glycosaminoglycan aminidase [23]. As in galactosialidosis, sialyloligosac- and a substrate for L-galactosidase, has been docu- charides are excreted in the urine, and it is impossible mented in the infantile form of GM1 gangliosidosis to distinguish the two disorders on this basis alone. but its impact on the severity and progression of the Analysis of cultured skin ¢broblasts is needed to dis- disease is regarded as minimal compared to that of criminate between the two. Early infantile and infan- GM1-ganglioside [7,11,31]. It is noteworthy that tile cases of sialidosis present with hydrops fetalis, both the degree of accumulation and the chemical and coarse facial features and are di¤cult to distin- composition of these keratan sulfate-like materials guish from comparable cases of GM1 gangliosidosis are di¡erent from those found in full-blown Morquio and galactosialidosis. As in GM1 gangliosidosis and disease. galactosialidosis, sialidosis occurs in clinically dis- In Morquio disease, type B, the major storage tinct forms but the major signs overlap considerably substance is keratan sulfate, characterized by the and complete delineation of these three distinct enti- repeating poly(N-acetyllactosamine) sequence of ties should include both enzyme assays and separa- ^3GalL14GlcNAc1^ which is sulfated on the C6 po- tion of urinary oligosaccharides (Table 1). sition of both residues but more commonly on the GlcNAc. Two types of keratan sulfates (KS1 and KS2) are known [32^34]. KS1 found in the cornea 4. Storage substances is longer (Mr 10 000^26 000) and the polysaccharide is linked to the amide N of asparagine while KS2 4.1. GM1-gangliosidosis and Morquio disease, type B (Mr about 5000) found in skeletal tissues and carti- lage is O-linked via N-acetylgalactosamine to either GM1 ganglioside is one of the major gangliosides serine or threonine. Both skeletal and corneal forms in both the newborn and mature brain and usually of keratan sulfate are present in the urine of Mor- comprises about 25% of the total ganglioside sialic quio disease patients. There have been few detailed acid. In types 1 and 2, GM1 gangliosidosis, the total analyses of Morquio disease brain and additional ganglioside content in gray matter increases 3^5-fold neuropathological studies are needed to con¢rm and GM1 makes up about 80% of the total; thus the that neither keratan sulfate nor GM1-ganglioside accumulation of this ganglioside is about 12^20-fold are stored in the brain [15]. over the control values [7,11,24,25]. GM1 ganglioside has been isolated from puri¢ed myelin, and the ele- 4.2. Galactosialidosis and sialidosis vated content in white matter likely arises from this membrane and the neuronal elements contaminating Urinary excretion of sialyloligosaccharides is the white matter preparations [25]. In adult, chronic biochemical hallmark of both galactosialidosis and GM1 gangliosidosis, mild GM1 ganglioside accumu- sialidosis, and this is totally ascribable to the neur- lation has been documented in the gray matter aminidase de¢ciency, be it primary or a secondary [26,27] and in the basal ganglia particularly the cau- enzyme defect. These substances arise largely from date nucleus and the putamen [26^28]. In the visceral the lysosomal degradation of asparagine-linked gly- organs, particularly the liver, GM1 ganglioside con- coproteins [17,18]. Sialyloligosaccharides arising tent is markedly elevated over the normal concentra- from degradation of the serine and threonine-O- tion but ganglioside content in this tissue is small linked oligosaccharides have not been characterized compared to the brain [25]. Concordant with GM1 [17,18]. The abundance of the respective mono-, di-, ganglioside accumulation is the storage of the non- tri-, and tetrasialylated oligosaccharides found in sialylated glycolipid GA1 in all three types of GM1 urine and other body £uids re£ect the relative distri- gangliosidosis [11,28]. bution of these polyantenna-containing glycoprotein At least 42 di¡erent glycoprotein-derived galac- precursors produced in the body. Both K2^6 and K2^ tose-terminal oligosaccharides have been isolated 3 linked sialylated termini have been characterized,

BBADIS 61880 10-9-99 90 J.W. Callahan / Biochimica et Biophysica Acta 1455 (1999) 85^103 with the K2^6 structure being relatively more abun- been de¢ned and a long intronic region occurs be- dant in sialidosis. Although some galactose-terminal tween exons 1 and 2 (22 kb in the human). The oligosaccharides have been characterized in galacto- mouse Gal gene (80 kb) and that of the dog also sialidosis, it is likely that they have arisen from in- contain 16 exons [42^44]. Morreau et al. [40] de- complete chain elongation during biosynthesis rather scribed two transcripts in human ¢broblasts, where- than from partial digestion due to residual neurami- by the larger (2.4 kb) and 12-fold more abundant nidase activity. mRNA encoded the full length lysosomal Gal while One of the most surprising ¢ndings to date is the the minor form (2.0 kb) was an alternately spliced relative lack of storage of the polysialogangliosides in Gal-like protein (which we have named S-Gal). The the brain in both galactosialidosis and sialidosis. This general structural features of both proteins are suggests that the neuraminidase de¢ciency character- shown in Fig. 1. Yamamoto et al. [39] and ourselves ized in these disorders has a higher degree of specif- [45] have independently con¢rmed the existence of icity for sialyloligosaccharides attached to glycopro- this alternate transcript and we have described its teins than the comparable structures attached to the properties [45^52]. ceramide backbone of gangliosides. The sialyl K2^3 linkage to galactose is more abundant in ganglio- 5.1. Biosynthesis and processing of Gal sides, compared to the ganglioside K2^8 linkage, and may be both terminal and internal whereas in The Gal gene encodes a 677 amino acid prepropo- glycoproteins the sialyl K2^3 linkage to galactose is lypeptide, where a 23 amino acid N-terminal signal always terminal. Since the ceramide backbone is de- sequence is cleaved on entry into the ER to generate graded only after the carbohydrate linkages have a propolypeptide of 654 residues. Gal is glycosylated been removed whereas in the case of glycoproteins, co-translationally to an 84 kDa precursor and when the protein backbone can be largely degraded inde- phosphorylated is 88 kDa. It has 8 cysteine residues pendent of the digestion of the carbohydrate, it is (likely forming 4 cystines), and 7 consensus putative conceivable that a ganglioside speci¢c neuraminidase exists that is both separate and distinct from the glycoprotein or oligosaccharide neuraminidase involved in these two diseases. There has been some recent evidence that this is the case and it would account for the lack of ganglioside storage in the brain in these diseases [35]. There is evidence for slight accumulations of the major monosialogangliosides, and GD1a ganglioside in spinal cord neurons in galactosialidosis patients but these accumulations may re£ect non-speci¢c disease-related changes as accumulations of similar gangliosides, particularly the monosialogangliosides have been documented in a variety of other disorders, such as Niemann^Pick disease, Hurler disease and other non-ganglioside, non-neuraminidase related disorders [36]. Fig. 1. Features of Precursor Gal, mature, fully-processed Gal and S-Gal. The downward arrows identify the putative N-glycosylation sites (at N26, N247, N464, N498, N542, N545, and 5. L-Galactosidase and elastin binding protein arise N555, where only one of N542 and N545 is likely to be occu- from the Gal gene by alternate splicing pied). The upward arrows show 14 sites of missense mutations and the horizontal bars identify the two duplication sites. Arg530 has been proposed as the C-terminal of the mature fully The human Gal gene, localized on chromosome processed Gal protein, which would have resulted in the loss of 3p21.33 [37^39], is 62.5 kb in length and is composed 123 residues, 3 putative N-glycosylation sites, and two cysteine of 16 exons [40,41]. The intron/exon junctions have residues (which are postulated to form a disul¢de bond).

