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FEBS Letters 584 (2010) 1895–1900

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Review Acid sphingomyelinase, cell membranes and human disease: Lessons from Niemann–Pick disease

Edward H. Schuchman *

Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, Icahn Medical Institute, Floor 14 Room 14-20A, 1425 Madison Avenue, New York, NY 10029, USA

article info abstract

Article history: Acid sphingomyelinase (ASM) plays an important role in normal membrane turnover through the Received 29 October 2009 hydrolysis of , and is one of the key responsible for the production of cera- Accepted 24 November 2009 mide. ASM activity is deficient in the genetic disorder Types A and B Niemann–Pick disease (NPD). Available online 26 November 2009 ASM knockout (ASMKO) mice were originally constructed to study this disorder, and numerous defects in -related signaling have been shown. Studies in these mice have further sug- Edited by Sandro Sonnino gested that ASM may be involved in the pathogenesis of several common diseases through the reor- ganization of membrane microdomains. This review will focus on the role of ASM in membrane Keywords: biology, with a specific emphasis on what a rare genetic disorder (NPD) has taught us about more Sphingomyelin Ceramide common events. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Membrane microdomain

1. Introduction Numerous reviews are available on the function of ASM in cell signaling, as well as on its involvement in specific human diseases The fluid mosaic model of the cell membrane, first proposed in (e.g. [5–7]). The purpose of this review is to summarize informa- the early 1970s, suggested that membranes exist in a disorder sta- tion regarding the role of ASM in membrane biology. In order to tus without significant selectivity [1]. This concept rapidly estab- provide a biological context, a systems approach will be used. lished itself as dogma, although in recent years a body of Much of this information comes from studies using ASMKO mice, literature has shown that the cell membrane is, in fact, composed which were originally created as a model of the human genetic dis- of small ‘‘microdomains” that exist in a liquid-ordered phase [2]. order, Types A and B Niemann–Pick disease (NPD) [8]. A brief back- These domains are static within the membrane, and can coalesce ground on ASM and NPD is provided below, followed by a and reorganize in response to various stimuli. Several laboratories summary of studies using the ASMKO mice that reveal the function also have shown that sphingolipids and cholesterol associate with of ASM on cell membranes. Despite the fact that NPD was de- these microdomains, and that these associations are integral to scribed nearly a century ago and ASM was identified over 40 years membrane function. The sphingolipid and cholesterol-enriched ago, this literature has mostly emerged during the past decade. It is membrane microdomains have been referred to as lipid ‘‘rafts” therefore an evolving field, but one that has already integrated di- [3,4]. Despite a growing literature, the concept of membrane verse scientific disciplines, ranging from physicians, biophysicists, microdomains has remained controversial, principally because lipid biochemists and signal transduction biologists, and identified data demonstrating the existence of these domains in vivo is lim- ASM as a target for numerous, common diseases. ited. As summarized below, studies of one sphingolipid , acid sphingomyelinase (ASM), specifically those using ASM knock- 1.1. Acid sphingomyelinase: historical perspective out mice (ASMKO), have provided some of the strongest evidence to date supporting the concept of membrane microdomains in Acid sphingomyelinase (ASM; EC 3.1.4.12) is one member of a vivo. They also have highlighted the important role of this enzyme family of enzymes that catalyzes the breakdown of sphingomyelin in normal cell function and the pathogenesis of many common by cleavage of the phosphorylcholine linkage, thereby producing diseases. ceramide. The existence of such a ‘‘sphingomyelin cleaving en- zyme” was first demonstrated in 1938 by the pioneering work of Thannhauser, Reichel and colleagues [9]. During the ensuing 25

* Fax: +1 212 849 2447. years, several similar enzymatic activities were identified that dif- E-mail address: [email protected] fered mostly in their tissue distribution and pH optimum. The first

