Nonvesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids

Phosphorylated sphingolipids [ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P)] have emerged as key regulators of cell growth, survival, migration, and inflammation1–5. C1P (Fig. 1a) produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production3,6–9, which contributes to disease pathogenesis in asthma/airway hyper-responsiveness, cancer, atherosclerosis, and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking, and presentation of C1P to specific cellular sites. Vesicular trafficking is likely10 but nonvesicular mechanisms for C1P sensing, transfer, and presentation remain unexplored11,12. Moreover, the molecular basis for selective recognition and binding among signaling lipids with phosphate headgroups, namely C1P, phosphatidic acid (PA) or their lyso-derivatives, remains unclear. Herein, an ubiquitously-expressed lipid transfer protein (CPTP) is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding via a novel surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated ‘sandwich’ topology identifies CPTP as the prototype for a new GLTP-fold13 subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi/TGN, nucleus, and plasma membrane. RNAi-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

selective recognition and binding among signaling lipids with phosphate headgroups, namely C1P, phosphatidic acid (PA) or their lyso-derivatives, remains unclear. Herein, an ubiquitouslyexpressed lipid transfer protein (CPTP) is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding via a novel surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, αhelically-dominated 'sandwich' topology identifies CPTP as the prototype for a new GLTP-fold 13 subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi/TGN, nucleus, and plasma membrane. RNAi-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA 2 α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.
The crystal structure of human CPTP/16:0-C1P complex (1.9 Å, Table S1) revealed a twolayered, all α-helical topology (Fig. 1f,g) homologous with GLTP-fold 13 . αN, α1 and α2 form one layer, α4, α5 and α8 form another layer, and α3, α6 and α7 localize along the periphery of the two-layer core. A positively-charged surface cavity for anchoring the lipid headgroup ( Fig. 1h) extends through a gateway portal, transforming into a deep interior hydrophobic cavity that accommodates the sphingosine and acyl chains. αN-helix (Leu10 and Leu14) impart pocket-like features by sealing the bottom. A triad of cationic residues (K60, R106, R110) in the surface cavity recognizes and binds the C1P phosphate headgroup (Fig. 2a). The anchoring H-bond network is complex, involving bifurcated hydrogen bonding by K60 (α2-helix) with the O1 and O2 atoms, bidentate hydrogen bonding by R106 (α4-helix) with the O2 and O3 atoms, and bidentate hydrogen bonding by R110 (α4-helix) directly and via water bridging to O3. Point mutation supports key roles for K60 and R106 in C1P headgroup recognition with K60A and R106L showing almost no C1P transfer, while R110 mutation (R110L) shows ~40% transfer (Fig. 2b). The positive-charge of this site also is enhanced by R97 (α3-4 loop) which hydrogen bonds to the O2 atom of phosphate via water-bridging. R97 is almost fully active when mutated to L (R97L), but mutation to acidic E (R97E) reduces C1P transfer to ~55%, supporting its role for attracting the lipid phosphate headgroup. Cation-pi interaction between R113 (α4-helix) and Y149 (α5-6 loop) provides stabilizing underpinning for the site (Supplementary Fig. S2c), as R113 mutation (R113L) strongly diminishes activity. Mutants Y149A, R113E, and R113L show poor C1P transfer, as expected by conformational destabilization (Fig. 2b). All key residues of the phosphate recognition site appear to be conserved in eukaryotes ( Supplementary Fig. S2a).
The functional consequences of CPTP hydrophobic pocket structural adaptability become clear upon transfer analyses. Pocket expansion accommodates ceramide aliphatic chains in 'molecular ruler'-like fashion with CPTP adaptability limits optimized for 16:0-or 18:1-C1P species which are particularly effective competitors at slowing the AV-C1P transfer rate (Fig. 3g), consistent with maximal pocket expansion and optimal fit (Table S4). It is noteworthy that C1P containing long lignoceryl (24:0) acyl chains are not very effective competitors, suggesting poor accommodation in the hydrophobic pocket because of adaptation limitations. Also 16:0-C1P with dihydrosphingosine base competes less effectively than 16:0-C1P with naturally-prevalent sphingosine base.  Table S1). PA occupies the same binding site and its phosphate group interacts with the same positively-charged residues as C1P (Supplementary Fig. S5b-d). Yet, K60 hydrogen bonding is single rather than bifurcated, and the lack of the acyl-amide moiety results in no hydrogen bonding with D56, distorting the position of the phosphate headgroup and both lipid chains and loosening PA binding. The distorted interaction mitigates PA transfer by CPTP ( Supplementary Fig S5e-h and Discussion).
CPTP architecture not only represents a novel motif for specific binding of phosphosphingolipids, but is previously unknown for any phosphate-modified biomolecule [15][16][17] . In CPTP, the fixed cationic residues of the phosphate recognition site undergo minimal conformational change upon C1P binding. B-factor distribution analyses show the regions between α1-α2 and α5-α6 are most flexible ( Supplementary Fig. S6a,b), consistent with a cleft-like gating mechanism facilitating ceramide chain entry/exit. The conserved lipid orientation in the pocket, with the nonpolar acyl chain always inside regardless of sphingosine being in or out, supports a concerted mechanism of action in which the acyl chain enters first and leaves last during membrane interaction ( Supplementary Fig. S6c,d and Discussion).
The conformational adaptability of the inherently flexible, single-cavity, hydrophobic pocket of CPTP contrasts with lipid cavities in fatty acid binding proteins which use β-barrels/βcups to generate a large, solvent-filled binding site that remains conformationally fixed whether or not occupied by fatty acid 18 . A single, fixed, lipid binding cavity also is characteristic of START lipid binding domains in PC transfer protein 19 and CERT 20 , which uses an α/β fold built around an incomplete U-shaped β-barrel to bind ceramide 21 ( Supplementary Fig. S7 and Discussion).
In the human genome, the differing origins of CPTP and GLTP are clear. CPTP (214 aa) is encoded by a 3-exon transcript originating from GLTPD1 on chromosome 1 (locus 1p36.33). GLTP (209 aa) is encoded by a 5-exon transcript originating from GLTP on chromosome 12 (locus 12q24.11) 22 . The shared folding topology encoded by GLTPD1 and GLTP, despite only limited sequence homology ( Supplementary Fig. S8a-e) and different lipid specificity, provides a striking example of evolutionary convergence and emphasizes the structural premium placed by eukaryotes on conservation of this fold [23][24][25] . The related architectures of CPTP and GLTP, but with naturally-evolved and remarkably different lipid headgroup specificity (Supplementary Discussion), suggest that 'Sphingolipid Transfer Protein (SLTP) Superfamily" might better reflect the existence of the two major subfamilies: CPTP, with selectivity for ceramide-linked phosphates; and GLTP, with selectivity for ceramide-linked sugars.
Figure 4h depicts a model showing how CPTP could regulate pro-inflammatory eicosanoid generation. In mammals, the only established pathway for C1P production is via phosphorylation of ceramide by CERK at the cytoplasmic surface of the trans-Golgi/ TGN 3,28 . CERK also contains nuclear localization/export signals and traffics to the plasma membrane via microtubule-driven vesicles in response to hyperosmotic shock 28 . To produce C1P, CERK uses ceramide delivered from its ER synthetic site to the Golgi by either CERT 27 or possibly by vesicular trafficking 10 . C1P elevation by CERK is known to activate soluble cPLA 2 α by enhancing translocation to the trans-Golgi/TGN 3,26 where cPLA 2 α action releases AA needed by eicosanoid producers such as COX-1 or COX-2. siRNAinduced CPTP depletion elevates C1P in the Golgi complex and nucleus, but lowers C1P plasma membrane levels. We propose that CPTP prevents excess C1P accumulation after production by CERK, thereby regulating cPLA 2 α action, diminishing AA release and downstream generation of pro-inflammatory eicosanoids. One destination for CPTP-cargo is the plasma membrane. Models involving CPTP in catabolic C1P generation by sphingomyelinase D are less plausible because mammalian cells lack this enzyme 3 (Supplementary Discussion).
Previously, the only identified mechanism for regulating CERK-mediated production of C1P was by control of ceramide availability via ceramide transfer protein 27 . It is noteworthy that siRNA-induced CPTP depletion yields the highest increase in endogenous C1P reported to date, mostly as 16:0-C1P, and dramatically alters Golgi cisternal stack morphology suggesting CPTP-mediated transport is essential for maintaining proper Golgi organization by safeguarding localized C1P levels. The ensuing stimulation in eicosanoid production triggered by elevated C1P in the Golgi complex potentially implicates CPTP in as of yet unidentified disease states associated with inflammation.

