SPHINGOLIPIDS INTRODUCTION TO SPHINGOLIPIDS AND RAFTS. 1. Sphingolipids

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1 SPHINGOLIPIDS INTRODUCTION TO SPHINGOLIPIDS AND RAFTS 1. Sphingolipids The sphingolipids comprise complex rnge of lipids in which ftty cids re linked vi mide bonds to long-chin bse or sphingoid. The root term sphingo- ws first coined by J.L.W. Thudichum in 1884 becuse the enigmtic nture of the molecules reminded him of the riddle of the sphinx. The term sphingolipide ws introduced by Herbert Crter nd collegues in While they re perhps less enigmtic thn they once were, sphingolipids re extremely verstile molecules nd surprises re certinly expected s new knowledge is gined of their functions in helthy nd disesed niml nd plnt tissues. They re lso found in one bcteril genus (Sphingomons). The complex sphingolipids re locted minly in the plsm membrne of mmmlin cells where they hve structurl function, lthough they lso serve s dhesion sites for proteins from the extrcellulr tissue. Similrly, they hve nlogous intrcellulr functions in ll cellulr comprtments, including the nucleus. In ddition, it hs become evident tht complex sphingolipids nd their metbolites hve importnt roles in signl trnsduction. OH CH 2 OH R.CHOH.CH.CH 2 OH NHOC.R' R.CHOH.CH.CH 2 O NHOC.R' O P _ O sphingosine NH 2 cermide + O CH 2 CH 2 N(CH 3 ) 3 OH sphingomyelin OH CH 3 (CH 2 ) 12 CH CH.CHOH.CH.CH 2 O O OH R'CO NH CH 2 OH glctosylcermide A long-chin bse, such s sphingosine, is the simplest possible functionl sphingolipid, but cermides, which contin ftty cid linked by n mide bond, re not only importnt molecules in their own right, but re the precursors of phospholipids nd glycolipids with n immense rnge of functions in tissues. These re quite distinct from the properties of the complex glycerolipids. For exmple, sphingomyelin hs structurl similrities to phosphtidylcholine, but hs very different physicl nd biologicl properties, while the complex oligoglycosylcermides nd gngliosides hve no true prllels mong the glycerolipids. In recent yers, it hs become pprent tht sphingolipids re involved in mny of the more common humn diseses including dibetes, mny different cncers, microbil infections, Alzheimer's disese nd other neurologicl syndromes, nd diseses of the crdiovsculr nd respirtory systems. Sphingolipids nd their metbolism re therefore likely to prove of ever incresing interest to scientists. W.W. Christie 1

2 2. Ftty cid Components of Sphingolipids The ftty cids of sphingolipids re very different from those of glycerolipids, consisting of verylong-chin (up to C 28 ) odd- nd even-numbered sturted or monoenoic nd relted 2-D-hydroxy components. In plnts, 2-hydroxy cids predominte sometimes ccompnied by smll mounts of 2,3-dihydroxy cids. Polyunsturted ftty cids re only rrely present, lthough sphingomyelins of testes nd spermtozo re exceptions in tht contin polyunsturted ftty cids, which re even longer in chin-length (up to 34 crbon toms), including 28:4(n-6) nd 30:5(n-6). Skin cermides lso contin unusul ftty cids, while yest sphingolipids re distinctive in contining minly C 26 ftty cids. Very-long-chin sturted nd monoenoic ftty cids re produced by specific elongses, but there is only limited informtion on how this is coordinted with cermide biosynthesis (see our webpge on long-chin bses). In plnts, it seems probble tht 2-hydroxyl groups re inserted into ftty cyl chins while they re linked to cermide, s cermide synthse does not ccept hydroxy ftty cids in vitro t lest. On the other hnd, in the brin of mice, experimentl evidence hs been obtined tht is consistent with 2-hydroxyltion occurring t the ftty cid level prior to incorportion into cermides. Although the ftty cids re only occsionlly considered in terms of the biologicl functions of sphingolipids, their