Lipid Transfer Assays Research Articles

Characterization of two Plasmodium falciparum lipid transfer proteins of the Sec14/CRAL-TRIO family

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is followed by dramatic modifications of erythrocytes properties, including de novo formation of new membrane systems. Lipid transfer proteins from both the parasite and the host cell are most likely an important part of those membrane remodeling processes. Using bioinformatics and in silico structural analysis, we have identified five P. falciparum potential lipid transfer proteins containing cellular retinaldehyde binding – triple functional domain (CRAL-TRIO). Two of these proteins, C6KTD4, encoded by the PF3D7_0629900 gene and Q8II87, encoded by the PF3D7_1127600 gene, were studied in more detail. In vitro lipid transfer assays using recombinant C6KTD4 and Q8II87 confirmed that these proteins are indeed bona fide lipid transfer proteins. C6KTD4 transfers sterols, phosphatidylinositol 4,5 bisphosphate, and, to some degree, also phosphatidylcholine between two membrane compartments. Q8II87 possesses phosphatidylserine transfer activity in vitro. In the yeast model, the expression of P. falciparumQ8II87 protein partially complements the absence of Sec14p and its closest homologue, Sfh1p. C6KTD4 protein can substitute for the collective essential function of oxysterol-binding related proteins. According to published whole genome studies in P. falciparum, absence of C6KTD4 and Q8II87 proteins has severe consequences for parasite viability. Therefore, CRAL-TRIO lipid transfer proteins of P. falciparum are potential targets of novel antimalarials, in search for which the yeast model expressing these proteins could be a valuable tool.

The ceramide transport protein CERT is involved in alkylacylglycerol transfer from the ER to the Golgi for the biosynthesis of ether phospholipid

Ether phospholipids are synthesized by a series of enzymes localized in peroxisomes, the endoplasmic reticulum (ER), and the Golgi apparatus. During this process, the lipid intermediate alkylacylglycerol (AAG) synthesized in the ER is transferred from the site of its synthesis to the Golgi apparatus. In this study, we determined whether ceramide transport protein (CERT) is a candidate for AAG transfer. A lipid transfer assay revealed that CERT can mediate AAG transfer between phospholipid liposomes. AAG transport activity was markedly inhibited by the CERT inhibitor HPA-12 and reduced when the lipid transport domain of CERT was deleted. Suppression of CERT in HEK293 cells resulted in increased levels of plasmanyl-PC, which is synthesized by the ER-residing choline/ethanolamine phosphotransferase 1 (CEPT1). The mRNA levels and enzymatic activity of plasmanyl-PC synthesizing enzymes were not increased in CERT-deficient cells, indicating that the increase in plasmanyl-PC results from AAG accumulation in the ER. Re-introduction of CERT into CERT-deficient cells caused a decrease in plasmanyl-PC. Taken together, our findings suggest for the first time that CERT is involved in the transfer of AAG from the ER to the Golgi apparatus and plays a role in the biosynthesis of ether phospholipids.

Structural insights on the deformations induced by various mutations on cholesteryl ester transfer protein

Cholesteryl Ester Transfer Protein (CETP) is a plasma glycoprotein that intervenes the reverse cholesterol transport (RCT) by equimolar exchange of Cholesteryl esters (CE) and Triglycerides (TGs) between anti-atherogenic High-Density Lipoproteins (HDLs) and pro-atherogenic Low-Density Lipoproteins (LDLs) resulting in the increased concentration of CEs in LDL. This is a potential cause for the formation of atherosclerotic plaques in blood vessels leading to fatality. Therefore, blocking the function of CETP has emerged as a novel strategy for suppressing atherosclerotic plaques. The crystal structure of CETP revealed two Cholesteryl esters (CEs) in the hydrophobic tunnel and two phospholipids (PLs) plugged on the concave surface. Previous lipid transfer assay experimental studies have shown a substantial reduction in the neutral lipid transfer in [R201S] and [I443W, V198W] mutants. However, the protein conformational arrangements due to the mutations present in the CETP system leading to a decrease in the transfer rate of neutral lipids is not explored. Thus, I explored the reason behind the decreased transfer rate in mutants using molecular dynamics (MD) simulations and free energy calculations. Resulting evidences show that R201S mutant induces unfavorable bending angle to CETP with a decreased binding efficiency between N-terminal phospholipid of CETP with S201. Also, an unfavorable conformation state of TGs is formed which makes them difficult to transfer across CETP. Likewise, [I443W, V198W] mutant induces unfavorable CE, TG, and bending angle conformation to CETP impeding neutral lipid transfer. Thus, my results provide sufficient insights on the causation for a decreased transfer rate as reported earlier. The detailed understanding obtained here could help in developing a new strategy in preventing the function of CETP by blocking the role of potential hot spot residues.