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.11.083 1896 E.H. Schuchman / FEBS Letters 584 (2010) 1895–1900 clear description of a sphingomyelin hydrolase that worked opti- totic effects of diverse stimuli, including but not limited to Fas/ mally at acidic pH (i.e., ASM) was made by Gatt and colleagues in CD95 [36], ischemia [37], radiation [35,38], chemotherapy [39], 1963 [10]. In addition to ASM, at least three other sphingomyelin- and TNFalpha [40]. This effect of ASM has been attributed to local ases have been described in mammalian cells that vary in their pH changes in sphingomyelin, ceramide and cholesterol content, and optimum and co-factor dependence [11–13]. While these enzymes ultimately reorganization of membrane microdomains. and an existing de novo synthetic pathway are alternative mecha- Another significant advancement in ASM research was the nisms for ceramide generation, each with their own implications large-scale purification of the recombinant enzyme from the media on cell signaling and disease, this review will specifically focus of genetically engineered Chinese hamster ovary cells [41]. With on the contributions of ASM. the availability of recombinant ASM, good antibodies against the By the late 1960s, researchers reported that the deficiency of enzyme also soon became available, stimulating new avenues of ASM was responsible for the rare, recessively inherited lysosomal research. Importantly, several investigators showed by immunocy- storage disorder, Niemann–Pick disease (Types A and B NPD; see tochemistry that when cells were exposed to various forms of below), stimulating intensive efforts to purify and characterize it stress, the location of this protein changed from primarily lyso- [14–18]. Early investigations identified ASM as a glycoprotein, somal/endosomal to the cell surface (e.g. Ref. [42]). This observa- and because the pH optimum of the enzyme in vitro was between tion marked a watershed in ASM biology, as it is at the outer 4.5 and 5.0, coupled with the fact that the majority of storage leaflet of the cell membrane where ASM initiates cell signaling, material in NPD patients was found in and/or late endo- and thus exerts its impact on the pathogenesis of a diverse group somes, the enzyme was classified as a lysosomal protein [19]. The of diseases. cDNA and gene encoding ASM (designated SMPD1) were cloned in The precise mechanism by which ASM, which normally resides 1989 and 1992, respectively [20,21]. They predicted a 629 amino within lysosomes, is translocated to the cell surface remains un- acid polypeptide that included a 46 amino acid signal peptide re- known. Recently, Zeidan et al. showed that phosphorylation of a gion and two in-frame ATG initiation sites. Mutation analysis in specific serine residue on ASM (S508) by PKCdelta is required for NPD patients showed that both ATG initiation sites were functional its activation in response to UV irradiation and movement to the in vivo [22]. membrane [43,44]. These investigators suggested that the phos- SDS–PAGE analysis of ASM purified from various sources re- phorylation occurs within the lysosomes, although it is also possi- vealed an estimated molecular weight of 72 kDa; enzymatic de- ble that a cytosolic pool of ASM serves as the substrate for glycosylation reduced the molecular weight to 60 kDa [14,23]. PKCdelta, or that phosphorylation occurs within some other com- Processing studies performed in COS-1 cells further showed that partment at or near the cell surface. Deciphering the precise traf- ASM was synthesized as a 75-kDa ‘‘prepro” protein that was traf- ficking and processing of ASM during cell signaling is likely to be ficked to lysosomes. Interestingly, in these same studies, a 57 kDa a fruitful area for future research. secreted form of ASM also was identified [24]. Subsequent charac- terization studies revealed that 5 of 6 N-glycosylation sites of the 1.2. Types A and B NPD: brief overview enzyme were occupied [25], and that the oligosaccharide side chains contained mannose-6-phosphate residues, typical of lyso- As noted above, until recently, interest into the biology of ASM somal proteins [26]. In addition, the disulfide bond structure of was due to its role in the genetic disorder, Types A and B NPD. Both ASM was characterized [27], and it was found the terminal cys- forms of this disorder are caused by recessive mutations in the teine at amino acid residue 629 was the only cysteine not involved SMPD1 gene encoding ASM. Type A NPD is the infantile form of in intra-molecular disulfide linkages. In fact, this terminal cysteine ASM deficiency, characterized by a rapidly progressive neurode- residue must be removed to obtain full ASM activity [28], and it is generative course that leads to death by age 2–3. In contrast, Type thought that the retention of this residue in the mature protein B NPD is the later-onset form