11 Sphingolipid Hydrolysis

Sphingolipid Hydrolysis ROSCOE 0. BRADY I. Introduction and Perspective . . . . . . . . . . . . . . . . . . 11. Purification and Properties . . . . . . . . . . . ..... . A. Hydrophobic Chromatography . . . . . . . . . . .. B. Lectin Affinity Column Chromatography , . . . . . . . . . . C. Affinity Chromatography with Ligands That Interact with Active Sites . . . . , . . . . . . . . . . . . . . . . . . . . D. High-pressure Liquid Chromatography . . . . . . . . . . . E. Comments on Purification of the Sphingolipid Hydrolases . . . . F. Size of Sphingolipid Hydrolases and Processing . . . . . . . 111. The Reactions Catalyzed . . . . . . . , . . . . . . . . . . . . A. Site and Nature of Catalytic Reactions . . . . . . . . . . B. Requirements . , . . . . . , . . . . . . . . . . . . . C. Kinetics. . . . . . . . . . . . . . . . . . . . . . . . D. Inhibitors ., . .. . . . . .... . . . . ...... IV. Biological Role . .. . . . . . . . . . . . . . .. . V. Research Applications . . . . . . . . . . . . . .

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409 411 411 412

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413 413 414 414 415 415 416 419 420 422 423

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I. Introduction and Perspective Since the demonstration in the mid- 1960s that subnormal activity of lipid-catabolizing enzymes was the biochemical basis of sphingolipid storage disorders in humans, the enzymes that catalyze the hydrolysis of these lipids have come under increasingly intensive investigation. Biochemists and biologists concerned with the pathogenesis and therapy of patients with these metabolic diseases have had to become familiar with the special properties of these lipids and enzymes, about which little information was previously available. Sphingolipids are primarily mem409 THE ENZYMES, VOL. XVI Copyright 8 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-122716-2

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brane components. They contain a common lipid portion called ceramide, which is composed of the long-chain amino alcohol sphingosine [CHI-(CHz) ,Z-CH=CH-CH(OH)-CH(NH~)-CH~OHI

to which a long-chain fatty acid is linked by an amide bond to the nitrogen atom on carbon-2 of sphingosine. Hydrophilic residues such as hexoses and phosphocholine are joined to carbon atom 1 of the sphingosine moiety of ceramide. The principal enzymes involved in sphingolipid catabolism are indicated in Figs. 1 and 2. Specific enzyme deficiency disorders in humans that have been identified for reactions in Fig. 1 are: generalized (GMJ gangliosidosis (Reaction 8) and Tay-Sachs and Sandhoff’s diseases (Reaction 11). Insufficient activity of the neuraminidase that catalyzes Reaction 5 has been implicated in the hereditary condition known as mucolipidosis IV. Disorders linked with reactions shown in Fig. 2 are as CER - GIs

- GAL - GALNAc - GAL I

NwNAc - NsvNAc

I

CER

I

NsuNAc- NavNAc

- GIE - GAL - GALNAc - GAL

I

CER

CER - Glc

I

NsuNAs - NsvNAc

NauNAc

- GIc - GAL - GALNAc - GAL

I

@

- GAL - GALNAe - GAL

lGDlb‘

NeuNAc

I

- GIc - GAL - GALNAc - G AL I NarNAc

- GAL - GALNAc - GAL

lGM1l

CER - G I s -G A L

I

I

NeuNAc

CER - 0 1 s - G AL - GALNAc

ICERAMIDELACTDSIDEI

I

“Tlb’

NwNAc - NwNAs

NwNAc

CER

CER - GIc

CER - Glc

I

NeuNAc

- GAL - GALNAe - GAL I I NauNAc - NeuNAc NwNAc

- GALNAc

CER -01s -G A L

IGM21

-h N A c

CER -GI= - GAL

Fro. 1. Pathways involved in the catabolism of gangliosides. The numbers in circles refer to the sites of action of sphingolipid hydrolases. Abbreviations used are: CER = ceramide; Glc = glucose; GAL = galactose; NeuNAc = N-acetylneuraminic acid; GALNAc = N-acetylgalactosarnine. GTET, GTla,etc., is a widely used convention to designate gangliosides. Reproduced with permission from Ref. ( l a ) .

41 1

11. SPHINGOLIPID HYDROLYSIS H-ISOANTIGEN

CER-GLc-GAL-GLcNAc-GAL-

Fuc

@

Fuc

4

CER-GLc-GAL-GLcNAc-GAL GAL

CER-GLc-GAL-GAL-GALNAc

@

@

CER-GLc-GAL-GLcNAc

SULFATIDE

CER-GAL-SO4

%+O

GALACTOCEREBROSIDE

1

CER-GAL GAL

@

GLOBDSIDE

GALNAc

CER-GLc-GAL-GAL

t

OF

CER-GLc-GAL

CERAMIDETRIHEXOSIDE

CERAMIDE LACTOSIDE

GAL GLUCOCEREBROSIDE

CE R-G Lc GLc

1-

P-CHOLINE

CER L C E R - P - C H O L i N E FATTY ACID

0

63

SPHlNGOMvELlN

SPHINGOSINE

FIG.2. Pathways involved in the catabolism of sphingolipids other than gangliosides. Abbreviations are the same as in Fig. 1, plus GlcNAc = N-acetylglucosamine; Fuc = fucose; P-choline = phosphocholine. Reproduced with permission from Ref. (la).