influence is considerble, especilly but not only in reltion to their physicl properties nd function in membrnes (see the comments on rfts below). For exmple, synthetic glycerolipids contining very-long-chin ftty cids (C 26 ) specificlly llow growth in yest mutnts lcking sphingolipids, probbly by stbilizing the proton-pumping enzyme H + -ATPse. Similrly, cermides contining different ftty cids cn be used in highly specific wys. For exmple, in fungi, C 16 or C 18 hydroxy cids re used exclusively for synthesis of glucosylcermide, while those contining very-long-chin C 24 nd C 26 hydroxy cids re used only for synthesis of glycosyl inositol phosphorylcermide nchors for proteins. 3. Generl Comments on Sphingolipid Metbolism The biosynthesis nd ctbolism of sphingolipids involves lrge number of intermedite metbolites, mny of which hve distinctive biologicl ctivities. In nimls the reltionships between these metbolites hve been rtionlized in terms of sphingomyelin cycle. glucosylcermide sphingomyelin sphingnine dihydrocermide cermide cermide-1-phosphte glctosylcermide sphingosine N,N-dimethylsphingosine hexdecenl etc. sphingosine-1-phosphte The 'sphingomyelin cycle' W.W. Christie 2

3 Ech of the vrious compounds in these pthwys hs chrcteristic metbolic properties, nd these re discussed in more detil on the web pges on the individul compounds. Thus, free sphingosine nd other long chin bses, which re the primry precursors of cermides nd thence of ll the complex sphingolipids, function s meditors of mny cellulr events, for exmple by inhibiting the importnt enzyme protein kinse C. Cermides re involved in cellulr signlling, nd especilly in the regultion of poptosis, nd cell differentition, trnsformtion nd prolifertion, nd most stress conditions. In contrst, sphingosine-1-phosphte nd cermide-1- phosphte promote cellulr division (mitosis) s opposed to poptosis, so tht the blnce between these lipids nd cermide nd/or sphingosine levels in cells is criticl. Similrly, the structurl sphingolipids, such s sphingomyelin, monoglycosylcermides, oligoglycosylcermides, gngliosides nd sulftides, ll hve unique nd chrcteristic biologicl functions, most of which re due to their physicl properties nd loction within membrnes (see below). Metbolic pthwys tht re comprble to those of the sphingomyelin cycle re believed to occur in plnts, lthough they hve not been studied s extensively s those in nimls (sphingomyelin itself does not occur in plnts). However, sphingolipid metbolites such s sphingosine-1- phosphte hve been linked to progrmmed cell deth, signl trnsduction, membrne stbility, host-pthogen interctions nd stress responses, for exmple. cermide phosphorylinositol sphingnine 4-hydroxysphingnine glucosylcermide cermide cermide-1-phosphte sphingnine hexdecenl etc. sphingnine-1-phosphte Sphingolipid pthwys in plnts Plnts lso contin unique rnge of complex lipids in their membrnes, such s cermide phosphorylinositol nd the phytoglycosphingolipids, nd these re now known to constitute higher proportion of the totl lipids thn hd hitherto been supposed. The functions of these hve hrdly been explored. 4. Rfts nd Cveole It is impossible to understnd the functions of sphingolipids without some understnding of their distinctive physicl loction within membrnes. Sphingolipids re locted only in the outer (exoplsmic) leflet of the plsm membrne bilyer, while glycerophospholipids such s phosphtidylinositol, phosphtidylserine nd phosphtidylethnolmine occur only in the inner (cytoplsmic) leflet. Cholesterol is believed to occur in roughly equl proportions in both leflets. Further, sphingomyelin nd other sphingolipids together with cholesterol re locted in n intimte W.W. Christie 3