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N-glycosylation sites although it is unlikely that all are occupied [53] as two are side-by-side (Asn542Ser- SerAsnTyrThr547) and steric hindrance may block glycosylation of the one if the other is occupied (Fig. 1). Zhang et al. [53] puri¢ed this Gal precursor to apparent homogeneity and characterized its structural and kinetic properties. It displayed the same speci¢c activity (0.7 mmol/h per mg) as the mature form of the enzyme puri¢ed from placenta, it was a monomer on molecular sieve chromatography and had an M of 88 000 on sodium dodecyl sulfate^poly- r Fig. 2. Posttranslational processing of the Gal precursor and acrylamide gels. It had the same pH optimum (pH the proposed role of protective protein. The propolypeptide pre- 4.3) and displayed kinetic constants with synthetic cursor is folded and N-glycosylated in the endoplasmic reticu- substrates identical to the mature placental enzyme, lum. The precursor is fully active (with synthetic substrates) although since no studies have appeared it is unclear and may account for the small amount of residual activity de- whether the precursor has the capacity to hydrolyze tected in most cells, although there is a small amount of immu- noreactive material that migrates like the mature form of the natural substrates. The puri¢ed precursor was endo- enzyme. Normal processing requires Gal to enter into a com- cytosed from the medium and corrected the defect in plex with protective protein and neuraminidase whereupon the GM1-gangliosidosis ¢broblasts, indicating the re- protein is processed at its C-terminal end to form the mature combinant precursor is phosphorylated and man- enzyme. The enzyme is degraded by resident proteases, with the nose-6-phosphate targeted to lysosomes, was con- appearance of 18 kDa and 50 kDa fragments. Since the same verted to a 72 kDa polypeptide upon digestion with 18 kDa fragment is found in both normal ¢broblasts and in galactosialidosis ¢broblasts, it can be assumed that this poly- N-glycanase, and to a 50 kDa form on digestion with peptide arises upon digestion of the heretofore unprocessed N- pepsin (no thiol reagents; Withers and Callahan, un- terminal end of the protein. published observations). Pulse-chase studies in human ¢broblasts have shown the Gal precursor is translocated to the endo- tease cleavage site at Arg595 (a cathepsin B site re- somal^lysosomal compartment via the mannose-6- quires a proximal hydrophobic residue; in this case phosphate receptor and is processed in a few hours Ala is at 594). In vivo, normal maturation of L-gal- to the mature enzyme, the monomer of which is 64 actosidase requires its association in a complex (this kDa [54^60]. Maturation to the 64 kDa enzyme oc- is discussed below) that protects this susceptible site curs by processing only at the C-terminal end of the but does not prevent a non-thiol protease from gen- protein in normal ¢broblasts both in the presence erating the mature, fully-processed Gal. Mature Gal and absence of leupeptin, a potent inhibitor of thi- is subsequently degraded and we have identi¢ed 18 ol-proteases such as cathepsin B, S, H, and L, indi- kDa and 50 kDa digestion products. Interestingly, in cating that normal maturation does not depend on I-cell ¢broblasts, only the Gal precursor and the 18 this class of cathepsins (Fig. 2). In galactosialidosis kDa fragment could be detected suggesting that a ¢broblasts, little normal maturation of L-galactosi- portion of the 18 kDa fragment could have arisen dase occurs. We have detected two abnormal early by non-lysosomal proteolysis [59]. cleavage steps in L-galactosidase C-terminal processing, resulting in the appearance of either an 80 kDa 5.2. Gal: substrates, the role of saposin B, and its species alone or together with a 72 kDa species [59]. active site Generation of the 80 kDa species is inhibited in the presence of leupeptin, but the appearance of the 72 Gal releases terminal L,1^4 or L,1^3 linked galac- kDa species is not a¡ected by leupeptin (Fig. 2). The tose residues from the glycosphingolipids (GM1 gan- precise locations for this inappropriate processing glioside, a major acidic lipid of brain gray matter, has not been de¢ned but examination of the deduced lactosylceramide, and gangliotetraosylceramide (asia- L-galactosidase sequence reveals a putative thiol pro- loGM1 ganglioside)), galactose-terminal neutral oli-