follows: fucosidosis (Reaction 1); Sandhoff’s disease (Reaction 4, as well as Reaction 11 in Fig. I); Fabry’s disease (Reaction 5); Gaucher’s disease (Reaction 7); metachromatic leukodystrophy (Reaction 8); Krabbe’s disease (Reaction 9); Niemann-Pick disease (Reaction 10); and Farber’s disease (Reaction 11). The majority of these disorders are accompanied by severe mental retardation, enlargement of the spleen and liver, and impairment of function of a number of other organs. The notable exception to nervous system damage occurs in patients with Type 1 Gaucher’s disease. Here, peripheral organomegaly and skeletal damage predominate. Because there are significant differences in the clinical manifestations in patients with the various disorders, appropriate references should be consulted for details (2-3). 11. Purification and Properties

A. HYDROPHOBIC CHROMATOGRAPHY Comparatively few of the enzymes indicated in Figs. 1 and 2 have been isolated in a homogeneous state. One of the principal difficulties is that a 1. Brady, R. 0. (1978). Annu. Rev. Biochem. 47, 687. la. Brady, R. 0. (1982). In “Pediatrics” (A. M. Rudolph, ed.), 17th ed., p. 311. Appleton, New York. 2. Brady, R. 0. (1982). Annu. Rev. Neurosci. 5 , 33. 3. Stanbury, J. B.,Wyngaarden, J. B., Fredrickson, D. S., Brown, M. S., and Goldstein, J. L. eds. (1983). “The Metabolic Basis of Inherited Disease,” 5th ed. McGraw-Hill, New York.

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large number of these catalysts are firmly bound to membranes. This requires extensive use of detergents for the extraction of the enzymes and the inclusion of detergents in column chromatography steps. Hydrophobic column chromatography, when it became available, provided a major aid in the purification of several of these enzymes. The enzymes are fractionally eluted from phenyl- or octyl-Sepharose or decyl-agarose columns with comparatively high concentration gradients of ethylene glycol. This adaptation provided a major breakthrough for the isolation of glucocerebrosidase from human placental tissue in a degree of purity and yield that has permitted enzyme replacement trials in Gaucher’s disease [Ref. (4); also see Chapter 201. Hydrophobic chromatography has also been used to isolate sphingomyelinase (5-7). Similarly, chromatography on phenyl-Sepharose was used in the isolation of GM1-/3-galactosidase(8).

B. LECTINAFFINITY COLUMNCHROMATOGRAPHY The discovery that sphingolipid hydrolases were highly enriched in lysosomes (9) and that lysosomal enzymes were glycoproteins (ZO) has led to the frequent use of covalently bound lectin column chromatography as a major step in the isolation of these enzymes (7, ZI-14). The most frequently employed lectin is concanavalin A bound to Sepharose. Many sphingolipid hydrolases bind tightly to such columns and they can usually be eluted with solutions of a-methylmannoside in a range of 0.2 to 0.5 M ; 1 M NaCl is often included in the eluting mixture. The process is frequently performed at room temperature. This step is particularly helpful since nonglycoproteins pass through the column, or are eluted with dilute concentrations of a-methylglucoside or a-methylmannoside. However, care must be exercised when using this procedure for the preparation of enzymes that are to be used in humans because some concanavalin A frequently leaches from such columns (4). Concanavalin A is a potent mitogen and can react with a number of cell receptor sites. It appears 4. Furbish, F. S., Blair, H. W., Shiloach, J . , Pentchev, P. G . , and Brady, R. 0. (1977). PNAS 74, 3560. 5. Jones, C. S., Shankaran, P., and Callahan, J. W. (1981). BJ 195, 373. 6. Yarnanaka, T., and Suzuki, K. (1982). J . Neurochem. 38, 1753. 7. Sakuragawa, N. (1982). J . Biochem. (Tokyo)92,637. 8. Yamarnoto, Y., Fujie, M., and Nishimura, K. J. (1982). J . Biochem. (Tokyo)92, 13. 9. Weinreb, N. J., Brady, R. O., and Tappel, A. L. (1968). BBA 159, 141. 10. Goldstone, A. P., Konecky, P., and Koenig, H. (1971). FEBS Lett. 13, 58. 11. Dale, G. L., and Beutler, E. (1976). PNAS 73, 4672. 12. Kusiak, J. W., Quirk, J. M., and Brady, R. 0. (1978). JBC 253, 184. 13. Fiddler, M. B., Ben-Yoseph, Y., and Nadler, H. L. (1979). BJ 177, 175. 14. Saralian, T. W., Fluharty, A. L., Kihara, H., Helfan, D. G . , and Edmond, J. (1982). J . Appl. Biochem. 4, 661.

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SPHINGOLIPID HYDROLYSIS

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advisable to ensure that no concanavalin A is present in enzyme preparations intended for human administration.

C. AFFINITYCHROMATOGRAPHY WITH LIGANDS THAT INTERACTWITH ACTIVESITES The demonstration that a number of enzymes could be considerably enriched by affinity column chromatography (15) suggested that ligands consisting of hexoses attached via unhydrolyzable thiohemiacetal bonds might be used for the purification of glycosidases that are involved in the degradation of sphingolipids. Distler and Jourdian successfully applied this technique to isolation of P-galactosidase from bovine testis (16). This approach has been extraordinarily useful for the purification of P-galactosidases from human liver (17-19) and from feline liver and brain tissue (20). A similar technique has been used in the purification of ceramidetrihexosidase, an a-galactosidase (21). An affinity column consisting of 2acetamido-N-(~-am~nocaproyl)-2-deoxy-~-~-glucopyranosylam~ne linked to Sepharose 4B has been helpful in isolating large amounts of human placental hexosaminidase A (22). A novel ligand consisting of sphingosylphosphocholine coupled to Sepharose 4B has been used in the isolation of sphingomyelinase (5). Although the enrichment obtained during this step was minimal, it was stated that this procedure was required in the purification scheme in order to obtain enzyme with high specific activity.