in which patients exhibit little or no may lead to the formation of inactive, higher molecular weight neurological symptoms, but may have severe and progressive vis- aggregates. ceral organ abnormalities, including hepatosplenomegaly, pul- Around this time, Tabas and co-workers identified a zinc- monary insufficiency and cardiovascular disease [45]. The dependant, secreted form of ASM that also was encoded by the different clinical presentations of Types A and B NPD are likely SMPD1 gene [29]. They proposed that the apparent differences in due to small differences in the amount of residual, functional zinc-dependence of the lysosomal and secreted ASM forms was ASM activity [46]. due to differential cellular trafficking that either exposed or The first patient with NPD (Type A) was described in 1914 by sequestered the enzyme from cellular pools of zinc [30]. It is note- the German pediatrician, Albert Niemann, and by the 1930s the worthy that a zinc-activated, secreted form of ASM was first iden- primary lipid accumulating in these individuals was identified as tified in plasma in the late 1980s, although its biological function sphingomyelin [47,48]. It is now known that secondary to sphingo- remained unknown [31]. There is now growing evidence suggest- myelin storage, other lipids, including cholesterol and gangliosides, ing that this ASM form may play a role in , cell sur- also accumulate in these patients, leading to many cellular abnor- face signaling and host inflammation (see below) [5,6,32]. malities [49]. While most of the clinical findings in NPD are likely Interestingly, the SMPD1 gene is within an ‘‘imprinted” region of related to lipid storage in lysosomes and/or endosomes, recent the human genome (chromosomal region 11p15.4), and is prefer- data revealing the important role ASM in membrane formation entially expressed from the maternal chromosome (i.e., paternally and function [42] suggests that defective function of the enzyme imprinted) [33,34]. This form of genetic regulation is typical of at the cell surface also could contribute to the pathophysiology genes that play an important role in development. of NPD as well. Until the mid-1990s, interest in ASM was limited primarily to Due to the fact that NPD is an extremely rare genetic disorder, researchers studying NPD. At this time, ASM knockout (ASMKO) ASMKO mice were created in the mid-1990s to further understand mice were constructed [8], and were found to be resistant to radi- the biology of this disease [8]. These animals have been extensively ation [35] and other forms of stress-induced apoptosis (see below). used for the pre-clinical evaluation of enzyme replacement ther- These observations introduced ASM to many new investigators and apy, gene therapy and stem cell transplantation for NPD patients, opened new avenues of research. To date, ASM inhibition (using leading to the first clinical trials of enzyme replacement using re- siRNA, pharmacologic inhibitors, or using NPD cells or KO mice) combinant ASM. In addition, the ASMKO mice have been an invalu- has been shown to render cells and animals resistant to the apop- able tool for studying sphingolipid metabolism, revealing the E.H. Schuchman / FEBS Letters 584 (2010) 1895–1900 1897 important and unexpected role of this enzyme in diverse cellular major influence on neuronal function. However, due to the paucity events [5]. The remainder of this review will focus on studies from of human materials for research, until recently the precise cellular these mice. and biochemical abnormalities in the brains of NPD patients, including changes in the cell membranes, have not been studied 2. ASM and cell membranes – overview in detail. Recently, the ASMKO mice have provided a new resource to study neural pathology related to ASM, shedding new light on The role of ASM in cell signaling is tightly linked to its ability to the enzyme’s function in neurons and other neural cells. For exam- reorganize the plasma membrane. As noted above, the central the- ple, using these mice Scandroglio et al. showed that in addition to sis of the classic, fluid mosaic model of membranes is that proteins sphingomyelin, ASMKO brain tissue and cultured cerebellar gran- float freely in the lipid bilayer [1]. However, protein interactions ule neurons had increased gangliosides, mainly GM2 and GM3. within the membrane are far more complex than originally pro- Gangliosides have diverse and important functions in the brain, posed, and sphingolipids (particularly sphingomyelin and cera- and changes in these lipids were not intuitively expected as a re- mide) are important membrane components that provide sult of ASM deficiency. In