FULL METHODS (for online version) Protein Expression and Purification
GLTPD1 ORFs encoding human and mouse (GenBank JN542538 & NP_077792.2) CPTP were cloned in pET-SUMO vector (Invitrogen) and expressed in BL21 (DE3) Star cells (Invitrogen). Soluble CPTP tagged N-terminally with His 6 -SUMO was affinity-purified by Ni-NTA chromatography followed by ubiquitin-like protein 1 (Ulp1) SUMO protease digestion overnight at 4°C to release His 6 -SUMO tag. Affinity repurification by Ni-NTA chromatography was followed by FPLC gel filtration chromatography. L-selenomethionine (Se-Met)-labeled protein for ab initio phasing was produced by feedback inhibition of the methionine synthesis pathway. Mutants were constructed by QuikChange Site-Directed Mutagenesis (Stratagene) and verified by sequencing.

Crystallization and Structure Determination
Crystallization hits from initial screens were optimized by the hanging drop vapor diffusion method and systematically varying pH and individual component concentrations (Table S3). For data collection, crystals were flash frozen (100 K) in reservoir solutions containing 20% (v/v) ethylene glycol. Diffraction data sets were collected on 24-ID-C and 24-ID-E beamlines at the Advanced Photon Source (APS) and X29 beamline at Brookhaven National Laboratory. All crystals belonged to different crystal forms. For phasing, single-wavelength anomalous dispersion (SAD) data were collected for Se-Met-labeled, CPTP/8:0-C1P crystal complex at Se peak wavelength (Table S2; see Supplementary Methods). Use of Se-CPTP structure enabled other structures to be solved by molecular replacement (Supplementary Methods). Statistics for data collection, refinement and SAD phasing are provided in Tables S1 and S2.