D.

HIGH-PRESSURE

LIQUIDCHROMATOGRAPHY

High-pressure liquid chromatography (HPLC) is increasingly being used in the isolation of peptides and proteins. This procedure has been extraordinarily helpful in obtaining homogeneous glucocerebrosidase in quantities in the range of tens of milligrams (23). Pure enzyme in this amount is useful for many studies that would otherwise be very difficult if 15. Cuatrecases, P., and Anfinsen, C. B. (1971). “Methods in Enzymology,” Vol. 22, p. 345. 16. Distler, J. J . , and Jourdian, G . W. (1973). JBC248, 6772. 17. Norden, A. G. W., Tennant, L. L., and O’Brien, J. S . (1974). JBC 249, 7969. 18. Miller, A. L., Frost, R. G., and O’Brien, J. S. (1977). BJ 165, 591. 19. Frost, R. G., Holmes, E. W., Norden, A. G. W . , and O’Brien, J. S.(1978). BJ 175, 181. 20. Anderson, J. K . , Mole, J. E . , and Baker, H. J . (1978). Biochemistry 17, 467. 21. Bishop, D. F . , and Desnick, R. J. (1981). JBC256, 1307. 22. Geiger, B . , Ben-Yoseph, Y., and Arnon, R. (1974). FEBS Lett. 45, 276. 23. Furbish, F. S., Barranger, J. A., Murray, G . J., Oliver, K., Rands, P., Ginns, E. I., Stowens, D. W., and Brady, R. 0. (1982). FP 41, 629.

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not impossible to undertake. It is expected that HPLC will eventually be used in the isolation of a number of the sphingolipid hydrolases, such as sphingomyelinase, galactocerebroside-P-galactosidase,and ceramidase; these compounds have been especially refractory to purification to homogeneity by conventional methods.

E. COMMENTS ON PURIFICATION OF THE SPHINGOLIPID HYDROLASES Several deductions seem apparent in considering the isolation of sphingolipid hydrolases. The fact that many of these enzymes are extremely hydrophobic originally hindered, but subsequently helped, in their purification after this property was appreciated and hydrophobic column chromatography became available; it is being used with increasing frequency for the purification of these enzymes. It seems likely that this procedure will play a major role in the isolation of galactocerebroside-pgalactosidase, sphingomyelinase, and ceramidase, in addition to glucocerebrosidase. Note that these enzymes catalyze the hydrolysis of components that are a part of (ceramide), or immediately adjacent to, the extremely hydrophobic portion of these molecules. It therefore seems reasonable that the enzymes involved in their catalysis would have domains unusually rich in hydrophobic amino acids. This has been shown to be true in the case of glucocerebrosidase, the only enzyme of this group that has been convincingly demonstrated to be homogeneous (23). In contrast, affinity columns consisting of unhydrolyzable sugar or amino sugar derivatives seem to be much more effective for the purification of enzymes that catalyze the cleavage of carbohydrate residues more remote from the ceramide core. The observation that this technique was particularly inefficient with regard to the purification of sphingomyelinase (5) is consistent with this deduction. Purification by bound ligands appears particularly applicable in the case of hexosaminidases A and B and ganglioside GM1P-galactosidase. Here two- and three-sugar residues, respectively, separate the nonreducing terminal molecules from the hydrophobic ceramide portion. It is conceivable that appropriately designed spacer arms will permit similar enrichment of enzymes that hydrolyze sphingolipids containing fewer components. The combination of hydrophobic chromatography and an appropriate affinity ligand could be useful for the isolation of these enzymes. F. SIZEOF SPHINGOLIPID HYDROLASES AND PROCESSING Most of the sphingolipid hydrolases have molecular weights in the range of 50,000 to 60,000. These are considered to be the mature tissue

1 1 . SPHINGOLIPID HYDROLYSIS

415

forms that have arisen from the processing of larger precursors (24, 25). The mature forms of many of these enzymes have a tendency to form higher-molecular-weight polymers (26, 27). Hexosaminidase A is composed of polypeptide subunits termed (Y and P (28).The larger, (Y, has a M, of 50,000. Polypeptides in the P-subunit have been reported to be dissimilar in size (29). The proposed structure for hexosaminidase A is aPaPb. Hexosaminidase B is a tetramer of P chains linked by a disulfide bridge (30, 31). Its structure may actually be 2(PaPb)(29). The genetic code for the (Y chain is located on chromosome 15, and that for the P chain(s) on chromosome 5 (32, 33). 111. The Reactions Catalyzed