contrast, cholesterol and glycerophos- increasing ‘‘order” to isolated membrane regions [50]. The most pholipids, which are more metabolically related to prevalent lipid in the outer leaflet of the membrane is sphingomy- sphingomyelin, remained unchanged in the brains of these animals elin, which is hydrolyzed to ceramide by ASM and other sphing- [49]. omyelinases [42]. Ceramide molecules in the lipid bilayer are Of specific relevance to this review, these investigators also known to interact with each other at the exclusion of other lipids, found that a higher detergent to protein ratio was required to leading to the formation of isolated lipid ‘‘microdomains” [51]. prepare detergent-resistant membrane fractions in ASMKO mouse Subsequently, either by changing the physical properties of the brains as compared to wild-type animals. This finding suggested a membrane or by direct ceramide–protein interactions, these cera- reduction in fluidity of specific membrane areas due to the mide-enriched microdomains are thought to enhance the density accumulation of sphingolipids, and provided some of the first di- of proteins, which promotes receptor dimerization as well as other rect evidence that ASM deficiency alters the lipid composition of protein–protein interactions [52]. Current theory postulates that the plasma membrane [49]. Similarly, Galvan et al. demonstrated ASM serves to reorganize the cell surface and activate signaling increased sphingomyelin in detergent-resistant membranes of proteins within these microdomains, thus enhancing, or possibly cultured ASMKO neurons that led to an aberrant distribution of lowering, the threshold for downstream signaling (for review, see GPI-anchored proteins, providing a direct link between lipid Ref. [53]). changes, ASM function and membrane embedded signaling pro- Data showing that ASM functions at the cell surface were ini- teins. Importantly, increasing sphingomyelin in wild-type neurons tially considered counter-intuitive since its housekeeping role re- mimicked these defects, whereas reducing this lipid in ASMKO sides within lysosomes and its pH optimum in vitro was clearly neurons corrected them [56]. Bianco et al. have also shown that acidic. However, an important observation was made in 1998, following activation of the ATP receptor P2X7, microparticle shed- when it was shown that the secreted form of ASM could degrade ding and IL-1beta release from microglia in ASMKO mice was sphingomyelin to ceramide within LDL particles at physiologic markedly reduced as compared to normal mice [57], and Camolet- pH, suggesting that the in vitro pH optimum might not predict to et al. reported that ASMKO synaptic membranes had higher lev- in vivo function [54]. In addition, there have been recent reports els of sphingomyelin and sphingosine that was associated with demonstrating acidified microenvironments at the cell surface, enhanced interaction of the docking molecules, Munc18 and syn- and some of these reports have linked such microenvironments taxin1 [58]. Overall, these findings provide the first molecular data to lipid microdomains [55], the very site of ASM action. demonstrating the importance of ASM in neural cell membrane organization and function, and shed new light into the pathogenic mechanisms underlying Type A NPD and other neurodegenerative 3. ASM and cell membranes: a systems approach diseases. Given the importance of membranes in neural cell com- munication, migration and survival, and the fact that ASM is ubiq- As noted above, during the past decade a body of literature has uitously expressed at high levels throughout the brain, new and emerged, mostly from studies in the ASMKO mice, that clearly dem- important roles for this enzyme in neural function are likely to onstrates a function for ASM at the cell surface and/or in the reorga- be uncovered in the future. nization of membrane microdomains. These findings have important implications for many diseases, including NPD, and have suggested new treatment options. In the section below recent findings from 3.2. Lung the ASMKO mice will be summarized according to five major organ systems: brain, lung, heart, gonads, and skin, since these five systems By its nature, infection requires a close interaction of pathogens represent the majority of the literature published to date with an (e.g. bacteria and virus) with membranes of the target cell. During obvious link to membrane biology and/or disease mechanisms. In the past several years, a large literature has evolved using the ASM- the future it is likely that the involvement of ASM in other organs will KO mice illustrating the important role of ASM in this process. Ini- continue