Insights into membrane association of the SMP domain of extended synaptotagmin

The Synaptotagmin-like Mitochondrial-lipid-binding Protein (SMP) domain is a newly identified lipid transfer module present in proteins that regulate lipid homeostasis at membrane contact sites (MCSs). However, how the SMP domain associates with the membrane to extract and unload lipids is unclear. Here, we performed in vitro DNA brick-assisted lipid transfer assays and in silico molecular dynamics simulations to investigate the molecular basis of the membrane association by the SMP domain of extended synaptotagmin (E-Syt), which tethers the tubular endoplasmic reticulum (ER) to the plasma membrane (PM). We demonstrate that the SMP domain uses its tip region to recognize the extremely curved subdomain of tubular ER and the acidic-lipid-enriched PM for highly efficient lipid transfer. Supporting these findings, disruption of these mechanisms results in a defect in autophagosome biogenesis contributed by E-Syt. Our results suggest a model that provides a coherent picture of the action of the SMP domain at MCSs.

An intermolecular hydrogen bonded network in the PRELID-TRIAP protein family plays a role in lipid sensing

The PRELID-TRIAP1 family of proteins is responsible for lipid transfer in mitochondria. Multiple structures have been resolved of apo and lipid substrate bound forms, allowing us to begin to piece together the molecular level details of the full lipid transfer cycle. Here, we used molecular dynamics simulations to demonstrate that the lipid binding is mediated by an extended, water-mediated hydrogen bonding network. A key mutation, R53E, was found to disrupt this network, causing lipid to be released from the complex. The X-ray crystal structure of R53E was captured in a fully closed and apo state. Lipid transfer assays and molecular simulations allow us to interpret the observed conformation in the context of the biological role. Together, our work provides further understanding of the mechanistic control of lipid transport by PRELID-TRIAP1 in mitochondria.

ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry

The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point invitro lipid transfer assay that allows direct tracking of [14C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.

Calcium-dependent and -independent lipid transfer mediated by tricalbins in yeast

Membrane contact sites (MCSs) formed between the endoplasmic reticulum (ER) and the plasma membrane (PM) provide a platform for nonvesicular lipid exchange. The ER-anchored tricalbins (Tcb1, Tcb2, and Tcb3) are critical tethering factors at ER–PM MCSs in yeast. Tricalbins possess a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and multiple Ca2+-binding C2 domains. Although tricalbins have been suggested to be involved in lipid exchange at the ER–PM MCSs, it remains unclear whether they directly mediate lipid transport. Here, using in vitro lipid transfer assays, we discovered that tricalbins are capable of transferring phospholipids between membranes. Unexpectedly, while its lipid transfer activity was markedly elevated by Ca2+, Tcb3 constitutively transferred lipids even in the absence of Ca2+. The stimulatory activity of Ca2+ on Tcb3 required intact Ca2+-binding sites on both the C2C and C2D domains of Tcb3, while Ca2+-independent lipid transport was mediated by the SMP domain that transferred lipids via direct interactions with phosphatidylserine and other negatively charged lipid molecules. These findings establish tricalbins as lipid transfer proteins, and reveal Ca2+-dependent and -independent lipid transfer activities mediated by these tricalbins, providing new insights into their mechanism in maintaining PM integrity at ER–PM MCSs.

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein.