BBADIS 61880 10-9-99 92 J.W. Callahan / Biochimica et Biophysica Acta 1455 (1999) 85^103 gosaccharides (either attached to a protein core as in glycoproteins or free in solution), and keratan sulfate-derived oligosaccharides [11,61^64] accumulat- ing in GM1 gangliosidosis and Morquio disease, type B. Substrate speci¢city studies have used mature forms of Gal puri¢ed from human liver or placenta, rabbit brain, or other tissue sources. In vitro hydrolysis of GM1 ganglioside and lactosylceramide required detergents (such as cutscum, or taurocholate). In vivo, a small 80 amino acid N-glycosylated polypeptide, saposin B (Fig. 3), which binds to the ceramide (lipid) moiety of GM1 ganglioside stimulates conversion of GM1 to GM2 ganglioside (for reviews, see [65,66]). Zschoche et al. [64] recently showed that both saposin B and C stimulated hydrolysis of lip- osomal lactosylceramide by Gal. Saposins B and C arise from proteolytic intralysosomal digestion of the parent, prosaposin, a 69 kDa prepropolypeptide (Fig. 3). Saposin B interacts directly with the ceramide moiety to form a 1:1 water soluble lipid^protein complex. It also binds sulfatide and stimulates its Fig. 4. Catalytic mechanism of a L-con¢guration retaining fam- hydrolysis by arylsulfatase A [65,66]. Neither deter- ily 35 L-galactosidase. gents, saposin B nor prosaposin, are known to be involved in the hydrolysis of galactose-terminal oligosaccharides. Of the three mutations known to oc- with Morquio, type B, the residual Gal hydrol- cur in saposin B, none give rise to GM1 gangliosi- yzes substrates with Km values severalfold higher dosis or Morquio disease phenotypes. In patients than normal [67,68]. Yutaka et al. [69] showed in 1982 that Gal-L-N-acetylglucosamine-(6-sulfo)-L- [1-3H]galactitol, a trisaccharide derived from keratan sulfate, was cleaved by crude ¢broblast Gal but there have been no further studies with this substrate. The Gal precursor cleaves the usual £uorogenic and colorimetric synthetic substrates and undergoes rapid inactivation upon cleaving the mechanism- based suicide substrate 2,4-dinitrophenyl-2-£uoro-2- deoxy-L-D-galactoside (DNP-FDG) [53]. These results demonstrated that Gal cleaves substrates by a Koshland double-displacement mechanism with a nucleophilic attack by an acidic catalytic site nucleophile (Glu or Asp) with L-con¢guration retention and formation of a galactosyl-enzyme intermediate [53] (Fig. 4). There have been no studies using the precursor Gal with natural substrates. Using the sui- Fig. 3. Saposin B, a posttranslational proteolytic product of cide substrate, electrospray ionization mass spec- Prosaposin. The Prosaposin prepropolypeptide of 524 amino trometry after pepsin digestion (+thiol agents) and acids, after removal of a 16 amino acid signal peptide is N-glycosylated and directed to the endosomal^lysosomal compart- HPLC separation of peptides [70], we [71] identi¢ed ment whereupon it is processed by lysosomal proteases into a the galactosylated-nucleophile as Glu268, a residue series of smaller products which have been called saposin A^D. which is completely conserved among 6 other known

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Gal sequences (Fig. 5). Analogous to the acid-acid mechanism of other glycosidases including Escheri- chia coli Gal [72,73], the residue required to cleave this bond and restore Gal to an active state is likely a conserved acidic residue. Of the other acidic residues in mature human Gal, only Glu186, Glu188, Glu339, and Asp332 are completely conserved among the same six enzymes (Fig. 5). We converted Glu268 to Gln and this form of Gal, puri¢ed from permanently-transfected CHO cells, was catalytically inac- tive con¢rming the role of Glu268 in the mechanism (Callahan and Zhang, unpublished observations). We have now identi¢ed a naturally-occurring mutation in Asp332 (it is converted to Asn332) and using the same CHO expression system have evidence that this residue is involved in the active site although it is too early to assign it a role in the catalytic mechanism (such as for cleavage of the galactosyl^Glu268 bond to release free galactose and restore the enzyme to an active state [74]).

5.3. S-Gal, a component of the elastin binding (EBP) receptor? Fig. 5. Conserved acidic residues of homologous L-galactosi- dases. The second product of the Gal gene, S-Gal, arises by alternate splicing of the primary Gal transcript whereby exons 3, 4 and 6 are skipped, and there is ligand binding studies we found that human S-Gal a frameshift in exon 5 (which encodes a unique 32 and sheep EBP were related. Sheep EBP contains a amino acid sequence) [40]. The reading frame is re- lactose (i.e., a L-galactoside) binding site and it binds stored at the start of exon 7. S-Gal has 546 amino to an a¤nity column containing the ligand, acids, a predicted mass of 60 000 and an apparent GSPSAQDEASPL, a portion of the unique sequence mass in the range 67 000^69 000 (depending on the found in human S-Gal [49^51]. Sheep EBP is one of number of occupied N-glycosylation sites). Three three proteins (the others are 54 kDa and 61 kDa) cysteines are removed during the alternate splicing that form a cell surface receptor the major function process but a new Cys occurs at position 87 in the of which is to facilitate the formation of extracellular frameshifted unique part of the sequence to restore elastin matrix by binding tropoelastin, the elastin an even number. Except for the area where splicing monomer, and deliver it to the cell surface where it occurs, S-Gal is identical to the wild-type Gal and is released in the presence of galactosugars (Fig. 6). although there is no di¡erence in the number of N- The 54 kDa and 61 kDa proteins serve as the mem- glycosylation sites between Gal and S-Gal, the latter brane anchors while the EBP can be dissociated from was localized to the perinuclear area of the cyto- and re-associate with the rest of the receptor. Neither plasm but not to lysosomes [40,45]. It is unclear if of the 54 kDa or 61 kDa proteins has been well S-Gal occurs in the mouse [43] but an alternately characterized at least in sheep. Inside cells, EBP is spliced transcript was not detected in the dog [44]. postulated to serve a `chaperone-like' function for Elastin binding glycoprotein (also called EBP), a correct targeting of tropoelastin, the elastin mono- 67 kDa glycoprotein, was originally identi¢ed in mer. Human S-Gal has functional similarity to the sheep and found to be a component of the elastin EBP from sheep aortic smooth muscle cells. Our af- binding receptor [46]. Based on immunological and ¢nity-puri¢ed anti-S-Gal antipeptide antibody recog-

