Synthesis of a novel photoactivatable glucosylceramide cross-linker

The biosynthesis of glucosylceramide (GlcCer) is a key rate-limiting step in complex glycosphingolipid (GSL) biosynthesis. To further define interacting partners of GlcCer, we have made a cleavable, biotinylated, photoreactive GlcCer analog in which the reactive nitrene is closely apposed to the GlcCer head group, by substituting the native fatty acid with d, l-2-aminohexadecanoic acid. Two amino-GlcCer diastereomer cross-linkers (XLA and XLB) were generated. XLB proved an effective lactosylceramide (LacCer) synthase substrate while XLA was inhibitory. Both probes specifically bound and cross-linked the GlcCer binding protein, glycolipid transfer protein (GLTP), but not other GSL binding proteins (Shiga toxin and cholera toxin). GlcCer inhibited GLTP cross-linking. Both GlcCer cross-linkers competed with microsomal nitrobenzoxadiazole (NBD)-GlcCer anabolism to NBD-LacCer. GLTP showed marked, ATP-dependent enhancement of cell-free intact microsomal LacCer synthesis from endogenous or exogenous liposomal GlcCer, supporting a role in the transport/membrane translocation of cytosolic and extra-Golgi GlcCer. GLTP was specifically labeled by either XLA or XLB GlcCer cross-linker during this process, together with a (the same) small subset of microsomal proteins. These cross-linkers will serve to probe physiologically relevant GlcCer-interacting cellular proteins. The biosynthesis of glucosylceramide (GlcCer) is a key rate-limiting step in complex glycosphingolipid (GSL) biosynthesis. To further define interacting partners of GlcCer, we have made a cleavable, biotinylated, photoreactive GlcCer analog in which the reactive nitrene is closely apposed to the GlcCer head group, by substituting the native fatty acid with d, l-2-aminohexadecanoic acid. Two amino-GlcCer diastereomer cross-linkers (XLA and XLB) were generated. XLB proved an effective lactosylceramide (LacCer) synthase substrate while XLA was inhibitory. Both probes specifically bound and cross-linked the GlcCer binding protein, glycolipid transfer protein (GLTP), but not other GSL binding proteins (Shiga toxin and cholera toxin). GlcCer inhibited GLTP cross-linking. Both GlcCer cross-linkers competed with microsomal nitrobenzoxadiazole (NBD)-GlcCer anabolism to NBD-LacCer. GLTP showed marked, ATP-dependent enhancement of cell-free intact microsomal LacCer synthesis from endogenous or exogenous liposomal GlcCer, supporting a role in the transport/membrane translocation of cytosolic and extra-Golgi GlcCer. GLTP was specifically labeled by either XLA or XLB GlcCer cross-linker during this process, together with a (the same) small subset of microsomal proteins. These cross-linkers will serve to probe physiologically relevant GlcCer-interacting cellular proteins. Glycosphingolipid (GSL) accumulation is the basis of the pathology of lysosomal GSL storage diseases (1.Lingwood CA. Glycosphingolipid functions.Cold Spring Harb. Perspect. Biol. 2011; 3: a004788Crossref PubMed Scopus (96) Google Scholar). In addition, aberrant GSL synthesis plays a key cofactor role in the pathology of many other human diseases (1.Lingwood CA. Glycosphingolipid functions.Cold Spring Harb. Perspect. Biol. 2011; 3: a004788Crossref PubMed Scopus (96) Google Scholar), and in models of such disease, GSL blockade ameliorates symptoms (2.Zhao H. Przybylska M. Wu I.H. Zhang J. Maniatis P. Pacheco J. Piepenhagen P. Copeland D. Arbeeny C. Shayman J.A. et al.Inhibiting glycosphingolipid synthesis ameliorates hepatic steatosis in obese mice.Hepatology. 