The Brichos domain-containing C-terminal part of pro-surfactant protein C binds to an unfolded poly-val transmembrane segment

Native lung surfactant protein C (SP-C) is a 4.2-kDa acylpeptide that associates with alveolar surfactant phospholipids via a transmembrane alpha-helix. This helix contains mainly Val, although poly-Val is inefficient in helix formation, and helical SP-C can spontaneously convert to beta-sheet aggregates and amyloid-like fibrils. SP-C is cleaved out from a 21-kDa integral membrane protein, proSP-C, in the alveolar type II cell. Recently several mutations localized in the endoplasmic reticulum-lumenal (C-terminal) part of proSP-C (CTproSP-C) have been associated with intracellular accumulation of toxic forms of proSP-C, low levels of mature SP-C, and development of interstitial lung disease. CTproSP-C contains a approximately 100-residue Brichos domain of unknown function that is also found in other membrane proteins associated with amyloid formation, dementia, and cancer. Here we find that recombinant CTproSP-C binds lipid-associated SP-C, which is in beta-strand conformation, and that this interaction results in an increased helical content. In contrast, CTproSP-C does not bind alpha-helical SP-C. Recombinant CTproSP-C(L188Q), a mutation associated with interstitial lung disease, shows secondary and quaternary structures similar to those of wild type CTproSP-C but is unable to bind lipid-associated beta-strand SP-C. Transfection of CTproSP-C into HEK293 cells that express proSP-C(L188Q) increases the amount of proSP-C protein, whereas no effect is seen on cells expressing wild type proSP-C. These findings suggest that CTproSP-C binds nonhelical SP-C and thereby prevents beta-sheet aggregation and that mutations in CTproSP-C can interfere with this function.     

The transmembrane domain of surfactant protein C precursor determines the morphology of the induced membrane compartment in CHO cells

Surfactant protein C (SP-C) is a small lipopeptide of which the main part consists of a typical valyl-rich transmembrane domain. The protein is expressed as a propeptide (proSP-C) which is processed and sorted via the regulated secretory pathway to the lamellar body, where mature SP-C is stored before secretion into the alveolar space. In this study we investigated the identity of the compartment to which proSP-C is sorted in cells that do not have a regulated secretory pathway, such as CHO cells. By electron microscopy we determined that proSP-C was localized in an uncommon membrane compartment with very regular morphology, which was not present in control cells. This membrane compartment is not influenced by the palmitoylation of proSP-C and is probably derived from the endoplasmic reticulum. However, proSP-C chimeras with artificial transmembrane domains induced a membrane compartment with a different morphology. Therefore we propose that the typical amino acid sequence of the transmembrane domain of proSP-C plays a role in membrane formation and morphology, which may be relevant under physiological conditions.     

Surfactant protein C: its unique properties and emerging immunomodulatory role in the lung

Surfactant protein C (SP-C) is a highly hydrophobic protein found in pulmonary surfactant. SP-C is synthesized exclusively in alveolar type II cells as a 21 kDa integral membrane precursor protein and subsequently proteolytically processed to a 3.7 kDa secretory protein. SP-C enhances the adsorption and spreading of phospholipids at the air-liquid interface thereby promoting the surface tension-lowering properties of surfactant. The importance of SP-C in normal lung function is underscored by the recent findings of inflammatory lung diseases associated both with absence of alveolar SP-C and with cellular expression of mutant SP-C isoforms. This review examines our current understanding of the role of SP-C in maintaining alveolar epithelial homeostasis and the potential role of abnormal SP-C expression in the development of lung diseases with particular emphasis on microbial pulmonary infection and inflammation.     

N-terminal 1-54 amino acid sequence and Armadillo repeat domain are indispensable for P120-catenin isoform 1A in regulating E-cadherin

P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1-54 amino acids and the Armadillo repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1-54 amino acid sequence and Armadillo repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin.     