4 ssocition in specific sub-domins or rfts (or relted structures termed cveole ) of membrnes. These re lterlly segregted regions tht form s result of selective ffinities between sphingolipids nd membrne proteins, which ct to comprtmentlize the ltter nd thereby seprte different biochemicl functions. The pcking of cholesterol with the sturted cyl chins of sphingolipids is thermodynmiclly fvoured over tht with unsturted cyl chins, nd cholesterol is essentil to the process of rft formtion. If either the sphingolipid or cholesterol is depleted by ny mens, the other follows nd vice vers. Indeed, there is evidence tht in niml tissues sphingomyelin regultes the cpcity of membrnes to bsorb cholesterol nd thereby controls its flux between the plsm membrne nd regultory pthwys in the endoplsmic reticulum. A forml definition of rfts hs been proposed, i.e. "Membrne rfts re smll ( nm), heterogeneous, highly dynmic, sterol- nd sphingolipid-enriched domins tht comprtmentlize cellulr processes. Smll rfts cn sometimes be stbilized to form lrger pltforms through protein-protein nd protein-lipid interctions." Erlier definitions hve tended to depend on the methods used experimentlly to isolte rft preprtions nd especilly their resistnce to non-ionic detergents, i.e. their insolubility in cold 1% Triton X-100, for exmple. They hve then been described s detergent-resistnt membrnes or DRM. This hs resulted in much confusion nd controversy in the literture. DRM certinly contin rft mteril, but it is considered tht they should not be treted s rfts per se. As much s 50% of the plsm membrne my consist of such rfts. As sphingolipids contining long, lrgely sturted cyl chins, they pck more tightly together, thus giving sphingolipids much higher melting tempertures thn membrne glycerophospholipids. This tight cyl chin pcking is essentil for rft lipid orgniztion, since the differentil pcking fcility of sphingolipids nd cholesterol in comprison with glycerophospholipids is believed to led to phse seprtion in the membrne, giving rise to sphingolipid-rich regions ('liquid-ordered' phse) surrounded by glycerophospholipid-rich domins ('liquid-disordered' phse). This ordering is responsible for the resistnce to ttck by detergents. As these rfts re reltively smll (pproximtely 50 nm dimeter nd contining roughly 3000 sphingomyelin molecules) nd mobile, they re not esy to study by microscopic methods. They re believed to be thicker thn norml membrnes (46 versus 40 ngstroms). Sphingolipids tend to hve more free hydroxyl groups, both in the long-chin bses nd ftty cid components thn glycerolipids, nd these enter into hydrogen bonding nd contribute to the stbility of rfts. The presence of very-long-chin ftty cid components (e.g. C 26 ) is believed to be essentil. Cholesterol intercts prticulrly strongly with sphingomyelin, nd much less with glycosphingolipids. Membrne proteins re believed to ply n importnt prt in rft formtion, nd n importnt result of the process is tht rfts contin mny different proteins, including glycerophosphoinositol(gpi)- nchored proteins nd tyrosine receptor kinses. These provide much of the importnt biologicl properties of rfts, nd re lso essentil to mintin their stbility. Thus, the interply of lipidbsed rft units together with protein-medited ssembly of specific protein complexes genertes functionl domins with high biologicl ctivity in cell membrnes. From comprisons of membrne solubility in different detergents, it ppers tht there my exist subsets of membrne rft domins, which differ in their moleculr compositions. In prticulr, they my contin distinct gnglioside species or glycosyl phosphtidylinositol (not sphingolipid, of course) nchoring specific proteins. Those subdomins in the plsm membrne relted to rfts nd termed cveole lck glycosyl phosphtidylinositol-nchored proteins nd re stbilized by prticulr membrne-spnning proteins, the cveolins, fmily of plmitoylted hirpin-like proteins, which orgnize flsk-shped invgintions in membrnes. In ddition to the cveolins, cveole re known to contin some W.W. Christie 4