BBADIS 61880 10-9-99 94 J.W. Callahan / Biochimica et Biophysica Acta 1455 (1999) 85^103 nized sheep EBP both on western blots [50,51] and in transfected cells displayed increased adherence to sections of sheep aorta tissue and a similar protein in elastin-covered dishes, consistent with the cell surface human placenta, muscle and kidney (Hinek and Call- distribution of the newly produced S-gal-encoded ahan, unpublished observations). Thus we were the protein. Transfection of DA SMC additionally cor- ¢rst to show that S-Gal is related to the elastin bind- rected their impaired elastic ¢ber assembly. These ing protein (EBP). We have recently shown that the results conclusively identify the 67 kDa splice variant majority of EBP residing on the cell surface can be of L-galactosidase as EBP. Further immunological internalized to endocytic compartments but not to studies show the human and sheep elastin binding lysosomes and can be recycled back to the cell sur- receptor contain in addition to S-Gal, the same two face within 45^60 min. To de¢ne further the genetic major proteins; namely, protective protein/cathepsin origin of human EBP, we isolated and characterized A and neuraminidase [52]. a full length human S-gal cDNA clone, and expressed it in COS-1 cells and in the EBP-de¢cient smooth muscle cells (SMC) from sheep ductus arte- 6. Neuraminidase riosus (DA) [45]. In vitro translation yielded an un- glycosylated form of S-gal which immunoreacted Lysosomal neuraminidase is a 45.5 kDa prepropoly- with anti-L-galactosidase antibodies and bound to peptide of 415 amino acids, with a signal peptide of elastin and laminin a¤nity columns. S-gal cDNA 45^47 amino acids (consisting of a positively charged transfections into COS-1 and DA SMC increased amino terminal region (residues 1^18) and a central expression of a 67 kDa protein that immunolocalized hydrophobic area (residues 19^38)), and is posttrans- intracellularly and to the cell surface and, when ex- lationally-glycosylated (it contains three potential N- tracted from the cells, bound to elastin. The S-gal glycosylation sites) and processed to a mature 48.3

Fig. 6. The role of S-Gal (elastin binding protein) in formation of extracellular matrix.

BBADIS 61880 10-9-99 J.W. Callahan / Biochimica et Biophysica Acta 1455 (1999) 85^103 95 kDa protein [75,76]. It is not clear if the neuramini- upon it becomes mature cathepsin A). PPCA was dase precursor is active or whether it requires a pro- originally thought to function only to `protect' Gal teolytic activating event but results to date indicate and Neuraminidase from intralysosomal digestion by that minimal posttranslational protein processing oc- forming a complex with these enzymes in lysosomes curs, only at the N-terminal. Homology predictions but site directed mutagenesis and expression studies indicate signi¢cant relatedness of the human enzyme [79^81] have shown that the serine protease activity to viral, bacterial, protozoan, and rodent neuramini- can be abolished without a¡ecting its ability to `pro- dases and consists of six four-stranded antiparallel L- tect' Gal and Neuraminidase from intralysosomal sheets arranged around a central axis of symmetry proteolysis. Studies on related proteases, yeast car- containing the active site. Neuraminidase is found in boxypeptidase Y, and mouse cathepsin L [82,83], both lysosomal and plasma membranes and it is suggest that the lack of protease activity in the pre- readily secreted from cells [77]. Sequence predictions cursor is partly due to a N-terminal propeptide intra- suggest it does not contain a hydrophobic sequence molecular chaperone which promotes folding of the that resembles a transmembrane domain. However, protein (for a review, see [84]) such that the propep- expression studies have shown that shortly after tide region blocks proteolytic cleavage at the site neuraminidase arrives in the ER, it associates with between the 32 and 20 kDa peptides until a confor- PPCA which serves as a protein-speci¢c ER chaper- mational change occurs presumably upon its associ- one for neuraminidase [76,77] and accompanies it ation with either Gal or neuraminidase. Elsliger and through the Golgi and endosomal-lysosomal com- Potier [85], using model structures, speculated that partments. Metabolic labeling studies have indicated protective protein was a dimer with the N-terminal that at least a portion of neuraminidase is poorly propeptides near the surface consistent with its abil- phosphorylated and may not be recognized by the ity to serve as an intramolecular chaperone [83]. Ru- mannose-6-phosphate receptor [78]. Association denko et al. [86,87] also used an atomic model para- with PPCA in the ER obviates this relative lack of digm of the PPCA to examine the structure-function lysosomal targeting capability. Mutations described relationships in the protein and predicted that 9 of to date which have been identi¢ed in either type I or the currently de¢ned 11 mutations [88^92] in PPCA type II forms of sialidosis include: a homozygous 4 (Table 2) drastically altered the folding and stability base pair duplication (+ACTG8^11) resulting in an of the protein, while the remaining two caused less early frameshift, a single base deletion from codon severe alterations of the protein. None of the muta- Ser403 which shifts the reading frame to include an tions identi¢ed to date involves the active site ser- additional 69 amino acids beyond the usual termina- ine150 or the two other required residues (His429, tion after codon 415, a chain terminating mutation and one of Asp370 or Asp372) [18,87]. Since severe (G1258T, Arg377ter) and three missense mutations de¢ciencies of both Gal and neuraminidase always (T401G, Leu91Arg; T779A, Phe260Tyr and accompanied these mutations, and since PPCA and T1088C, Leu303Pro) [75,76]. The gene for neurami- neuraminidase associate in the ER [78] it can be cor- nidase has been localized to chromosome 6p21.3. Ex- rectly assumed that PPCA carrying these mutations pression in COS-1 cells of the frameshifted Ser403 does not exit the ER either alone or in conjunction and the Arg377ter mutations resulted in misfolded with neuraminidase. proteins that did not exit the ER.