Adhesion of acidic lipid vesicles by 21.5 kDa (recombinant) and 18.5 kDa isoforms of myelin basic protein

Myelin basic protein (MBP) is thought to be responsible for adhesion of the intracellular surfaces of compact myelin to give the major dense line. The 17 and 21.5 kDa isoforms containing exon II have been reported by others to localize to the cytoplasm and nucleus of murine oligodendrocytes and HeLa cells while the 14 and 18.5 kDa isoforms lacking exon II are confined to the plasma membrane. However, we show that the exon II(-) 18.5 kDa form and a recombinant exon II(+) 21.5 kDa isoform both caused similar aggregation of acidic lipid vesicles, indicating that they should have similar abilities to bind to the intracellular lipid surface of the plasma membrane and to cause adhesion of those surfaces to each other. The circular dichroism spectra of the two isoforms indicated that both had a similar secondary structure. Thus, both isoforms should be able to bind to and cause adhesion of the cytosolic surfaces of compact myelin. The fact that they do not could be due to differences in post-translational modification in vivo, trafficking through the cell and/or subcellular location of synthesis, but it is not due to differences in their lipid binding.     

Biosynthesis of surfactant protein C (SP-C). Sorting of SP-C proprotein involves homomeric association via a signal anchor domain

Rat surfactant protein C (SP-C) is synthesized as a 194-amino acid propeptide (SP-C-(1-194)) that is directed to the distal secretory pathway and proteolytically processed as an integral membrane protein to yield its mature form. We had shown previously that trafficking of proSP-C is mediated both by a signal anchor domain contained within the mature SP-C sequence and by a targeting domain in the NH(2)-flanking propeptide. Based on evidence from other integral membrane proteins, we hypothesized that proSP-C targeting is effected by oligomerization of proSP-C monomers. To evaluate this in vitro, cDNA constructs encoding for either wild type proSP-C (pcDNA3/SP-C-(1-194)) or heterologous fusion proteins containing green fluorescent protein (EGFP) linked to SP-C-(1-194) (EGFP/SP-C-(1-194)), to mutant proSP-C lacking the NH(2) targeting domain (EGFP/SP-C-(24-194)), or to mature SP-C alone (EGFP/SP-C-(24-58)) were produced. In transfected A549 cells, fluorescence microscopy revealed that pcDNA3/SP-C-(1-194) and EGFP/SP-C-(1-194) were each expressed in CD63 (+), EEA1 (-) cytoplasmic vesicles. Expression of EGFP/SP-C-(24-194) or EGFP/SP-C-(24-58) resulted in translocation but retention in early compartments. When co-transfected with pcDNA3/SP-C-(1-194), both EGFP/SP-C-(24-194) and EGFP/SP-C-(24-58) were directed to CD63 (+) vesicles that also contained SP-C-(1-194). In contrast, trafficking of a folding mutant that forms juxtanuclear aggregates, EGFP/SP-C(C122/186G), was not corrected by cotransfection with pcDNA3/SP-C-(1-194). Chemical cross-linking studies of transfected cell lysates with bismaleimidohexane produced multimeric forms of both EGFP/SP-C-(1-194) and EGFP/SP-C-(24-58). These results indicate that sorting involves oligomeric association of proSP-C monomers mediated by the mature SP-C domain. Heteromeric assembly allows wild type proSP-C to facilitate trafficking of SP-C mutants with intact transmembrane domains but lacking targeting signals. We speculate that heterotypic oligomerization of wild type with SP-C folding mutants produces a dominant negative thus contributing to the pathology of chronic lung disease associated with patients heterozygous for mutant SP-C alleles.     

Genetic replacement of surfactant protein-C reduces respiratory syncytial virus induced lung injury

Background

Individuals with deficiencies of pulmonary surfactant protein C (SP-C) develop interstitial lung disease (ILD) that is exacerbated by viral infections including respiratory syncytial virus (RSV). SP-C gene targeted mice (Sftpc -/-) lack SP-C, develop an ILD-like disease and are susceptible to infection with RSV.

Methods

In order to determine requirements for correction of RSV induced injury we have generated compound transgenic mice where SP-C expression can be induced on the Sftpc -/- background (SP-C/Sftpc -/-) by the administration of doxycycline (dox). The pattern of induced SP-C expression was determined by immunohistochemistry and processing by Western blot analysis. Tissue and cellular inflammation was measured following RSV infection and the RSV-induced cytokine response of isolated Sftpc +/+ and -/- type II cells determined.