2009; 50: 85-93Crossref PubMed Scopus (73) Google Scholar, 3.Marshall J. McEachern K.A. Chuang W.L. Hutto E. Siegel C.S. Shayman J.A. Grabowski G.A. Scheule R.K. Copeland D.P. Cheng S.H. Improved management of lysosomal glucosylceramide levels in a mouse model of type 1 Gaucher disease using enzyme and substrate reduction therapy.J. Inherit. Metab. Dis. 2010; 33: 281-289Crossref PubMed Scopus (40) Google Scholar, 4.Natoli T.A. Smith L.A. Rogers K.A. Wang B. Komarnitsky S. Budman Y. Belenky A. Bukanov N.O. Dackowski W.R. Husson H. et al.Inhibition of glucosylceramide accumulation results in effective blockade of polycystic kidney disease in mouse models.Nat. Med. 2010; 16: 788-792Crossref PubMed Scopus (133) Google Scholar). Understanding the synthesis of complex GSL is therefore crucial in generating the means for selective therapeutic correction of GSL levels. Glycosyltransferase knockout studies in mice identify central roles for GSLs in embryology and differentiation, particularly in the peripheral and central nervous system (5.Allende M.L. Proia R.L. Simplifying complexity: genetically resculpting glycosphingolipid synthesis pathways in mice to reveal function.Glycoconj. J. 2014; 31: 613-622Crossref PubMed Scopus (44) Google Scholar). However, differences are observed for the same deletion in different studies (6.Biellmann F. Hulsmeier A.J. Zhou D. Cinelli P. Hennet T. The Lc3-synthase gene B3gnt5 is essential to pre-implantation development of the murine embryo.BMC Dev. Biol. 2008; 8: 109Crossref PubMed Scopus (32) Google Scholar, 7.Togayachi A. Kozono Y. Ikehara Y. Ito H. Suzuki N. Tsunoda Y. Abe S. Sato T. Nakamura K. Suzuki M. et al.Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains, facilitating B cell activation.Proc. Natl. Acad. Sci. USA. 2010; 107: 11900-11905Crossref PubMed Scopus (31) Google Scholar, 8.Kuan C.T. Chang J. Mansson J.E. Li J. Pegram C. Fredman P. McLendon R.E. Bigner D.D. Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase–a key enzyme in lacto-neolacto ganglioside synthesis.BMC Dev. Biol. 2010; 10: 114Crossref PubMed Scopus (18) Google Scholar), indicating that other factors in the regulation of GSL biosynthesis remain to be determined. One such factor is the relationship between the synthesis of the acidic and the several neutral GSL subclasses from lactosylceramide (LacCer) (9.De Rosa M.F. Sillence D. Ackerley C. Lingwood C. Role of multiple drug resistance protein 1 in neutral but not acidic glycosphingolipid biosynthesis.J. Biol. Chem. 2004; 279: 7867-7876Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). GSL synthesis is complicated by the fact that the common precursor, glucosylceramide (GlcCer), is made on the outer membrane of the Golgi (10.Futerman A.H. Pagano R.E. Determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver.Biochem. J. 1991; 280: 295-302Crossref PubMed Scopus (246) Google Scholar, 11.Jeckel D. Karrenbauer A. Burger K.N. van Meer G. Wieland F. Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.J. Cell Biol. 1992; 117: 259-267Crossref PubMed Scopus (259) Google Scholar), while complex GSL synthesis occurs within the Golgi luminal membrane. The mechanism by which GlcCer translocation is achieved is still largely a matter of conjecture (12.D'Angelo G. Polishchuk E. Di Tullio G. Santoro M. Di Campli A. Godi A. West G. Bielawski J. Chuang C.C. van der Spoel A.C. et al.Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.Nature. 2007; 449: 62-67Crossref PubMed Scopus (329) Google Scholar). Phosphatidylinositol-four-phosphate adapter protein 2 (FAPP2)-facilitated cytosolic GlcCer traffic is implicated in neutral GSL synthesis, while vesicular GlcCer traffic is involved in ganglioside biosynthesis (13.D'Angelo G. Uemura T. Chuang C.C. Polishchuk E. Santoro M. Ohvo-Rekila H. Sato T. Di Tullio G. Varriale A. D'Auria S. et al.Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi.Nature. 