5 specific proteins not present in other rft microdomins. Other thn glycosyl phosphtidylinositol, the lipid composition of cveole is similr to tht of rfts in generl in tht they contin pprecible mounts of sphingolipids nd cholesterol. However, the gngliosides GM 1 nd to some extent GM 3 pper to be concentrted in cveole. It is believed tht there my be different clsses of cveole with different metbolic functions. Cveole pper to hve role in controlling the level of free cholesterol in cells, nd thence my ffect signlling processes. They re especilly bundnt in dipocytes where they my regulte the flux of ftty cids cross the plsm membrne. Also in dipocytes, insulin is the min hormone tht ffects metbolism, nd the receptor t the plsm membrne is locted in cveole with possible implictions for dibetes, obesity nd other metbolic disorders. In ddition, cveole function s n importnt route by which nutrients, such s folte nd glucose, re ble to cross the plsm membrne, lthough the mechnism is still obscure. It should be recognized tht lipid rfts in generl re dynmic structures, which cn be formed or undergo compositionl chnges during signlling events nd re short-lived (milliseconds or less). There my lso be some form of cross-tlk between different rft popultions which cn colesce during ctivity. Thus in resting cells, sphingolipids my exist in smll nd highly dynmic domins, which on stimultion cn stbilize nd grow; in the process, they initite biochemicl rections by promoting interctions between proteins. Lipid rfts re believed to modulte signlling events in number of different wys ccording to the composition of the specific subpopultions. Thus, the loction of signlling molecules within one such micro-domin might in itself be control mechnism, s protein ctivted by phosphoryltion within the rft nd might be prevented from intercting with n inctivting phosphtse in nother region of the membrne, for exmple. By concentrting ll of the components of prticulr signlling pthwys within one domin, lipid rfts could promote signlling in response to stimuli, while movement of signlling molecules in nd out of the rft could control whether cells re ble to respond to stimuli. Similrly, communiction between different signlling pthwys could be simplified if the relevnt molecules were concentrted in the sme lipid rft. In contrst, rfts might lso regulte signls in negtive mnner by sequestering signlling molecules in n inctive stte. The physicl properties of rfts my be key fctors in these interctions. Thus, in response to receptor ctivtion or other stimuli, sphingolipid compositions in rfts my be ltered with effects on membrne rchitecture or morphology producing further downstrem events. Rft formtion ppers to be prticulrly importnt to the ctivity of T cells, i.e. lymphocytes derived from the thymus glnd tht re intimtely involved in ntibody production. Lipid rfts re key element of membrne orgniztion tht pper to be crucil for the initition of T cell signlling by enbling efficient interction between ntigens nd receptors. There is evidence tht certin pthogens ctivte the cid sphingomyelinse tht releses cermide in membrne rfts trnsforming them into lrger units. These cn medite the internliztion of bcteri, viruses nd prsites into host cells, to initite progrmmed cell deth (poptosis) nd relese signlling molecules. They my lso ssist in the budding of viruses from infected cells. In effect, rfts nd cveole re-orgnize the receptor nd intrcellulr signlling molecules in the cell membrne nd enble the interction of pthogens with cells. The lipids of viruses re derived from the host membrnes, nd for exmple, it hs recently been demonstrted tht the lipids of the HIV virus re enriched in sphingolipids tht pper to be derived very specificlly from rfts. Further, these lipids re essentil for the infectivity of the virus. In contrst, rfts cn ssist cells to defet infection, by ctivtion of trnscription fctors nd the relese of cytokines. Specific micro-domins or rfts tht re enriched in the chrcteristic plnt sterols, sterol glucosides nd sphingolipids such s glucosylcermide hve lso been detected in the plsm W.W. Christie 5