A. SITEA N D NATURE OF CATALYTIC REACTIONS

The catabolism of sphingolipids is initiated through the action of hydrolytic enzymes that cleave nonreducing terminal molecules of neuraminic acid, hexoses, or hexosamines from sphingoglycolipids. Related enzymes catalyze the removal of sulfuric acid from sulfatide (Fig. 2, Reaction 8), phosphocholine from sphingomyelin (Fig. 2, Reaction lo), and fatty acid from ceramide (Fig. 2, Reaction 11). The removal of these components occurs in a stepwise fashion. Catabolism stops and the sphingolipid accumulates if there is inadequate activity of any of the enzymes. No evidence has been obtained for an endoglycosidase type of reaction involving sphingolipids. It has been speculated that the enzymic hydrolysis of these lipids may occur in a concerted fashion, perhaps while the substrate is associated with an array of enzymes on lysosomal membranes (1). Some support for this notion is seen in the pathology that occurs in many sphingolipid storage disorders. Generally there is an intracellular accumulation of materials in the form of multilamellated membranous bodies. 24. Hasilik, A., and Neufeld, E. F. (1980). JBC 255, 4937. 25. Ginns, E. I . , Erickson, A . , Bameveld, R., Brady, R. O., Tager, J . , and Barranger, J. A. (1983). Isozymes: Curr. Top. Biol. Med. Res. (in press). 26. Pentchev, P. G., Brady, R. O . , Gal, A. E., and Hibbert, S. R. (1977). BBA 488,312. 27. Gatt, S. (1982). In “Phospholipids in the Nervous System, Vol I . Metabolism” (L. A. Horrocks, G . B. Ansell, and G. Porcellati, eds.), p. 181. Raven Press, New York. 28. Beutler, E. (1979). Am. J . Hum. Genet. 31, 105. 29. Mahuran, D. J . , Tsui, F., Gravel, R. A., and Lowden, J. A. (1982). PNAS 79, 1602. 30. Mahuran, D . , and Lowden, J . A. (1980). Can. J . Biochem. 58, 287. 31. Mahuran, D., and Lowden, J. A. (1981). Can. J . Biochern. 59, 237. 32. Lalley, P. A., Rattazzi, M. C., and Shows, T. B. (1974). PNAS 71, 1569. 33. Gilbert, F., Kucherlapati, R., Creagan, R. P., Mumane, M. J., Darlington, G . J., and Ruddle, F. H. (1975). PNAS 72, 263.

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These cytoplasmic bodies, isolated from tissues of patients with sphingolipid storage disorders, contain the characteristic accumulating lipid. In addition, high specific activity of a number of lysosomal enzymes has been shown to be associated with these inclusions (34). Perhaps the uncatabolized lipid cannot dissociate from the lysosomal membrane. As a result the cell is required to produce more lysosomes to store the accumulating lipid and to continue to carry out other lysosomal functions. This increase in tertiary lysosomes (storage vacuoles) could account for another poorly understood phenomenon; that is, the frequently observed increase in activity of a number of related lysosomal enzymes in tissues obtained from patients with deficiency of a particular sphingolipid hydrolase (35).

B. REQUIREMENTS 1. Acidic Environment

A majority of the reactions involving sphingolipid hydrolysis take place under comparatively strong acidic conditions. For example, the optimal pH for rat brain ganglioside GM2 and GDlaneuraminidase is 4.4 (36),and for human placental GM2 hexosaminidase, 4.2 (37). p-Galactosidases with pH optima of 4.2 and 4.8 (38, 39), as well as more neutral P-galactosidase(s) that catalyze the hydrolysis of ceramidelactoside (40), have been reported. The optimal pH for glucocerebroside hydrolysis is 5.9 to 6.0 (4, 41). The apparently gradual increase in the pH optimum with a shortening of the sphingoglycolipid chain may be important. This gradient may only apply to sphingoglycolipids produced through the concerted sequential action of several hydrolases. The more neutral ceramidelactoside-P-galactosidases may be involved in the catabolism of sphingolipids derived from larger precursors. Note that the pH optima for the hydrolysis of sphingomyelin and galactocerebroside are 5.0 (26) and 4.6 (42), respectively. The catabolism of these components commences ab initio for 34. Tallman, J. F., Brady, R. O . , and Suzuki,K. (1971). J . Neurochem. 18, 1775. 35. Brady, R. O . , O'Brien, J. S., Bradley, R. M . , and Gal, A. E. (1970). BBA 210, 193. 36. Tallman, J. F., and Brady, R. 0. (1972). JBC 247,7570. 37. Tallman, J. F., Brady, R. O., Quirk, J. M., Villalba, M., and Gal, A. E. (1974). JBC 249, 4389. 38. Wenger, D. A. (1974). Chem. Phys. Lipids 13, 327. 39. Tanaka, H., and Suzuki, K. (1975). JBC 250, 2324. 40. Ben-Yoseph,Y., Shapira,E., Edelman, D., Burton, B. K., and Nadler, H. L. (1977). ABB 184, 373. 41. Brady, R. O . , Kanfer, J., and Shapiro, D. (1965). JBC 240, 39. 42. Wenger, D. A., Sattler, M., and Roth, S. (1982). BBA 712, 639.

11.

SPHINGOLIPID HYDROLYSIS

417

sphingomyelin and for galactocerebroside. However, a portion of the latter lipid is also derived from sulfatide through the action of sulfatase, the immediately preceding step. Several enzymes seem to be involved in the deacylation of ceramide, the penultimate product of all of these reactions. A ceramidase with a pH optimum of 4.8 appears to be associated with lysosomes (43, 44). Other ceramidases have been reported that are maximally active at neutral or alkaline conditions (45). One of these with a pH optimum of 7.6 has been demonstrated in the small intestine (46). Since it has been shown that sphingolipids are excreted from the liver via the bile (47) and that intestinal mucosa has comparatively high levels of sphingolipid hydrolytic activities (48,49),the catabolism of these substances in the small intestine may constitute an important metabolic pathway.