2013; 501: 116-120Crossref PubMed Scopus (115) Google Scholar). We have proposed the Golgi located MDR1(multidrug resistance protein 1) pump as a potential mechanism for flipping GlcCer into the Golgi (14.Lala P. Ito S. Lingwood C.A. Transfection of MDCK cells with the MDR1 gene results in a major increase in globotriaosyl ceramide and cell sensitivity to verocytotoxin: role of P-gp in glycolipid biosynthesis.J. Biol. Chem. 2000; 275: 6246-6251Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), but its role remains ill-defined and is unlikely the only mechanism. Furthermore, GlcCer is emerging as an important factor in intracellular membrane traffic (15.Shen W. Henry A.G. Paumier K.L. Li L. Mou K. Dunlop J. Berger Z. Hirst W.D. Inhibition of glucosylceramide synthase stimulates autophagy flux in neurons.J. Neurochem. 2014; 129: 884-894Crossref PubMed Scopus (27) Google Scholar) and membrane order (16.Varela A.R. Goncalves da Silva A.M. Fedorov A. Futerman A.H. Prieto M. Silva L.C. Effect of glucosylceramide on the biophysical properties of fluid membranes.Biochim. Biophys. Acta. 2013; 1828: 1122-1130Crossref PubMed Scopus (30) Google Scholar). GlcCer synthesis and trafficking are, in addition, regulated by statin-sensitive prenylation mechanisms (17.Binnington B. Nguyen L. Kamani M. Hossain D. Marks D.L. Budani M. Lingwood C.A. Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling.Glycobiology. 2016; 26: 166-180Crossref PubMed Scopus (21) Google Scholar). As a means to address the mechanism by which GlcCer is trafficked intracellularly and translocated into the Golgi lumen, we have designed a novel GlcCer-based photoaffinity probe, using a 2-amino fatty acid derivative (18.Schwarzmann G. Wendeler M. Sandhoff K. Synthesis of novel NBD-GM1 and NBD-GM2 for the transfer activity of GM2-activator protein by a FRET-based assay system.Glycobiology. 2005; 15: 1302-1311Crossref PubMed Scopus (42) Google Scholar). The cross-linker is converted to LacCer and competes with nitrobenzoxadiazole (NBD)-GlcCer for GSL synthesis in cell free studies and thus provides a potential means to define GlcCer binding proteins, which should include any GlcCer flippase. Sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethyl-1,3′-dithioproprionate (sulfo-SBED) biotin label transfer reagent (no-weigh format) and streptavidin horseradish peroxidase conjugate (SA-HRP) were purchased from Thermo Scientific. GlcCer (glucocerebrosides) was purchased from Matreya LLC. 2-Aminohexadecanoic acid, di-tert-butyl dicarbonate (BOC anhydride), Mg(OAc)2, UDP-galactose (UDP-Gal), pyridine, ethyl acetate, trifluoroacetic acid (TFA), NaOH, HCl, triethylamine (TEA), acetic acid, acetic anhydride, dimethylformamide (DMF), dichloromethane (DCM), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), sucrose, sodium bicarbonate, and cholera toxin B subunit (CTB) were purchased from Sigma-Aldrich. Succinimidyl 6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino)hexanoate (NBD-X SE) was purchased from AnaSpec Inc. MEM, Dulbecco's PBS 1X (D-PBS), FBS, and trypsin (0.05%)/EDTA were purchased from Wisent Inc. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), protease inhibitor cocktail, Tris, MgCl2, and BSA were purchased from BioShop. Chloroform, methanol, silica gel 60, and KCl were purchased from Caledon Laboratory Chemicals. C-18 silica gel and conduritol β epoxide (CBE) were purchased from Toronto Research Chemicals Inc. Precoated TLC sheets (Polygram SIL G/UV254) were purchased from Machery-Nagel. Sep-Pak Vac 6 cc (1 g) certified C18 cartridges were purchased from Waters. DU145 cells were kindly supplied by Dr. N. Fleshner, University of Toronto. 3H-UDP-Gal was purchased from American Radiolabeled Chemicals. Glycolipid transfer protein (GLTP) was kindly provided by Dr. Thorsten Lang, Department of Membrane Biochemistry at the Life & Medical Sciences (LIMES) Institute, University of Bonn, Germany. Verotoxin-1 B subunit (VTB) was made as described (19.Ramotar K. Boyd B. Tyrrell G. Gariepy J. Lingwood C. Brunton J. Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron.Biochem. J. 1990; 272: 805-811Crossref PubMed Scopus (66) Google Scholar). Twelve milligrams of GlcCer was dried under nitrogen and low heat and then lyophilized overnight. GlcCer was deacylated in 11.5 ml 1 M NaOH/methanol at 70°C for 4 days (20.Basta M. Karmali M. Lingwood C. Sensitive receptor-specified enzyme-linked immunosorbent assay for Escherichia coli verocytotoxin.J. Clin. Microbiol. 1989; 27: 1617-1622Crossref PubMed Google Scholar). Reaction products were neutralized with concentrated HCl, and dried by rotary evaporation. The reaction products were dissolved in water and desalted with C-18 reversed phase silica gel column chromatography and then purified with normal phase silica gel column chromatography (80% yield). To protect the amino function of 2-aminohexadecanoic acid before coupling to lyso-GlcCer, a mole ratio of 1:1.5:2 of 2-amino­hexadecanoic acid (121.9 mg), BOC anhydride, and sodium bicarbonate were initially dissolved in CH3OH/water then for 2 days at D. D. synthesis of Synthesis of acid, Google Scholar). were dried under dissolved in ethyl and with water using a Reaction acid, was purified by silica gel column chromatography and by and acid were dried in a The acid and reagent were dissolved in and at for under dissolved in was to the reaction and for at under nitrogen M. M. B. Lingwood C. a to the selective regulation of cellular glycosphingolipid Biol. Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). The reaction was to to and then with Reaction products were desalted by C-18 reversed phase silica gel column and then purified by silica gel column chromatography yield). The analog was lyophilized dissolved in and then at for D. D. synthesis of Synthesis of acid, Google Scholar). was to the reaction products to the and then dried under nitrogen and B were purified by silica gel column and purified products and B were by TLC The products were by Both and B were dissolved in acetic at for 2 and dried M. Lingwood C.A. of under Synthesis of as to for with binding PubMed Scopus Google Scholar). To the products were dissolved in and at M. Lingwood C.A. of under Synthesis of as to for with binding PubMed Scopus Google Scholar). and products were by TLC in and then purified by silica gel column was by Both and B were dissolved in and in the at with 1 analog for Reaction XLA and XLB XLA and XLB) were desalted using C18 purified by silica column chromatography and for XLA and and by The DU145 cell was to cells a of neutral and acidic DU145 with with FBS, were with by in an of to and with and were at Cell were in of MgCl2, M and with of a and were at for at The was and at for at The was and protein The protease inhibitor was to and were at is designed to cytosolic factors and and is a of that by Rosa et (9.De Rosa M.F. Sillence D. Ackerley C. Lingwood C. Role of multiple drug resistance protein 1 in neutral but not acidic glycosphingolipid biosynthesis.J. Biol. Chem. 2004; 279: 7867-7876Abstract Full Text Full Text PDF PubMed Scopus (89) Google was to reduction of the in XLA and and GlcCer, XLA or XLB were dried under and in and 1 and with DU145 and 3H-UDP-Gal at for S. E. 1 activity in human cells from normal and Biophys. Acta. PubMed Scopus Google Scholar). The assay is a of that of and S. E. 1 activity in human cells from normal and Biophys. Acta. PubMed Scopus Google Scholar). GlcCer was dried and in with and 1 MgCl2, 2 intact DU145 and 3H-UDP-Gal at for were at for 2 with 1 M in reaction was neutralized with normal and was to a the phase was with phase and the phase was and dried under were dissolved