to emerge, expanding our understanding of this enzyme, its tial studies by Grassme and Hauck showed that inhibition of ASM role in membrane biology, and identifying new molecular targets for (pharmacologically or genetically in ASMKO mice) prevented the therapy. It is not the intention of this review to comprehensively as- entry of Neisseria gonorrhoeae into epithelial [59] and phagocytic sess the role of ASM in the pathogenesis of disease, as this has been cells [60]. Interestingly, subsequent studies with Listeria monocyt- accomplished in other reviews (e.g. [5]). Rather, the goal below is to ogenes showed that ASMKO mice are 100-fold more sensitive to summarize specific, recent examples that provide direct evidence for infection with this bacterium than wild-type mice [61], presum- a function of ASM on the cell membrane. ably because the ASM-deficient macrophages were unable to kill the bacteria and restrict their growth. These latter data suggested 3.1. Brain a novel function of ASM in infectious biology that directly relates to its role in controlling the fusion of intracellular phagosomes Given the severe, neurodegenerative phenotype of Type A NPD, with lysosomes, a process that is inherently dependent on the it has been presumed for nearly a century that ASM should have a interaction of vesicle membranes. 1898 E.H. Schuchman / FEBS Letters 584 (2010) 1895–1900

The involvement of ASM has been shown in infection of other lial cells. The formation of this lipid raft platform was prevented bacteria as well, including Staphylococcus aureus [62], Salmonella by RNAi knockdown of ASM. Thus, reorganization of cell mem- typhimurium [63], Escherichia coli [64], Mycobacterium [65] and branes by ASM is likely to play diverse roles in the heart, and Pseudomonas aeruginosa [66]. Studies with this latter pathogen abnormalities in this process could be responsible for several have important implications for patients with systemic infections, cardiac pathologies. ventilator-associated pneumonia, and cystic fibrosis. Infection of lung epithelial cells with P. aeuroginosa normally leads to a rapid 3.4. Gonads activation of ASM that correlates with translocation of the enzyme to the extracellular leaflet of the cell membrane and the site of bac- Fertilization, like infection, is inherently dependent on complex terial infection [67]. The activity of ASM at this site leads to the for- membrane interactions, and sperm are known to secrete large mation of ceramide-enriched rafts, which are critical for the amounts of hydrolytic enzymes, including ASM, presumably to internalization of P. aeurginosa into the cells, the induction of cell reorganize the membrane of the oocyte and facilitate fertilization death, and the gradual release of inflammatory cytokines. Cera- [69]. However, little is known about the specific role of ASM in mide-enriched membrane domains may regulate internalization germ cell function and fertilization. Of note, Butler et al. performed of P. aeurginosa by clustering of the CFTR protein, as it has been one of the first analyses of ASM in gametes using sperm from ASM- shown that CFTR moves into rafts after infection and that internal- KO mice, and showed elevated levels of sphingomyelin and choles- ization of the pathogen can be prevented by disruption of these terol that resulted in morphologic abnormalities such as kinks and membrane structures [68]. Compromised host response in ASMKO bends at the midpiece-principle piece junction, leading to reduced mice further corroborated the importance of ASM in effective motility [70]. Flow cytometric analysis further revealed that af- phagocytosis and eradication of pathogens via membrane modula- fected spermatozoa had disrupted acrosomal membranes and did tion [66]. Clearly, the mechanisms by which ASM participates in not undergo proper capacitation, as assessed by nitric oxide release infection are variable and pathogen-specific, and highly dependent and bilayer translocation of phosphatidylserine. In addition, these on membrane interactions. This literature is rapidly expanding, sperm exhibited compromised plasma membranes and mitochon- and will likely reveal ASM as a potential drug target for numerous drial membrane depolarization. Notably, spermatozoa from the infectious diseases. ASMKO mice regained normal morphology upon incubation in mild detergent, demonstrating that these defects were a direct 3.3. Heart consequence of membrane lipid accumulation [70]. These results provided in vivo evidence that normal sphingomyelin and choles- For many years it has been known that Types A and B NPD pa- terol metabolism within the sperm membrane is essential for tients have abnormal