2. Actiuators a. Detergents. It has long been known that assays of enzymes involved in the hydrolysis of sphingolipidsgenerally require the presence of detergents so that the water-insoluble substrate can interact with the enzyme. Nonionic detergents such as Cutscum (isooctylphenoxypolyoxyethanol)or Triton X-100are frequentlyemployed.However,it is also known that the inclusion of various bile salts in the reaction mixture greatly increases the activity of many of these enzymes, such as gluco- and galactocerebrosidase (48) and ceramidetrihexosidase (49). Important studies on the mechanism of activation by bile salts have been carried out by Gatt and his co-workers, particularly with regard to the hydrolysis of sphingomyelin (50). The inclusion of bile salts in the assays, alone or in combination with other detergents, is not a trivial matter. Only through the judicious optimization of galactocerebroside-p-galactosidaseactivity with a combination of bile salts and other detergents was it possible to demonstrate the enzymatic defect in Krabbe’s disease (52). The field has been clouded by the use of bile salts of varying purity. “Crude” bile salt preparations frequently provide greater stimulation than individual salts 43. Gatt, S. (1966). JEC 241, 3724. 44. Yavin, E., and Gatt, S. (1969). Biochemistry 8, 1692. 45. Chen, W. W., Moser, A. B., and Moser, H. W. (1981). ABB 208,444. 46. Nilsson, A. (1969). BEA 176, 1969. 47. Pentchev, P. G., Gal, A. E., Wong, R . , Morrone, S., Neumeyer, B. A., Massey, J., Kanter, R . , Sawitsky, A., and Brady, R. 0. (1981). EEA 665,615. 48. Brady, R. O., Gal, A. E., Kanfer, J. N., and Bradley, R. M. (1965). JEC 240, 2766. 49. Brady, R. O., Gal, A. E., Bradley, R. M., and Martensson, E. (1967). JBC 242,1021. 50. Gatt, S., Dinur, T., Yedgar, S., and Leibovitz-Ben Gershon, Z. (1978). Adu. Exp. Med. B i d . 101, 487. 51. Suzuki, K., and Sukuki, Y. (1970). PNAS 66,302.

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or synthetic mixtures. However, in order to maximize reproducibility, most investigators now use chemically defined products in these assays. b. “Heat-stable” proteins. Given the artificiality of in vitro assays that required the use of detergents and/or bile salts for the interaction of sphingolipid substrates with isolated enzymes, a primary concern of biochemists was whether there were naturally occurring counterparts of these substances. The earliest indication that such materials existed was provided by Mehl and Jatzkewitz, who demonstrated that the activity of arylsulfatase A on sulfatide was enhanced by the addition of a noncatalytically active protein of M,21,500 (52, 53). Futher studies revealed that this activator was localized in lysosomes (54) and that it bound to sulfatide in a 1 to 1 complex (55). A similar substance that was stable to heating was found to enhance the activity of glucocerebrosidase(56).This material was shown to be a rather acidic glycoprotein with an apparent M, of 11,000, and binding between enzyme and activator required the presence of phospholipid (57,58). Berent and Radin prepared an activator of glucocerebrosidase from bovine spleen that required the addition of phosphatidylserine for maximal activation (59). The specificity of the effect of this factor on glucocerebrosidase was questioned by Wenger et al. (60), who demonstrated that it stimulated the hydrolysis of galactocerebroside and sphingomyelin as well as glucocerebroside; however, this material did not accelerate the degradation of sulfatide or ganglioside GM,. Another important contribution in this area was the discovery of Li and co-workers that liver contains a heat-stable nondialyzable factor that promotes the catabolism of ganglioside GM2 by purified hexosaminidase A (62). This report and subsequent studies by Conzelmann and Sandhoff (62) and by Hechtman and co-workers ( 6 3 , which indicate a role in substances of this type in the pathogenesis of human sphingolipid storage 52. Mehl, E., and Jatzkewitz, H. (1968). BBA 151, 619. 53. Fischer, G., and Jatzkewitz, H. (1975). Hoppe-Seyler’s Z . Physiol. Chem. 356,605. 54. Mraz, W., Fischer, G . , and Jatzkewitz, H. (1976). Hoppe-Seyler’s Z. Physiol. Chem. 357, 1181. 55. Fischer, G . , and Jatzkewitz, H. (1977). BBA 481, 561. 56. Ho, M. W., and OBrien, J. S. (1971). PNAS 68, 2810. 57. Ho, M. W., and Light, N. D. (1973). BJ 136, 821. 58. Ho, M. W., and Rigby, M. (1975). BBA 397, 267. 59. Berent, S. L., and Radin, N. S . (1981). AEB 208, 248. 60. Wenger, D. A., Sattler, M., and Roth, S. (1982). BBA 712, 639. Mazzotta, M. Y., Wan, C.-C., Orth, R., and Li, S.-C. (1973). JBC 248, 61. Li, Y.-T., 7512. 62. Conzelmann, E., and Sandhoff, K. (1978). PNAS 75, 3979. 63. Hechtman, P., Gordon, B. A., and Ng Ying Kin, N. M. K. (1982). Pediatr. Res. 16, 217.

11. SPHINGOLIPID HYDROLYSIS

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diseases (see Section IV), have stimulated considerable research in this area. In particular, it has been known for a decade that the cleavage of N-acetylgalactosamine from ganglioside GM2by purified hexosaminidase A is extraordinarily slow compared with the activity of this enzyme on artificial substrates such as 4-methylumbelliferyl-~-~-galactosaminide (or 4-methylumbelliferyl-~-~-glucosaminide since the enzyme does not discriminate between these epimers) (37).It was therefore believed that an effector required for the interaction of the glycolipid had been removed in the purification of the enzyme. The data support this contention and provide a degree of insight into the interaction of such a cohydrolase with enzyme and substrate (64).A similar heat-stable activator protein has been reported to be involved in the pathogenesis of a variant form of metachromatic leukodystrophy (65). c. Phospholipids. Although stimulation of numerous enzymes by the addition of phospholipids is amply documented, the mechanism of phospholipid activation of sphingolipid hydrolases is not well understood. By and large the increase caused by the addition of phospholipid is significant but not great. Phosphatidylserine appears to be the most active in this regard, having been shown to augment glucocerebrosidase (66)and galactocerebroside-p-galactosidase(67)activity. It is presumed that the association of the enzyme with lipid results in an interaction of substrate and catalyst in a configuration more closely resembling physiological conditions than that obtained by the enzyme alone. However, glucocerebrosidase from which lipid has been removed by extraction with butanol retains complete catalytic activity in the presence of Cutscum and sodium taurocholate (4). Additional experimentation on the physical chemistry of sphingolipid catalysis is needed to clarify these uncertainties.