8. The `complexes' formed by Gal and S-Gal with 7. Protective protein/cathepsin A protective protein/cathepsin A and neuraminidase

Protective protein/cathepsin A is synthesized as a 8.1. The lysosomal complex 54 kDa propeptide zymogen which when processed in lysosomes is cleaved into 32 kDa and 20 kDa Upon arriving in the endosomal-lysosomal com- components (held together via interchain disul¢de partment, Gal associates with the pre-associated bonds) and displays serine protease activity (where- PPCA and neuraminidase to form a complex that

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Table 2 Protective protein/cathepsin A mutations Residue/mutationa Sequence conservedb Other allele Mutation e¡ect Ref. GROUP 1: Group 1 mutations give severe disease except when the second allele is from Group 2, but none involve active site residues; they destabilize protein folding and stability, and the resultant protein is unlikely to exit the ER Q49R/152A s G F440V Mild [87,88] Q49R/152A s G SpDEx7 Mild [87,88] S51Y/158C s A H,M,C,W,Y +grp1 Severe [87,88] W65R/199T s C H,M,C,W,Y F411V Mild [87,88] W65R/199T s C H,M,C,W,Y SpDEx7 Mild [87,88] S90L/274-5TC s CT H,M,C,W,Y +grp1 Severe [87,88] V132M/400G s A H,M,C,W,Y +grp1 Severe [87,89] L236P/713T s C H,M,C,W,Y +grp1 Severe [87,89] Y395C/1190A s G H,M,C,W,Y +grp1 Severe [87,88,92] M406T/1223T s C F440V Mild [87,89] M406T/1223T s C SpDEx7 Mild [87,89] G439S/1321G s A H,M,C,W,Y +grp1 Severe [87,89]

GROUP 2: a¡ect dimerization; monomer is rapidly degraded Y249N/751T s A H,M,C, +grp2 Mild [87,88] F440V/1324T s G H,M,C, +grp2 Mild [87,89]

GROUP 3: a¡ect RNA stability and abundance; markedly decreased amounts of PPCA SpDEx7 SpDEx7 Mild [87,88] +grp2 Mild [87] +grp1 Not known [87] dC118 Y249N/751T s A Not known [89] c517delTT IVS+9C s G Mild [91] aAll amino acids are numbered from the initiating methionine and from the ¢rst base of the codon which is position +7 of the cDNA. bThese residues are conserved in the human (H), mouse (M), and chicken (C) PPCAs, and wheat (W) and yeast (Y) serine carboxy- peptidases.

is absolutely essential for its stabilization and for its terminal end of Gal which contains the most hydro- normal posttranslational processing to its mature philic sequence of the entire protein and a postulated form (Fig. 2). Recently, Pshezhetsky and Potier [93] `intramolecular chaperone' that is essential for cor- isolated a 1.27 MDa form of the complex that con- rect folding in the ER and Golgi, but is not required tained, in addition, N-acetylgalactosamine-6-sulfate for catalytic activity [59,60]. This region of the en- sulfatase, the enzyme de¢cient in Morquio disease zyme (comprising residues 653^666) is relatively pro- type A, and showed that its activity was also reduced line-rich and may serve to facilitate protein-protein in galactosialidosis. This correlates with elevated lev- interaction with protective protein, thus blocking a els of keratan sulfate in the urine of galactosialidosis putative thiol protease susceptible site at R595. patients. These ¢ndings suggest that storage of ker- When the C-terminal is lost after lysosomal process- atan sulfate in both types A and B Morquio disease ing (Fig. 2), this stabilizing function is replaced by result from the combined e¡ects of the direct muta- the `Complex' which also facilitates Gal dimerization tions in N-acetylgalactosamine-6-sulfate sulfatase and oligomerization. Since the 88 kDa Gal precursor and Gal, respectively, which a¡ect complex forma- is a monomer, and fully active, it must be folded into tion and stability [93,94]. Unlike neuraminidase an active conformation prior to its release from the which undergoes minimal posttranslational process- ER and does not require protective protein for its ing, substantial processing of Gal occurs at the C- transit through the Golgi, its lysosomal localization,

BBADIS 61880 10-9-99 J.W. Callahan / Biochimica et Biophysica Acta 1455 (1999) 85^103 97 nor for its secretion from cells [53,95,96]. The major- gether and reside in the plasma membrane [76,78] ity of studies on the complex to date have focused on and are in agreement with our earlier data [49,50]. the role played by the protective protein in the stabilization and oligomerization of Gal. However, based on our recent work [59,60] it seems clear that the 9. Genetic heterogeneity in GM1 gangliosidosis and fully processed PPCA is partly responsible for the Morquio disease, type B C-terminal processing of Gal precursor to the mature form but it is not known why processing stops when About 39 disease-causing mutations in Gal (Table Gal is about 64 kDa in size (Fig. 2). Although PPCA 3) and several polymorphisms have been described in and neuraminidase associate in the ER and travel the world population [10,11,97^105]. Of these delete- together through the intracellular compartments, it rious mutations, three are splice junction mutations, is unclear if neuraminidase displays catalytic activity two are duplications, three are insertions leading to prior to its localization in the acidi¢ed endosomal- frameshifts, and two are premature chain termina- lysosomal compartment. Vinogradova et al. [77] have tion mutations [11,105]. Of these, only the 641insT/ recently shown that activation of neuraminidase ac- 1627insG, the 896insC/1627insG, the dupl2/dupl2, tivity coincides with its occurrence in the mature high and Arg457ter/Arg457ter would be expected to result molecular mass complex found in lysosomes and in no functional protein. Twenty-nine missense mu- without this association, neuraminidase undergoes tations are distributed uniformly over the entire rapid and irreversible proteolysis. length of the gene. The active site catalytic nucleophile, Glu268, which is totally conserved amongst 8.2. The elastin binding receptor Gal species de¢ned to date (Fig. 5), has not been identi¢ed as a mutated residue in any patient. Muta- As mentioned above we have presented evidence tions in Glu268 would predict the occurrence of pa- that the alternately spliced form of Gal, S-Gal, is tients with a severe phenotype of combined GM1 identical to the 67 kDa elastin binding protein, the gangliosidosis and Morquio, type B. No such pa- major function of which is to assist in the delivery of tients have been described to our knowledge. Candi- tropoelastin to the cell surface [45,49^51] and that dates for the second acid/base residue in the catalytic the elastin binding receptor is formed from S-Gal, mechanism are those that are completely conserved PPCA and neuraminidase. Release of newly secreted in mature human Gal and the other family 35 hydro- tropoelastin molecules from the association with S- lases (only Glu186, Glu188, Glu339, and Asp332). Gal occurs upon interaction between it and galacto- Asp332Asn Gal, when expressed in CHO cells and sugar moieties protruding from the carbohydrate puri¢ed to homogeneity, results in a marked reduc- chains of glycoproteins thus facilitating formation tion in catalytic activity consistent with it having a of the micro¢brillar sca¡old of elastic ¢bers. Studies role in the active site [74]. Asp491Asn, which has not are now under way examining elastic ¢ber assembly been expressed, and Glu632Gly, which is near the C- by ¢broblasts from patients with GM1 gangliosido- terminal end of the protein and unlikely to play a sis, Morquio disease, type B, galactosialidosis, and role in catalysis, are the only other mutated acidic sialidosis. Our initial results in ¢broblasts have found residues known. In the sequence immediately adja- that most but not all GM1 cell lines have the ca- cent to Glu268, both Trp273 and Pro263 are highly pacity to assemble elastic ¢bers while cells from pa- conserved and are involved in Gal de¢ciencies. The tients with Morquio disease type B do not (all the most common mutation in Gal reported to date giv- Gal mutations in these cells have been de¢ned). In ing rise to Morquio disease, type B, involves conver- addition we noted that cells from patients with ga- sion of Trp273 to Leu and has been found in 12 (7 lactosialidosis and sialidosis also do not assemble homoallelic; 5 heteroallelic) of the 15 patients re- elastic ¢bers [52]. These data are in accord with the ported [106]. One Morquio, type B patient had the recent ¢ndings that newly synthesized PPCA and Trp273Leu mutation on one allele with an Arg- neuraminidase associate in the ER, are secreted to- 482His mutation on the other while Mosna et al.