Epifluorescence Microscopy Analyses
BSC-1 cells on coverslips were fixed in −20 °C methanol and labeled with anti-CPTP (Santa Cruz Biotechnology sc247014), and Golgi markers anti-TGN46, anti-GM 130, and anti-p230, or endosome markers anti-Rab5 and anti-Rab9 (Cell Signaling) followed by secondary antibodies coupled to Alexa-488, Alexa-594, or Alexa-660. Cells were counterstained with DAPI, mounted in 10% PBS, 90% glycerol, imaged using a Leica DM RXA2 microscope with a 63x 1.4 NA APO C objective, a Hamamatsu ORCA ER CCD camera, and Simple PCI software and analyzed as intensity scattergrams with measured correlation coefficients 30 . Time-lapse images of living cells expressing EGFP-CPTP were captured with a Leica DM RXA2 microscope stand equipped with a Yokagawa CSU-10 spinning disk confocal head and using illumination from a Coherent 488 nm 200mW "Sapphire" continuous wave optically pumped solid-state laser and 3I Slidebook software 31 (see Supplementary Methods for more details).

Immunoblot Analysis
BSC1 cells were grown to semi-confluence, collected by manual scraping, pelleted, and boiled in SDS-PAGE buffer. Proteins were separated on a 10% discontinuous SDS-PAGE gel, transferred to PDVF membrane, and immuno-labeled 32 . The immunoreactive band was detected by chemiluminescence (Image Quant system, GE Healthcare).

siRNA-mediated CPTP Down-regulation and Rescue in Cells Ectopically Expressing Wildtype and Mutant CPTP Constructs
Low passage A549 cells (5×10 5 ) were grown (10-cm plates) in appropriate medium under standard incubator conditions (SIC) overnight. Cells were treated with siRNA (Dharmacon) against CPTP (GLTPD1) or CERK as well as non-targeting siRNA sequence for control per manufacturer's protocol and incubated for 48 h under SIC. For rescue experiments, cells were transfected with either the empty pFLAG-CMV4 (Neo r ) plasmid or this vector containing wtCPTP, K60A-CPTP, K60N-CPTP, R106L-CPTP, or R110L-CPTP. Batch cultures of cells stably expressing the transfected constructs were obtained by selection for two weeks in regular medium containing G418 (genticin; 500μg/ml) under SIC. Following selection, cells (5×10 5 ) were transferred to 10 cm tissue culture plates and cultured overnight in regular media without G418 under SIC. Cells then were treated with either control siRNA or a mixture of 4 siRNA constructs (Dharmacon) designed against the 3′ UTR of endogenous CPTP (GLTPD1) mRNA following standard manufacturer's protocol (Supplementary Methods). The 3′UTR was not included in the ectopically-expressed constructs to ensure siRNA targeting only to endogenous CPTP. Cells were incubated 48 h in regular media without G418 under SIC. Full serum media was replaced with media containing 2% serum 15 h prior to harvest.

RNA Isolation, Reverse Transcription-PCR, and Quantitative PCR
To evaluate downregulation of CERK and CPTP, quantitative PCR was performed 26 . Briefly, total RNA was isolated using RNeasy kits (Qiagen). Total RNA (1 μg) was reversetranscribed using Superscript III reverse transcriptase (Invitrogen). The level of CERK transcript was monitored using quantitative PCR and TaqMan technology (Applied Biosystems) specific to CERK and CPTP with 18 S as control. cDNA was amplified using an ABI 7900HT with premixed primer-probe sets and TaqMan Universal PCR master mix (Applied Biosystems).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.    eicosanoid production. C1P is synthesized by ceramide kinase (CERK) which concentrates in the trans-Golgi network vicinity via its pleckstrin homology (PH) domain during stimulation. To produce C1P, CERK uses ceramide transported from the ER by ceramide transfer protein (CERT) which also contains a targeting PH domain. After synthesis, C1P is transported to subcellular destinations by CPTP and possibly by vesicular trafficking. RNAi knockdown of CPTP (red X) leads to accumulation and elevation of C1P at the Golgi complex, a condition that activates soluble cytosolic phospholipase A 2 α (cPLA 2 α), releasing AA needed for generation of downstream, pro-inflammatory eicosanoids. CPTP overexpression has the opposite effect on lipid levels. cPLA 2 α activation occurs by translocation from the cytoplasm and/or cis-Golgi (red arrows) via enhanced anchoring to C1P generated by CERK in TGN. COX-1 and inducible COX-2 which use AA to produce pro-inflammatory prostaglandins, also concentrate in the trans-Golgi/TGN vicinity during stimulation. For clarity, other eicosanoid generator pathways, i.e. LOX (e.g., cytoplasmic 5lipoxygenase) and CYP (ER-associated cytochrome P-450), are not depicted. Also not depicted is Golgi cisternal stack fragmentation induced by 24 h of CPTP RNAi.