C. KINETICS Probably the most detailed studies of the kinetics of sphingolipid metabolism have been carried out over the years by Gatt and his associates (50, 68, 69). They have analyzed in critical fashion the effect of substrate, detergents, bile salts, and pH on the activity of sphingolipid hydrolases. 64. Conzelmann, E., and Sandhoff, K. (1979). Hoppe-Seyler's Z. Physiol. Chem. 360, 1837. 65. Stevens, R. L., Fluharty, A. L., Kihara, H . , Kabach, M. M., Shapiro, L. J., Marsh, B., Sandhoff, K., and Fischer, G. (1981). Am. J . H u m . Genet. 33, 900. 66. Dale, G. L., Villacorte, D. G . , and Beutler, E. (1976). BBRC 71, 1048. 67. Hanada, E . , and Suzuki, K . (1979). BBA 575,410. 68. Gatt, S., and Barenholz, Y . (1973). Annu. Rev. Biochem. 42, 61. 69. Gatt, S., Dinur, T., and Desnick, R. J. (1982). Prog. Clin. B i d . Res. 95, 315.

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By and large, the V,, of these enzymes is rather low compared with that of many enzymes, although these substances are comparatively tightly associated with their catalysts, with K,’s in the range of 5 x lo-’ M. Specific enzymatic activity is in the range of 0.02mmole/mg of protein per minute for highly purified glucocerebrosidase and sphingomyelinase with the respective sphingolipid substrates (4, 6). Specific activity twenty to thirty times greater is found with purified hexosaminidase A using fluorogenic substrates (37). However, the maximal velocity of ganglioside G M 2 catabolism under optimal incubation conditions is thousands of times less than that with artificial substrates. The hydrolysis of ganglioside G M i by purified P-galactosidase occurs at about one-sixth the rate observed with 4-methyl-~-~-galactopyranoside (17). However, the hydrolysis of glucocerebroside by purified glucocerebrosidase actually exceeds the maximal velocity obtained with artificial substrates such as 4-methyl-P-~-glucopyranoside (70). Little progress has been made in identifying the cause of these wide discrepancies in catalytic properties, although the interaction of enzymes with monomeric lipid substrates or in the form of micelles has been extensively investigated. D. INHIBITORS The ceramide that is produced by several of these reactions (Fig. 2, Reactions 7, 9, 10) is moderately inhibitory (48). Cholesterol, which is often found in increased quantity in tissues of patients with Niemann-Pick disease, has been reported to inhibit the activity of sphingomyelinase (72).This finding may be important for the pathogenesis of Type D (Nova Scotia variant) of Niemann-Pick disease, where cholesterol accumulates in the tissues, although it has not been possible to demonstrate a deficiency of sphingomyelinase in v i m (72). Deacylated sphingosine derivatives, such as glucosylsphingosine and galactosylsphingosine (psychosines), are more potent inhibitors (48, 69). By and large the water-soluble products resulting from the action of these enzymes exert comparatively little effect on the reactions. However, a notable exception was found in an examination of inhibitors of placental a-galactosidases. Most human tissues contain two a-galactosidases. One of these, a-galactosidase A, is 70. Pentchev, P. G., Brady, R. O . , Balk, H. E., Britton, D. E., and Sorrell, S . H. (1978). PNAS 75, 3970. 71. Maziere, J. C., Wolf, C., Maziere, C., Mora, L., Bereziat, G . , and Polonovski, J. (1981). BBRC 100, 1299. 72. Brady, R. 0. (1983). I n “The Metabolic Basis of Inherited Disease” (J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, J. L. Goldstein, and M. S. Brown, eds.), 5th ed., p. 831. McGraw-Hill, New York.