in and was by TLC with a GSLs from The TLC was with and to at synthesis of and were dissolved in ml of and at for A. of a binding protein for of native PubMed Scopus Google Scholar). (80% was purified by silica gel column chromatography and at in the the assay is a of the of and S. E. 1 activity in human cells from normal and Biophys. Acta. PubMed Scopus Google Scholar). were (1 and XLA or XLB were dried under dissolved in and to a of 1 1 Mg(OAc)2, protease inhibitor 1 and DU145 were at as and by of 1 ml of and water for as of was by TLC The dried was using a purified protein XLA or XLB were dissolved in 2 of at for a and then in of GLTP and in were with cross-linker at for 1 microsomal protein XLA or XLB were dried in ml under dissolved in and at cross-linker were with 2 GLTP and GlcCer or water of at for 1 and GlcCer were with 1 1 Mg(OAc)2, protease inhibitor cocktail, and DU145 at for 1 and microsomal proteins were cross-linked with from a of for were by with or by using The was designed to closely native GlcCer with for membrane a in the to the head group, and a biotin for of synthesis is in GlcCer was deacylated to lyso-GlcCer, desalted on a C-18 phase silica gel and The amino function of 2-aminohexadecanoic acid was with which of the reaction by of the 2-amino fatty acid. TLC with was to reaction 2-Aminohexadecanoic acid and which to as distinct by the d, have closely to as was purified on silica column and was by to have the of 2-aminohexadecanoic acid was to acid to the Reaction was by TLC with GlcCer analog and by was the products and were purified and by TLC and and B were as by with a of and XLB synthesis were by of 2-aminohexadecanoic acid. TLC of 2-aminohexadecanoic acid, and reaction products were with coupling reaction to 2-aminohexadecanoic acid. TLC of reaction products with GlcCer analog and with TLC with of purified products from in A. and B with products by with of products is TLC with of purified cross-linkers XLA and XLB with and are not by TLC the amino on the fatty acid of the was to probe the TLC of the is to the of the was that of is to an Both and B an of and B were to to the cross-linkers XLA and XLB XLA and XLA and XLB were purified and by to have the of To the of XLA and to GlcCer binding protein GLTP was with GSL binding proteins and and binding protein was by XLA or XLB with an of and with GLTP was cross-linked by XLA or XLB with and other GSL binding proteins gel with an of and in XLA and XLB for GLTP was by of GlcCer. with GLTP was by in the of GlcCer for XLA and XLB with cross-linker GlcCer competes with cross-linkers as for GLTP binding XLA and XLB were as in a 3H-UDP-Gal assay with DU145 in GlcCer was as a substrate which a with the of the LacCer XLB a which with the of the LacCer The was that from GlcCer, but was observed for XLB is a substrate for XLA is not an The TLC the substrate XLA was not during the substrate assay was to and XLA or XLB for and GlcCer synthesis was in the of either XLA or with substrate with by was not in the of the the is in the Golgi and this assay an intact provides a potential of Golgi luminal to exogenous GlcCer, GLTP to transfer GSLs to model to glycolipid C.S. T. R.E. Glycolipid transfer protein with by J. 2005; Full Text Full Text PDF PubMed Scopus (44) Google Scholar), we that GLTP be to XLA and XLB into microsomal However, we to GLTP of GlcCer increase LacCer We 3H-UDP-Gal in to LacCer and synthesis from endogenous or exogenous GlcCer, in the and of GLTP and GLTP LacCer synthesis from endogenous and exogenous GlcCer. was in the of on LacCer synthesis but the observed of GlcCer. LacCer and synthesis were by exogenous GlcCer In the of exogenous GlcCer XLA and XLB was in DU145 GLTP to and the cross-linker into the microsomal XLA or XLB were GLTP and then with for 