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Table 3 Mutations in the L-galactosidase gene Exon Nucleotide Residue Allelic residue Phenotype Origin Ref. Res. act./expresseda 1;1^25 T29C L10P Polymorph. Common [97] ivs1, iT@75 Adult Danish [101] Mother has 37.5% of N activity, new spl site not expressed, see note 4 2;26^82 C145T R49C Unknown Infantile Japanese [99] Not stated 2;26^82 T152A I51T I51T Adult Japanese [97] expressed act 26.7% of N activity; serum, 3.4%, see note 1 2;26^82 T155C S52P Unknown Juvenile English/ [100] Not stated Scottish 2;26^82 G226A R59H R59H Infantile Brazilian [105] Not stated 2;26^82 C245T T82M Unknown Adult Danish [101] Father with T82M has 45.6% N activity, expression gave 3^4% of N activity, no WB 2;26^82 T297C Y83H Unknown [11] Not stated 2;26^82 nt254^276 Y316C Infantile Japanese [97] Not stated dupl 1 2;26^82 gGT s aGT, frameshift spl2/spl2 Morquio Not stated [102] spl. def., int 2 [11] 3;83^132 G413T R121S R208C Infantile Brazilian [105] Not expressed 3;83^132 G367A G123R G494C Infantile Japanese [97] Expression gave 0.3% N activity but no Western blot 4,133^153 C442A R148S D332N Infantile English [74,112] Expression of R148S gave N activity in COS cells, thus a folding mutation. Patient also polymorphic for S532G 6;185^245 C601T R201C Unknown LI/Juvenile Japanese [97] Expression gave 8.3% N activity, see note 2 6;185^245 C601T R201C R201C LI/Juvenile Japanese [97] 6.9%, 3.4% see note 2 [102] 6;185^245 G602A R201H P263S Adult Not stated [97] Expression gave 46.5% N activity, but no Western blot 6;185^245 G602A R201H N318H Juvenile/ Greek [102] Expression gave 46.5% N morquio activity, but no Western blot 6;185^245 C622T R208C R208C Infantile English/ [100] Expressed mutation gave 6 1% European activity but no Western blot R208C 1627insG Brazilian [105] 6;185^245 641insT frameshift 1627insG Infantile Brazilian [105] Not expressed 6;185^245 T647C V216A Unknown Infantile English [74,112] Not expressed 6;185^245 G768A V240M V240M Infantile Brazilian [105] Not expressed 6;185^245 caa s gAA spl. Unknown [11] Not stated def., int 6 7;246^264 CCC s TCC P263S R201H Adult Not stated [11,102] Expressed mutation gave 0.0% activity, but no Western blot 8;265^308 TG817/18CT W273L R482H Morquio American [104] See note 3; puri¢ed mutant enzyme low residual activity W509C Morquio American 8;265^308 896insC frameshift 1627insG Infantile Brazilian [105] Not expressed 9;309^318 A947G Y316C dupl1 Infantile Japanese [97] Not expressed 9;309^318 AAT s CAT N318H R201H Juvenile/ Greek [102] Not expressed morquio 10;319^356 GAC s AAC D332N R148S Infantile English [74,112] Expression of D332N gave,1% N activity in COS-1 and CHO cells active site mutation