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

In vitro lipid transfer assays of phosphatidylinositol transfer proteins provide insight into the in vivo mechanism of ligand transfer

Fluorescence resonance energy transfer (FRET) assays and membrane binding determinations were performed using three phosphatidylinositol transfer proteins, including the yeast Sec14 and two mammalian proteins PITPα and PITPβ. These proteins were able to specifically bind the fluorescent phosphatidylcholine analogue NBD-PC ((2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine)) and to transfer it to small unilamellar vesicles (SUVs). Rate constants for transfer to vesicles comprising 100% PC were slower for all proteins than when increasing percentages of phosphatidylinositol were incorporated into the same SUVs. The rates of ligand transfer by Sec14 were insensitive to the inclusion of equimolar amounts of another anionic phospholipid phosphatidylserine (PS), but the rates of ligand transfer by both mammalian PITPs were strikingly enhanced by the inclusion of phosphatidic acid (PA) in the receptor SUV. Binding of Sec14 to immobilized bilayers was substantial, while that of PITPα and PITPβ was 3–7 times weaker than Sec14 depending on phospholipid composition. When small proportions of the phosphoinositide PI(4)P were included in receptor SUVs (either with PI or not), Sec14 showed substantially increased rates of NBD-PC pick-up, whereas the PITPs were unaffected. The data are supportive of a role for PITPβ as functional PI transfer protein in vivo, but that Sec14 likely has a more elaborate function.

Structure-function insights into direct lipid transfer between membranes by Mmm1-Mdm12 of ERMES.

The endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12-Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1-Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes.

Regeneration of Streptavidin-coated Paramagnetic Beads for Multiple uses in Inter-membrane Lipid Transfer Assays

The specificity and the strong binding capacity of the biotin-streptavidin complex make the system one of the most widely used affinity pairs in molecular, immunological and cellular (in-vitro) assays. Previously reported regeneration process of the active Streptavidin solid support in DNA sequencing using water at temperatures above 70 oC, without denaturing the Streptavidin tetramer is shown in this study to be ineffective for a previously used Streptavidin-coated magnetic beads used in lipid transfer assay. Regeneration of the used beads in chloroform inactivated the beads while the use of 10 M NaOH destroyed the solid support of the bead. However, regenerating the paramagnetic beads used in lipid transfer assay with 25% aqueous ammonia only and with 25% aqueous ammonia in methanol (9:1) at 25 oC was very effective, with their reuse efficiency being 90% after recovery. This represents ca. 50 – 67 % research cost savings, especially in this era of economic depression.

Lipid transfers to HDL are predictors of precocious clinical coronary heart disease

Background High-density-lipoprotein (HDL) has several antiatherogenic properties and, although the concentration of HDL-cholesterol negatively correlates with incidence of coronary artery disease (CAD), this is not sufficient to evaluate the overall HDL protective role. The aim was to investigate whether precocious CAD patients show abnormalities in lipid transfers to HDL, a fundamental step in HDL metabolism and function. Methods Thirty normocholesterolemic CAD patients aged < 50 y and 30 controls paired for sex, age and B.M.I. were studied. Fasting blood samples were collected for the in vitro lipid transfer assay and plasma lipid determination. A donor nanoemulsion labeled with radioactive free-cholesterol, cholesteryl esters, phospholipids and triglycerides was incubated with whole plasma and after chemical precipitation of non-HDL fractions, supernatant was counted for radioactivity in HDL. Results LDL and HDL-cholesterol and triglycerides were equal in both groups. Transfers of free-cholesterol (3.8 ± 1.2%vs 7.0 ± 3.3%,p < 0.0001) and triglycerides (3.7 ± 1.7%vs 4.9 ± 1.9%, p = 0.0125) were diminished in CAD patients whereas cholesteryl ester transfer increased (6.5 ± 1.9%vs 4.8 ± 1.8%, p = 0.0008); phospholipid transfer was equal (17.8 ± 3.5% vs19.5 ± 3.9%). Conclusion Alterations in the transfer of lipids to HDL may constitute a new marker for precocious CAD and relation of this metabolic alteration with HDL antiatherogenic function should be investigated in future studies.