1 before with for with GLTP not of microsomal proteins with XLA or XLB protein of and were cross-linked by XLA and GLTP was cross-linked during the GLTP was in the of as as a cross-linked protein at in GLTP only in XLA and XLB were designed with to closely native GlcCer. The fatty acid of provides a for coupling to the cross-linker The of the the of the head of the analog to and is to head binding proteins. The involved in are and be with of different different fatty acid and different coupling the fatty acid to lyso-GlcCer, TLC These are from the and of 2-aminohexadecanoic acid in the coupling reaction with The of analog were in the same which in that on TLC but with which not is the of an between the and the of the in of the To address this the of the products were of the function the TLC indicating an from the amino of was for the TLC Both were to to XLA and which activity in The of XLA and XLB was using the GlcCer binding protein GLTP was cross-linked by cross-linkers as with GSL binding proteins and GLTP with XLA and XLB in the of GlcCer, which a common binding on GLTP and and GLTP be to transfer cross-linker into The assay in showed only XLB was converted into its LacCer not However, XLA still synthesis with in a for or the intact microsomal membrane remains a substrate membrane translocation to luminal LacCer of exogenous liposomal GlcCer to LacCer thus provides an of GlcCer synthesis in intact cell is translocated by a GlcCer to by to be a substrate for membrane flipping The is a for glucosylceramide and other J. 2005; PubMed Scopus Google Scholar). the microsomal cytosolic to was particularly by The of the in XLA to the of the for XLB is a substrate for effective GlcCer in in in XLA as an an provides a of of GSL function C.A. of glycolipid function.Glycoconj. J. PubMed Scopus Google Scholar, H. A.J. N. B. T. L. and in glycosphingolipid of J. 2014; 107: Full Text Full Text PDF PubMed Scopus Google Scholar). The role of GLTP in GSL synthesis was in the cell-free microsomal In the of GLTP LacCer synthesis from endogenous and exogenous GlcCer. is with the between GLTP and cellular GlcCer levels P. Glycolipid transfer protein is by glycosphingolipid 2013; 8: PubMed Scopus Google Scholar). The increase we observed was by but not LacCer to was by GLTP not to be an ATP-dependent step an ATP-dependent The of was for exogenous GlcCer, with a on the of extra-Golgi GlcCer for LacCer that using GLTP to XLA and XLB to microsomal to GlcCer binding proteins and XLA and XLB were cross-linked in DU145 using GLTP to and the cross-linker into the microsomal as GLTP to and GSLs in model C.S. T. R.E. Glycolipid transfer protein with by J. 2005; Full Text Full Text PDF PubMed Scopus (44) Google Scholar, R.E. with is by acid and 2013; Full Text Full Text PDF PubMed Scopus Google Scholar), and studies GLTP enhancement of microsomal GSL GLTP was cross-linked by XLA and XLB in DU145 but of microsomal proteins was not by GLTP translocation is not the rate-limiting of the was in with GLTP that this be a microsomal proteins were cross-linked in and cross-linking. studies are the of GlcCer binding proteins and and potential role in GSL The Li and from & for for and Dr. Thorsten from Department of Membrane Biochemistry at the Institute, University of Bonn, for kindly fluoride hydrochloride di-tert-butyl dicarbonate benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate conduritol β epoxide cholera toxin B subunit dichloromethane dimethylformamide glucosylceramide glycolipid transfer protein glycosphingolipid lactosylceramide LacCer synthase nitrobenzoxadiazole streptavidin horseradish peroxidase conjugate benzamido)-hexanoamido) ethyl-1,3′-dithioproprionate triethylamine trifluoroacetic acid B subunit

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