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Table 3 (continued) Mutations in the L-galactosidase gene Exon Nucleotide Residue Allelic residue Phenotype Origin Ref. Res. act./expresseda 10;319^356 CGA s TGA R351ter R351ter Infantile Venezuelan [74,112] 11;357^381 11/12 nt1069^1233 dupl2 Unknown Infantile Japanese [97] 11/12 nt1069^1233 dupl2 dupl2 Infantile Japanese [98] 0.7^1.6%, large mRNA 12;382^410 13;411^449 14;450^493 C1369T R457ter R457ter Infantile Japanese [99] 14;450^493 C1369A R457Q I51T Adult Japanese [97] Expressed mutation gave 0.6% N activity but no Western blot 14;450^493 G1445A R482H R482H Infantile Italian [107] Not expressed 14;450^493 G1445A R482H W273L Morquio American [102] Not expressed [104] Expressed mutation gave 0.4% N activity but no Western blot 14;450^493 G1521A D491N 1627insG Infantile Brazilian [105] Not expressed 15;494^578 G1480T G494C G123R Infantile Japanese [97] Not expressed 15;494^578 G1527T W509C W273L Morquio American [104] Not expressed 15;494^578 A1594G S532G several None Common [74,112] Polymorphism giving 75^95% of normal activity on expression 15;494^578 1627insG frameshift R59H Infantile Brazilian [105] Not expressed 15;494^578 A1733G K578R R208C Infantile English [100] Expressed mutation gave 6 4% of control activity, but no Western blot 15;494^578 A1733G K578R R208C Infantile Mexican [100] Expressed mutation gave 6 4% of control activity, but no Western blot 16;579^677 G1769A R590H R208C Juvenile English/ [100] Expressed mutation gave 6 1% German of control activity, but no Western blot 16;579^677 A1828G T610A R208C Juvenile Italian/Puerto[100] Not expressed Rican 16;579^677 A1895G E632G R208C Juvenile English/ [100] Expressed mutation gave 6 1% Scottish of control activity, but no Western blot 16;579^677 A1953G K655R Unknown Juvenile English [100] Not expressed Single-letter abbreviations are used to identify the amino acid residues. Abbreviations used: WB, Western blot; N, normal. Note 1: I51T is a common mutation in adults with GM1. The expressed precursor was not phosphorylated, there was no e¡ect of the co-expressed protective protein, enzyme was not processed and E64 had no e¡ect [94]. Case 7 in [97] same as A/C in [98]. Clinical data on adults found in [10]. I51T when expressed gave 13.8% of normal according to Ishii et al. [102]. Note 2: R201C is considered a common mutation in late infantile/juvenile patients. In one report, on transfection, there was a decreased amount of precursor (folding mutation?), the protective protein had no e¡ect on stability, and the presence of the thiol protease inhibitor, E64, increased the amount of precursor [93]. Ishii et al. [102] reported that this mutation gave a 3.4% residual activity. Note 3: The W273L mutation is considered a common mutation for Morquio B. Although there is reduced activity this mutation results in ample amounts of precursor, protective protein helps processing and there is an increase with E64 [94]. Ishii et al. [102] reported this mutation gave 7.9% residual activity. Note 4: The insertion of a T at position 75 alters the usual 5P splice donor site but activates a new downstream donor splice site. This results in a 20 bp insertion of intron sequence in the mRNA resulting eventually in a frameshift and premature termination of translation. aNucleotide positions use the D'Azzo clone, and are marked from the initiating ATG.

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[107] described a severely a¡ected type 1, GM1 pa- evidence to date) from the mature protein during tient who was homozygous for Arg482His. Therefore enzyme processing to its mature form (see above). the features of the Morquio, B phenotype must be Consistent with this notion, mutations in this region due to the least severe of the two mutations, namely have been found only in patients with the later onset Trp273Leu. We expressed the Trp273Leu mutation type 2, GM1 gangliosidosis. in permanently-transfected CHO cells and found a Approximately 16 mutations have been expressed markedly diminished catalytic activity in the puri¢ed by transfection into host cells. In addition to Asp- precursor (speci¢c activity 0.31 Wmol/h per mg pro- 332Asn, and the Trp273Leu, cited above, we found tein compared to the wild-type activity of 500^700 the Arg148Ser mutation produced a fully active en- Wmol/h per mg protein) [74] in accord with our ear- zyme that was su¤ciently misfolded that it could not lier prediction [71]. Fukuhara et al. [108] found an exit the ER and was likely rapidly degraded while the altered Km and Vmax for this mutant expressed in Ser532Gly polymorphism was fully active and was insect cells which con¢rms and extends these ¢nd- appropriately targeted as expected [112]. Oshima et ings. We postulated that Trp273 plays an important al. al [94] found that the expressed precursor contain- role in substrate binding [71] (Fig. 4) by promoting ing the I51T mutation was not phosphorylated prop- `stacking' between the planar indole ring and the erly. Many other mutations have been examined only pyranose backbone stabilizing the enzyme^substrate for their e¡ect on enzyme activity [10,11,97,100,105]. complex [109]. Based on sequence homologies, hu- In our hands, all of the approximately 20 GM1-gan- man Gal may have a structure similar to that of 6- gliosidosis and Morquio, B patients we have exam- phospho-L-galactosidase from Lactococcus lactis ined display markedly decreased levels of immuno- which has been crystallized and is also a member reacting material [59,112,113]. Since Glu268, Asp332 of the 4/7 superfamily. It hydrolyzes lactose-6-phos- and Trp 273 are the only residues known to date that phate (glucose-galactose-6-phosphate) [110,111]. The are involved in catalytic activity although many crystal structure shows it to contain a (LK)8-barrel others also likely play a role, it seems certain that structure with a central core, a catalytic mechanism the majority of the missense mutations have their identical to human Gal where unique conformational major e¡ect on enzyme folding resulting in degrada- features bring the two acidic residues close enough to tion in the ER such that only small amounts of pre- catalyze the reaction. This mechanism also docu- cursor arrive in the lysosome and are available for ments hydrophobic stacking roles for tryptophan processing to the mature form. and tyrosine residues analogous to that proposed for human Gal Trp273. Interestingly, the same unique conformational features may be provided by 10. Pathophysiological bases of GM1 gangliosidosis Pro263 in the human enzyme which is predicted to and Morquio, type B: a hypothesis facilitate substrate binding and may be consistent with the milder GM1 phenotype seen (Table 3). It can be predicted that if a 5^10% residual Gal Six mutations (5 occur in GM1, 1 in Morquio, B) activity as seen in Morquio disease, type B, gives rise introduce new cysteine residues which would a¡ect to keratan sulfate storage, then a 0^5% residual ac- cystine bond formation and would be predicted to tivity would be expected to give rise to storage of all result in a misfolded conformation (the mutant pre- natural substrates of the enzyme (GM1 ganglioside, cursor would not exit the ER). For example, Oshima galactose-terminal neutral oligosaccharides and ker- et al. [94] expressed the Arg201Cys mutation and atan sulfate). However, this does not seem to hap- showed it produced a misfolded protein, as expected, pen. Furthermore, infantile patients with GM1 gan- while the Arg201His mutation alone was reported to gliosidosis have unequivocal skeletal abnormalities at produce 46.5% of the normal level of activity. Four least as severe as seen in Morquio B disease, but they mutations occur in exon 16 near the C-terminal do not display the same massive storage of keratan (from residue 579^677), a region which is thought sulfate, while patients with a primary keratan sulfate to be essential for complex formation in the lysosome storage do not show massive accumulation of GM1 but which is presumed to be lost (there is no direct ganglioside in the brain. To reconcile the exclusiv-