Regulation of the NPC2 protein‐mediated cholesterol trafficking by membrane lipids

Recycling and turnover of cell membranes play a critical role in cell metabolism. The internalization of membranes through the different processes of endocytosis, phagocytosis, and autophagy deliver a considerable amount of membranes and lipids to the endosomal and lysosomal system which is tasked with its degradation. Its failure to do so leads to severe fatal neurodegenerative diseases. In order to better understand how membranes are degraded, we have to investigate the complex interactions that take place in this compartment between complex membrane lipids, enzymes and lipid binding and transfer proteins involved. To this end, we developed lipid transfer and fusion assays which allow us to quantify these interactions and assess their specificity. The published results of these investigations are summarized in this article. One of our main conclusions is that we have provided evidence for the hypothesis that acid sphingomyelinase stimulates Niemann pick disease protein type 2-mediated cholesterol export substantially by converting sphingomyelin to ceramide in the inner membranes of late endosomes.

Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins

Human microsomal triacylglycerol transfer protein (hMTP) is essential for apolipoprotein B (apoB)-lipoprotein assembly and secretion and is known to transfer triacylglycerols, cholesterol esters, and phospholipids. To understand the relative importance of each lipid transfer activity, we compared the ability of hMTP and its Drosophila ortholog (dMTP) to assemble apoB lipoproteins and to transfer various lipids. apoB48 secretion was induced when co-expressed with either hMTP or dMTP in COS cells, and oleic acid supplementation further augmented secretion without altering particle density. C-terminal epitope-tagged dMTP (dMTP-FLAG) facilitated the secretion of apoB polypeptides in the range of apoB48 to apoB72 but was approximately 50% as efficient as hMTP-FLAG. Comparison of lipid transfer activities revealed that although phospholipid transfer was similar in both orthologs, dMTP was unable to transfer neutral lipids. We conclude that the phospholipid transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB lipoproteins and may represent its earliest function evolved for the mobilization of lipid in invertebrates. Identification of MTP inhibitors, which selectively affect transfer of a specific lipid class, may have therapeutic potential.

Preparation of fluorescent tocopherols for use in protein binding and localization with the α-tocopherol transfer protein

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin α-tocopherol were prepared incorporating fluorophores at the terminus of ω-functionalized 2- n-alkyl-substituted chromanols ( 1a– d and 4a– d) that match the methylation pattern of α-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein’s binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (α-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2 R,4′ R,8′ R-α-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of α-TTP-mediated inter-membrane transfer of α-tocopherol and to investigate the mechanism of α-TTP function at membranes of different composition.

StarD10, a START Domain Protein Overexpressed in Breast Cancer, Functions as a Phospholipid Transfer Protein

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.

Microsomal triglyceride transfer protein expression in adipocytes: A new component in fat metabolism

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.

Phospholipid Transfer Protein Is Present in Human Tear Fluid

The human tear fluid film consists of a superficial lipid layer, an aqueous middle layer, and a hydrated mucin layer located next to the corneal epithelium. The superficial lipid layer protects the eye from drying and is composed of polar and neutral lipids provided by the meibomian glands. Excess accumulation of lipids in the tear film may lead to drying of the corneal epithelium. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. To gain insight into the formation of tear film, we investigated whether PLTP and CETP are present in human tear fluid. Tear fluid samples were collected with microcapillaries. The presence of PLTP and CETP was studied in tear fluid by Western blotting, and the PLTP concentration was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. Size-exclusion and heparin-affinity chromatography assessed the molecular form of PLTP. PLTP is present in tear fluid, whereas CETP is not. Quantitative assessment of PLTP by ELISA indicated that the PLTP concentration in tear fluid, 10.9 +/- 2.4 microg/mL, is about 2-fold higher than that in human plasma. PLTP-facilitated phospholipid transfer activity in tears, 15.1 +/- 1.8 micromol mL(-)(1) h(-)(1), was also significantly higher than that measured in plasma. Inactivation of PLTP by heat treatment (+58 degrees C, 60 min) or immunoinhibition abolished the phospholipid transfer activity in tear fluid. Size-exclusion chromatography of tear fluid indicated that PLTP eluted in a position corresponding to a size of 160-170 kDa. Tear fluid PLTP was quantitatively bound to Heparin-Sepharose and could be eluted as a single peak by 0.5 M NaCl. These data indicate that human tear fluid contains catalytically active PLTP protein, which resembles the active form of PLTP present in plasma. The results suggest that PLTP may play a role in the formation of the tear film by supporting phospholipid transfer.

Influence of acylation sites of influenza B virus hemagglutinin on fusion pore formation and dilation.

The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.