plasma lipid profiles, characterized by in- sperm maturation and function, and that ASM activity plays a crit- creased levels of LDL-cholesterol and triglycerides, and markedly ical role in these events. More recently, ASM also was found to be reduced HDL-cholesterol [72]. However, the precise role of ASM an important component of normal oocyte maturation and survival in lipoprotein assembly and metabolism is unclear, as is its role by modulating ceramide signaling (e.g. [71]), although the direct in normal cardiac function. An interesting concept of direct rele- effect of this enzyme on the oocyte membrane has not been eluci- vance to this review has been put forth by Tabas and colleagues, dated. There also have been sporadic reports in the NPD literature who suggested that the sphingolipid content of circulating lipopro- of reduced fertility among female NPD patients, although the teins controls their propensity to self-assemble and aggregate, mechanism remains unknown. leading to their association with cell membranes and retention within the arterial wall. For example, Marthe et al. [73] showed 3.5. Skin that the secreted form of ASM (referred to by these workers as S- SMase) was present in atherosclerotic lesions and bound to specific Ceramide plays a critical function in epidermal barrier homeo- components of the subendothelial extracellular matrix. Schissel stasis by constituting an integral component of the extracellular et al. [54] further showed that S-SMase could hydrolyze sphingo- lipid bilayer at the stratum corneum [77]. However, despite the myelin present in LDL at physiological pH, stimulating subendo- well documented role of ceramide in this process, the precise thelial retention and aggregation. This was an important function of ASM in the generation of epidermal ceramide has observation, as prior to this publication it was assumed that the not been studied in great detail. In 2000, Schmuth et al. showed only function(s) for ASM were within acidified vesicles such as that a subset of NPD patients with severe ASM deficiency demon- lysosomes. The fact that ASM could hydrolyze sphingomyelin at strated abnormal permeability barrier homeostasis, presumably physiological pH suggested new functions for the enzyme, includ- due to an abnormally low ceramide content [78]. To gain further ing its potential activity at the cell surface and on membranes. mechanistic insights into this finding, these same investigators Devlin et al. [74] later reproduced this finding by showing investigated the effects of ASM inhibitors, palmitoyldihydrosp- that ASM induced lipoprotein retention and accelerated athero- hingosine and desipramine, on the skin of hairless mice, and sclerotic lesion progression in vivo. Clearly, the pathogenesis of found that inhibitor treatment led to an increase in sphingomye- these events relates to remodeling of the lipoproteins in a way lin and a reduction of normal extracellular lamellar membrane that results in a propensity towards interactions with the suben- structures in the stratum corneum. In addition, they found a de- dothelial cell membrane. Inhibition of this LDL-membrane inter- layed barrier recovery after injury. This delay could be overcome action could have important implications in the treatment of by the topical application of ceramide, indicating that the finding atherosclerosis. in NPD patients was likely due to an altered ceramide–sphingo- ASM remodeling of the cell membrane also plays an impor- myelin ratio rather than sphingomyelin accumulation itself [78]. tant role in other cardiac cell types. For example, Jia et al. [75] Patients with atopic dermatitis also have reduced levels of ASM found that oxotremorine, a muscarinic type 1 receptor agonist, (and neutral sphingomyelinase) in their lesions, likely leading to increased lipid raft clustering in bovine coronary arterial myo- reduced ceramide levels and the permeability barrier abnormali- cytes, leading to formation of a complex of CD38 within ASM ties characteristic of this disease [79]. While it is apparent that and ceramide-enriched membrane domains. Jin et al. [76] also the normal function of skin relies heavily on the integrity of epi- showed a colocalization of lipid rafts with NADPH oxidase sub- dermal cell membranes, the involvement of ASM in this process units, gp91 and p47, in endostatin-stimulated coronary endothe- awaits further elucidation. E.H. Schuchman / FEBS Letters 584 (2010) 1895–1900 1899