BBADIS 61880 10-9-99 J.W. Callahan / Biochimica et Biophysica Acta 1455 (1999) 85^103 101 ity of storage substances in these diseases, one or Acknowledgements more of the following explanations seem correct. (i) Trp273 is important, if not essential, for binding to This work was supported by the Medical Research either the terminal galactose or the terminal disac- Council of Canada. charide of keratan sulfate, probably because this dis- accharide contains a 6-sulfated-N-acetylglucosamine, to secure it at the active site. Trp273 is less critical in References the binding of other substrates. There is some evidence in favor of this [67,68,74,108]. (ii) Binding of [1] T. Mutoh, M. Naoi, A. Takahashi, M. Hoshino, Y. Nagoi, keratan sulfate at the active site is favored over oli- T. Nagatsu, Neurology 36 (1986) 1237^1241. gosaccharides. Binding of GM1 ganglioside is least [2] T. Mutoh, M. Naoi, T. Nagatsu, A. Takahashi, Y. Matsuo- favored as it requires association with the activator ka, Y. Hashizume, N. Fujiki, Biochim. Biophys. Acta 964 (1988) 244^253. protein, saposin B. Any mutation a¡ecting the sap- [3] R.M. Norman, H. Urich, A.H. Tingey, R.A. Goodbody, osin binding site would reduce the ability to bind J. Pathol. Bacteriol. 72 (1959) 409^421. saposin^GM1 complex and abolishing hydrolysis [4] B.H. Landing, F.N. Silverman, M.M. Craig, M.D. Jacoby, GM1 ganglioside. GM1 gangliosidosis would result. M.E. Lahey, D.L. Chadwick, Am. J. Dis. Child. 108 (1964) In addition, many mutations found in GM1 ganglio- 503^522. sidosis are heteroallelic with one mutation being of [5] J.S. O'Brien, M.D. Stern, B.H. Landing, J.K. O'Brien, G.N. Donnell, Am. J. Dis. Child. 109 (1965) 338^346. the missense type, but none involve Trp273. (iii) Mis- [6] D.M. Derry, J.S. Fawcett, F. Andermann, L.S. Wolfe, Neur- sense mutations a¡ect folding but misfolded Gal may ology 18 (1968) 340^348. be catalytically active as we have shown for [7] L.S. Wolfe, J. Callahan, J.S. Fawcett, F. Andermann, C.R. Arg148Ser. In these instances, we assume su¤cient Scriver, Neurology 20 (1970) 23^44. enzyme exits the ER, reaches the lysosome and cata- [8] J.A. Lowden, J.W. Callahan, R.A. Gravel, M.A. Skomo- rowski, L. Becker, J. Groves, Neurology 31 (1981) 719^724. lyzes hydrolysis of the most favorably bound sub- [9] Y. Suzuki, N. Nakamura, K. Fukuoka, Y. Shimada, M. strates, i.e., keratan sulfate. Therefore, keratan sul- Uono, Hum. Genet. 36 (1977) 219^229. fate out-competes oligosaccharides and GM1 [10] K. Yoshida, A. Oshima, H. Sakuraba, T. Nakano, N. Ya- ganglioside for the residual enzyme present. Since nagisawa, K. Inui, S. Okada, E. Uyama, R. Namba, K. hydrolysis of keratan sulfate does not require saposin Kondo, S. Iwasaki, K. Takamiya, Y. Suzuki, Ann. Neurol. B, and the turnover rate for maintenance is predicted 31 (1992) 328^332. [11] Y. Suzuki, H. Sakuraba, A. Oshima, in: C.R. Scriver, A. to be lower than for GM1 ganglioside, the catalytic Beaudet, W.S. Sly, D. Valle (Eds.), Metabolic Basis of In- requirements are more easily met even by the lowest herited Disease, vol. 2, 7th ed., McGraw-Hill, New York, residual enzyme level and keratan sulfate metabolism 1995, pp. 2785^2823. can occur. (iv) No studies have been reported on the [12] J.S. O'Brien, E. Gugler, A. Giedion, U. Weissmann, N. role of S-Gal in these diseases. Our preliminary data Herschkowitz, C. Meier, J. Leroy, Clin. Genet. 9 (1976) 495^504. suggest it is involved in the formation of extracellular [13] A.I. Arbisser, K.A. Donnelly, C.I. Scott, N. DiFerrante, J. matrix and may be uniquely involved in extracellular Singh, R.E. Stevenson, A.S. Aynesworth, R.R. Howell, Am. keratan sulfate deposition. Expression in S-Gal of J. Med. Genet. 1 (1977) 195^205. the mutations which a¡ect Gal are required to an- [14] J.J. Van Gemund, M.A.H. Giesberts, R.F. Eerdmans, W. swer this question. Finally, some of the mutations Blom, W.J. Kleijer, Hum. Genet. 64 (1983) 50^54. listed in Table 3 (homozygous chain termination mu- [15] A. Koto, A.L. Horwitz, K. Suzuki, C.W. Ti¡any, K. Suzuki, Ann. Neurol. 4 (1978) 26^36. tations) are predicted to disrupt synthesis of viable [16] R. Giugliani, M. Jackson, S.J. Skinner, C.M. Vimal, A.H. forms of both Gal and S-Gal and where found the Fensom, N. Fahmy, A. Sjovall, P.F. Benson, Clin. Genet. 32 patients should manifest the features of both GM1 (1987) 313^325. gangliosidosis and Morquio B disease. However, the [17] Y. Okamura-Oho, S. Zhang, J.W. Callahan, Biochim. Bio- reports do not mention the coincidence of clinical phys. Acta 1225 (1994) 244^254. [18] A. D'Azzo, G. Andria, P. Strisciuglio, H. Galjaard, in: C.R. features of both diseases. Further work is therefore Scriver, A. Beaudet, W.S. Sly, D. Valle (Eds.), Metabolic needed at the clinical, biochemical and molecular Basis of Inherited Disease, vol. 2, 7th ed., McGraw-Hill, levels to resolve these questions. New York, 1995, pp. 2825^2837.

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