6 membrne nd Golgi pprtus of plnt cells. These contin distinct sets of proteins, including those nchored by glycosyl phosphtidylinositol or glycosyl inositol phosphorylcermide. As in niml cells, such rfts my ssist in positioning proteins in specific regions of the cell where they cn function in development nd signlling. Similrly, micro-domins enriched in ergosterol nd cermides hve been found in peroxisoml membrnes of yests. The problem of the ctul size nd lifetime of membrne rfts is not esily ddressed. While gret del of ttention hs been focused on rfts in the outer leflet of the plsm membrne, little is known of the structurl orgniztion nd properties of the corresponding inner leflet or how the two lyers interct. A corollry of the existence of rfts, which re rich in cholesterol nd with low levels of polyunsturted ftty cids, is tht microdomins must lso exist tht re depleted in cholesterol nd enriched in polyunsturted ftty cids. Indeed, the rigid structure of cholesterol nd the highly flexible chins of docoshexenoic cid, for exmple, re incomptible nd promote the lterl segregtion of membrnes into rfts. Microdomins tht re PUFA-rich/cholesterol-poor re techniclly less esy to study thn rfts, but these my lso contin distinctive proteins nd hve importnt biologicl functions. Suggested Reding o Fielding, C.J. nd Fielding, P.E. Membrne cholesterol nd the regultion of signl trnsduction. Biochem. Soc. Trns., 32, (2004). o Gulbins, E., Dreschers, S., Wilker, B. nd Grssmé, H. Cermide, membrne rfts nd infections. J. Mol. Med., 82, (2004). o Hirbyshi, T., Igrshi, Y. nd Merrill, A.H. (Editors) Sphingolipid Biology. (Springer, Heidelberg) (2006). o Mrtin, S.W., Glover, B.J. nd Dvies, J.M. Lipid microdomins - plnt membrnes get orgnized. Trends Plnt Sci., 10, (2005). o Merrill, A.H., Wng, M.D., Prk, M. nd Sullrds, M.C. (Glyco)sphingolipidology: n mzing chllenge nd opportunity for systems biology. Trends Biochem. Sci., 32, (2007). o Mishr, S. nd Joshi, P.G. Lipid rft heterogeneity: n enigm. J. Neurochem., 103, (2007). o Prton, R.G. nd Simons, K. The multiple fces of cveole. Nture Rev. Mol. Cell Biol., 8, (2007). o Riethmuller, J., Riehle, A., Grssme, H. nd Gulbins, E. Membrne rfts in host-pthogen interctions. Biochim. Biophys. Act, 1758, (2006). o Sengupt, P., Bird, B. nd Holowk, D. Lipid rfts, fluid/fluid phse seprtion, nd their relevnce to plsm membrne structure nd function. Seminrs Cell Developm. Biol., 18, (2007). o Sperling, P., Wrnecke, D. nd Heinz, E. Plnt sphingolipids. In: Lipid Metbolism nd Membrne Biogenesis. pp (ed. G. Dum, Springer-Verlg, Heidelberg) (2004). o Suzuki, T. nd Suzuki, Y. Virus infection nd lipid rfts. Biol. Phrm. Bull., 29, (2006). o Zeyd, M. nd Stulnig, T.M. Lipid Rfts & Co.: n integrted model of membrne orgniztion in T cell ctivtion. Prog. Lipid Res., 45, (2006). o Zheng, W., Kollmeyer, J., Symolon, H., Momin, A., Munter, E., Wng, E., Kelly, S., Allegood, J.C., Liu, Y., Peng, Q., Rmrju, H., Sullrds, M.C., Cbot, M. nd Merrill, A.H. Cermides nd other bioctive sphingolipid bckbones in helth nd disese: lipidomic nlysis, metbolism nd roles in membrne structure, dynmics, signling nd utophgy. Biochim. Biophys. Act, 1758, (2006). W.W. Christie 6

7 W.W. Christie Scottish Crop Reserch Institute (nd Mylnefield Lipid Anlysis), Invergowrie, Dundee (DD2 5DA), Scotlnd Lst updted: 17/3/2008 W.W. Christie 7