11. SPHINGOLIPID HYDROLYSIS

42 1

involved in the catabolism of ceramidetrihexoside and is lacking in patients with Fabry’s disease (73). The other, a-galactosidase B, may in fact actually be an a-N-acetylgalactosaminidasethat also catalyzes the hydrolysis of fluorogenic or chromomogenic a-galactopyranosides (74). Kusiak and co-workers found that the activity of a-galactosidase A was unaffected by the addition of N-acetyl-D-galactosamineto the incubation mixtures, whereas a-galactosidase B was strongly inhibited by this amino sugar (12). Mayes and his associates astutely applied this information to discriminate between a-galactosidase A and B activities in unfractionated tissue extracts, thereby increasing the specificity of these assays for the diagnosis of Fabry’s disease (75). Conduritol-/3-epoxide, a 1,Zanhydroinositol, has been found to be a potent inhibitor of glucocerebrosidase. It has been used to produce a model of Gaucher’s disease in mice, with modest but significant accumulation of glucocerebroside in the tissues of the treated animals (76). Another important application of this inhibitor has been its use to investigate the turnover of glucocerebrosidase (77). A similar active site-directed inhibitor has been synthesized that inhibits P-galactosidases (78). Several inhibitors of neuraminidase have also been described (79, 80). In contrast, changing the enantiomorphic configuration of hexoses apparently does not produce an inhibitory substance. This aspect was investigated by Gal and co-workers, who synthesized L-glucocerebroside (81). The L-enantiomorph was not hydrolyzed by purified glucocerebrosidase or by other tissue glucosidases. Investigation of the fate of this substance in uiuo led to the discovery of the biliary excretion of sphingoglycolipids (47). Compounds such as this are expected to play an important role in elucidating pathogenetic mechanisms in the sphingolipidoses since comparatively few animal models of these human disorders are available (82). 73. Brady, R. O., Gal, A. E., Bradley, R. M., Martensson, E., Warshaw, A. L., and Laster, L. (1967). N . Engl. J . Med. 276, 1163. 74. Dean, K. J., Sung, S.-S. J., and Sweeley, C. C. (1977). BBRC 77, 1411. 75. Mayes, J. S., Scheerer, J. B., Sifers, R. N., and Donaldson, M. L. (1981). Clin. Chim. Acta 1l2, 247. 76. Kanfer, J. N., Legler, G., Sullilvan, J., Raghaven, S. S., and Mumford, R. A. (1975). BBRC 67, 85. 77. Hara, A., and Radin, N. S. (1979). BBA 582, 412, 423. 78. Legler, G., and Herrchen, M. (1981). FEBS Lett. 135, 139. 79. Haskell, T. H., Peterson, F. E., Watson, D., Plessas, N. R., and Culbertson, T. (1970). J . Med. Chem. 13, 697. 80. Schauer, R., Veh, R. W., Sander, M., Corfield, A. P. and Wiegandt, H. (1980). Adu. Exp. Med. Biol. 125, 283. 81. Gal, A. E., Pentchev, P. G., Massey, J. M., and Brady, R. 0. (1979). PNAS76,3083. 82. Desnick, R. J., Patterson, D. F., and Scarpelli, D. G. (1982). “Animal Models of Inherited Metabolic Diseases.” Alan R. Liss, Inc., New York.

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IV. Biological Role

Sphingolipid hydrolases play an important function during the ontogeny and development of almost all tissues. This function is particularly evident during the maturation of the nervous system, where rapid turnover of gangliosides appears to be required to establish neuronal connections (83), to form synaptic junctions (84), and to develop the myelin sheath (85). Ganglioside turnover obviously requires catabolism of these lipids, and the array of enzymes indicated in Fig. 1 is maximally active during this period. Thereafter, when measured in whole brain extracts, ganglioside catabolism gradually diminishes to a constant level that is about 5% of that during the maturation of these structures. However, this figure may not reflect special activity of certain cells in the brain such as the Purkinje cells where lysosomal enzymes may continue to be highly active throughout life (86). This requirement for the continuous catabolism of gangliosides by specific cells may underlie the pathogenesis of the lateonset forms of Tay-Sachs disease, which stand in marked contrast with the usual cases where brain damage becomes evident within 5 or 6 months after birth (2, 3). Another particularly important function of sphingolipid hydrolases is their role in the catabolism of stroma and membranes from senescent red and white blood cells. Erythrocytes contain a variety of neutral sphingoglycolipids, of which globoside is the major component. They also contain a considerable quantity of ganglioside GM3in their stroma (87). Erythrocyte survival in the blood is on the order of 120 days. As these cells become senescent, they are removed from circulation by phagocytic cells of the reticuloendothelial system (monocyte-macrophage series) in the spleen, liver, lungs, and lymph nodes. Their membrane components, including sphingolipids , are catabolized by lysosomal sphingolipid hydrolases. Deficient activity of any of the enzymes results in the accumulation of sphingolipid. Two additional points should be made in this regard. First, much more sphingolipid appears to arise from the catabolism of leukocytes than from the catabolism of erythrocytes (88). The major neutral glycolipid of white cells is ceramidelactoside. It has been calculated that twenty to forty 83. Roth, S. (1978). Int. Congr. Ser.-Excerpta Med. 432, 196. 84. Brady, R. 0. (1976). Science 193, 733. 85. Brady, R. O., and Quarles, R. H. (1973). Mol. Cell. Biochem. 2, 23. 86. de Duve, C. (1963). Sci. Am. 208, (5)64. 87. Yamakawa, T., and Nagai, Y . (1978). Trends Biochem. Sci. 3, 128. 88. Kattlove, H. E., Williams, J. C., Gaynor, E., Spivack, M., Bradley, R . M . , and Brady, R . 0. (1969). Blood 33, 379.

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times more sphingolipid is derived from leukocytes than from red blood cells. “Gaucher cells” filled with sphingolipid that may actually be ceramidelactoside rather than glucocerebroside are frequently seen in the spleens of patients with myelocytic leukemia. Here there is no enzymatic defect, but the degradative capacity of the macrophages has been overwhelmed. Second, it appears that only a small fraction of the quantity of sphingolipid that arises from the daily turnover of red and white blood cells accumulates in patients with Gaucher’s disease, in spite of markedly reduced activity of glucocerebrosidase (89).This observation has several implications. One is that the residual glucocerebrosidase activity in the tissues of these patients, which is characteristically in the range of 6 to 15% of normal, may be sufficient to catabolize nearly all of the glucocerebroside that is presented. Another, and perhaps a more likely possibility, is that a considerable amount of this lipid is excreted in the bile (47). This excretory pathway suggests that there may be intercellular transfer of sphingolipids from the phagocytic Kupffer cells in the liver to hepatocytes. Some evidence for such a transfer of phospholipids is available (90) and investigations are under way with ~-[‘~C]glucosylceramide to determine whether sphingolipids follow a similar route. V. Research Applications