4. Summary deficient mice. a model of types A and B Niemann–Pick disease. Nat. Genet. 10, 288–293. [9] Thannhauser, S.J., Reichel, M. and Grattan, J.F. (1938) The effect of ascorbic acid The study of ASM is an excellent example of how diverse fields on beta-glycerophosphate. Biochem. J. 32, 1163–1165. of biology can co-exist for long periods of time without interaction, [10] Gatt, S. (1963) Enzymic hydrolysis and synthesis of ceramide. J. Biol. Chem. and then unexpectedly coalesce to open productive and insightful 238, 3131–3133. [11] Levade, T., Andrieu-Abadie, N., Ségui, B., Augé, N., Chatelut, M., Jaffrézou, J.P. new areas of research. The first case of NPD was described nearly a and Salvayre, R. (1999) Sphingomyelin-degrading pathways in human cells century ago, and the first isolation of ASM was achieved nearly five role in cell signalling. Chem. Phys. Lipids. 102, 167–178. decades ago. Simultaneous with this research, biochemists defined [12] Stoffel, W. 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In turn, Purification to homogeneity, antigenic properties and partial amino-acid these findings have suggested new therapeutic options based on sequences of the enzyme. Biol. Chem. Hoppe-Seyler 372, 215–223. [19] Fowler, S. (1969) Lysosomal localization of sphingomyelinase in rat liver. either inhibiting ASM function or overexpressing ASM at specific Biochim. Biophys. Acta 191, 481–484. target sites. It is likely that this will remain a fruitful area of re- [20] Schuchman, E.H., Levran, O., Pereira, L.V. and Desnick, R.J. (1992) Structural search for the foreseeable future, and will lead to new treatment organization and complete nucleotide sequence of the gene encoding human acid sphingomyelinase (SMPD1). Genomics 12, 197–205. options for these devastating disorders. While it is unfortunate that [21] Quintern, L.E., Schuchman, E.H., Levran, O., Suchi, M., Ferlinz, K., Reinke, H., these diverse areas of biology could not come together sooner, Sandhoff, K. and Desnick, R.J. (1989) Isolation of cDNA clones encoding human with new technologies and rapid sharing of literature it is likely acid sphingomyelinase: occurrence of alternatively processed transcripts. EMBO J. 8, 2469–2473. that such cross-fertilization will occur at a much faster pace in [22] Pittis, M.G., Ricci, V., Guerci, V.I., Marçais, C., Ciana, G., Dardis, A., Gerin, F., the future, and biomedical science will benefit greatly from it. Stroppiano, M., Vanier, M.T., Filocamo, M. and Bembi, B. (2004) Acid sphingomyelinase: identification of nine novel mutations among Italian Niemann Pick type B patients and characterization of in vivo functional in- Conflict of interest statement frame start codon. Hum. Mutat. 24, 186–187. [23] Lansmann, S., Ferlinz, K., Hurwitz, R., Bartelsen, O., Glombitza, G. and Dr. Schuchman is an inventor on a patent owned by the Mount Sandhoff, K. (1996) Purification of acid sphingomyelinase from human placenta: characterization and N-terminal sequence. FEBS Lett. 399, 227– Sinai School of Medicine that has been licensed to the Genzyme 231. Corporation for the development of enzyme replacement therapy [24] Ferlinz, K., Hurwitz, R., Vielhaber, G., Suzuki, K. and Sandhoff, K. (1994) for NPD. Also, Dr. Schuchman is a consultant and receives research Occurrence of two molecular forms of human acid sphingomyelinase. Biochem. J. 301, 855–862. grants from Genzyme for the study of ASM and NPD. [25] Ferlinz, K., Hurwitz, R., Moczall, H., Lansmann, S., Schuchman, E.H. and Sandhoff, K. (1997) Functional characterization of the N-glycosylation sites of Acknowledgements human acid sphingomyelinase by site-directed mutagenesis. Eur. J. Biochem. 243, 511–517. [26] Hurwitz, R., Ferlinz, K., Vielhaber, G., Moczall, H. and Sandhoff, K. (1994) E.H.S. would like acknowledge the contributions of the many Processing of human acid sphingomyelinase in normal and I-cell fibroblasts. J. students, fellows and scientists who have worked in his laboratory Biol. Chem. 269, 5440–5445. on the biology of ASM and NPD, as well as the patients and families [27] Lansmann, S., Schuette, C.G., Bartelsen, O., Hoernschemeyer, J., Linke, T., Weisgerber, J. and Sandhoff, K. (2003) Human acid sphingomyelinase. Eur. J. who have contributed valuable research materials. He would also Biochem. 270, 1076–1088. like to acknowledge the specific contribution of Dr. Ching-Yin Lee [28] Qiu, H., Edmunds, T., Baker-Malcolm, J., Karey, K.P., Estes, S., Schwarz, C., for assistance with the preparation of this manuscript. ASM and Hughes, H. and Van Patten, S.M. (2003) Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine. NPD research in Dr. Schuchman’s laboratory is supported by the Biol. Chem. 278, 32744–32752. National Institutes of Health, Genzyme Corporation, and National [29] Schissel, S.L., Schuchman, E.H., Williams, K.J. and Tabas, I. 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