The demonstration of the specific enzyme defects in patients with sphingolipid storage disorders was quickly applied to the development of diagnostic tests based on assays of the various enzymes in readily available tissue such as white blood cells (92), cultured skin fibroblasts (92), and serum (93).These assays were refined so that heterozygous carriers of the disorders could be identified (93, 94). This development provided the basis for reliable genetic counseling, and eventually for the widely used prenatal diagnosis of fetuses with any of these metabolic disorders. High priority is being devoted to developing procedures to obtain suf89. Pentchev, P. G., Brady, R. O., Gal. A. E., and Hibbert, S. R. (1975). J . Mol. M e d . 1, 73. 90. Roerdink, F., Dijkstra, J . , Hartman, G . , Bolscher, B . , and Scherphof, G. (1981). BBA 677,79. 91. Kampine, J . P., Brady, R. ‘O., Kanfer, J. N., Feld, M . , and Shapiro, D. (1967). Science 155, 86. 92. Sloan, H. R., Uhlendorf, B. W., Kanfer, J. N., Brady, R. O., and Fredrickson, D. S. (1969). BBRC 34, 582. 93. O’Brien,J. S., Okada, S., Chen, A . , Fillerup, D. L. (1970). N . Engl. J . Med. 283, 15. 94. Brady, R. O., Johnson, W. G . , and Uhlendorf, B. W. (1971). A m . J . M e d . 51, 423.

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1

2

3

4

5

6

FIG. 3a. Graphic representation of an irnmunochemical nitrocellulose blot radioautograph of NaDodS04-polyacrylamide gel electrophoresis of glucocerebrosidase isozymes in extracts of cultured skin fibroblasts, using a monospecific polyvalent antibody raised against homogeneous human placental glucocerebrosidase (96). Lane 1, normal control; lanes 2 , 3 , and 4 from patients with Types 1, 2, and 3 Gaucher’s disease, respectively; lanes 5 and 6 from Types 2 and 3 Gaucher heterozygotes.

ficient quantities of the sphingolipid hydrolases in pure form for the complete characterization of oligosaccharide residues of these glycoproteins. Comparatively large amounts of the enzymes are also required for bloodclearance and tissue-uptake studies in animals in order to put enzyme replacement trials in humans on a rational basis. Enzyme for human therapy will require the development of pilot-plant-size isolation methods in order to approximate or exceed the levels of the catalysts in normal individuals. Major research efforts in this area are directed toward obtaining an understanding of the molecular basis of these disorders. The chromosomes that carry some of these genes have been identified, the latest being chromosome 1 for glucocerebrosidase (95). Allelic mutations of glucocerebrosidase isozymes have been demonstrated in the various clinical forms of Gaucher’s disease (96). Confluent cultured skin fibroblasts from normal humans have two major isozymes with Mr’s of 63,000 and 56,000 and a minor component with an apparent M,of 61,000 (Fig. 3a). Patients with the most frequently encountered Gaucher’s disease, Type 1, without central nervous system disorders, have markedly diminished glucocerebrosidase activity in their cells and exhibit a major glucocerebrosidase 95. Shafit-Zagardo, B., Devine, E. A., andDesnick, R. J. (1981). Am. J . Hum. Genet. 33, 564.

96. Ginns, E. I., Brady, R. O., Pirmccello, S., Moore, C . , Sorrell, S., Furbish, F. S., Murray, G. J., Tager, J., and Barranger, J. A. (1982). PNAS 79, 5607.

11. SPHINGOLIPID HYDROLYSIS

425 STANDARDS

;B‘p

-=

-~,000

-

- 67,000

- 43.000

- 30,000

isozyme of M, 56,000 that cross-reacts with antibody against purified placental glucocerebrosidase. Minor bands at 63,000 and 61,000 are also apparent by immunoradiochemical blotting techniques. In contrast, patients with Gaucher’s disease with neurological involvement (Type 2 infantile and Type 3 juvenile forms) demonstrate cross-reacting material with only a M , of 63,000. The absence of the low-molecular-weight isozyme in these patients suggests that the mutation(s) has prevented proper processing of the 63,000 precursor to the mature form (25). A monoclonal antibody raised against homogeneous human placental glucocerebrosidase has been found to provide discrimination between the 63,000 isozymes in Type 2 and Type 3 Gaucher’s disease patients (25). This antibody recognizes an epitope in the Type 3 isozyme that is not present in Type 2 (Fig. 3b). Thus, a reliable rapid procedure is available to determine the specific phenotype in patients with this disorder. This development has important applications for genetic counseling and for devising strategies to treat patients with the various forms of this disorder. It may be predicted that similar techniques and applications will be attempted in many other sphingolipid storage disorders, such as NiemannPick disease with its varying clinical presentations (72), and in the various forms of metachromatic leukodystrophy (97). 97. Kolodny, E. H., and Moser, H. W. (1983). I n “The Metabolic Basis of Inherited Disease” (J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, J. L. Goldstein, and M. S. Brown, eds.), 5th ed., p. 881. McGraw-Hill, New York.

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Recombinant DNA techniques are expected to provide a large amount of information relevant to this entire group of disorders. Attempts to clone the genes for the various enzymes are under way in a number of laboratories. Sequencing peptides derived from homogeneous enzymes will permit the construction of nucleotide probes that should speed these efforts. It is hoped that introducing the gene into microorganisms may eventually provide a more economical supply of these enzymes. Investigation of the sphingolipid hydrolases has been time consuming and difficult, but the many practical applications that have arisen from these efforts provide incontrovertible testimony to the value of continuing research in basic enzymology.