Results

After 5 days of dox administration transgene SP-C mRNA expression was detected by RT-PCR in the lungs of two independent lines of bitransgenic SP-C/Sftpc -/- mice (lines 55.3 and 54.2). ProSP-C was expressed in the lung, and mature SP-C was detected by Western blot analysis of the lavage fluid from both lines of SP-C/Sftpc -/- mice. Induced SP-C expression was localized to alveolar type II cells by immunostaining with an antibody to proSP-C. Line 55.3 SP-C/Sftpc -/- mice were maintained on or off dox for 7 days and infected with 2.6x10⁷ RSV pfu. On day 3 post RSV infection total inflammatory cell counts were reduced in the lavage of dox treated 55.3 SP-C/Sftpc -/- mice (p = 0.004). The percentage of neutrophils was reduced (p = 0.05). The viral titers of lung homogenates from dox treated 55.3 SP-C/Sftpc -/- mice were decreased relative to 55.3 SP-C/Sftpc -/- mice without dox (p = 0.01). The cytokine response of Sftpc -/- type II cells to RSV was increased over that of Sftpc +/+ cells.

Conclusions

Transgenic restoration of SP-C reduced inflammation and improved viral clearance in the lungs of SP-C deficient mice. The loss of SP-C in alveolar type II cells compromises their response to infection. These findings show that the restoration of SP-C in Sftpc -/- mice in response to RSV infection is a useful model to determine parameters for therapeutic intervention.     

Mutations linked to interstitial lung disease can abrogate anti-amyloid function of prosurfactant protein C

The newly synthesized proSP-C (surfactant protein C precursor) is an integral ER (endoplasmic reticulum) membrane protein with a single metastable polyvaline alpha-helical transmembrane domain that comprises two-thirds of the mature peptide. More than 20 mutations in the ER-lumenal CTC (C-terminal domain of proSP-C), are associated with ILD (interstitial lung disease), and some of the mutations cause intracellular accumulation of cytotoxic protein aggregates and a corresponding decrease in mature SP-C. In the present study, we showed that: (i) human embryonic kidney cells expressing the ILD-associated mutants proSP-C(L188Q) and proSP-C(DeltaExon4) accumulate Congo Red-positive amyloid-like inclusions, whereas cells transfected with the mutant proSP-C(I73T) do not; (ii) transfection of CTC into cells expressing proSP-C(L188Q) results in a stable CTC-proSP-C(L188Q) complex, increased proSP-C(L188Q) half-life and reduced formation of Congo Red-positive deposits; (iii) replacement of the metastable polyvaline transmembrane segment with a stable polyleucine transmembrane segment likewise prevents formation of amyloid-like proSP-C(L188Q) aggregates; and (iv) binding of recombinant CTC to non-helical SP-C blocks SP-C amyloid fibril formation. These results suggest that CTC can prevent the polyvaline segment of proSP-C from promoting formation of amyloid-like deposits during biosynthesis, by binding to non-helical conformations. Mutations in the Brichos domain of proSP-C may lead to ILD via loss of CTC chaperone function.     

Structure and influence on stability and activity of the N-terminal propeptide part of lung surfactant protein C

Mature lung surfactant protein C (SP-C) corresponds to residues 24-58 of the 21 kDa proSP-C. A late processing intermediate, SP-Ci, corresponding to residues 12-58 of proSP-C, lacks the surface activity of SP-C, and the SP-Ci alpha-helical structure does not unfold in contrast to the metastable nature of the SP-C helix. The NMR structure of an analogue of SP-Ci, SP-Ci(1-31), with two palmitoylCys replaced by Phe and four Val replaced by Leu, in dodecylphosphocholine micelles and in ethanol shows that its alpha-helix vs. that of SP-C is extended N-terminally. The Arg-Phe part in SP-Ci that is cleaved to generate SP-C is localized in a turn structure, which is followed by a short segment in extended conformation. Circular dichroism spectroscopy of SP-Ci(1-31) in microsomal or surfactant lipids shows a mixture of helical and extended conformation at pH 6, and a shift to more unordered structure at pH 5. Replacement of the N-terminal hexapeptide segment SPPDYS (known to constitute a signal in intracellular targeting) of SP-Ci with AAAAAA results in a peptide that is mainly unstructured, independent of pH, in microsomal and surfactant lipids. Addition of a synthetic dodecapeptide, corresponding to the propeptide part of SP-Ci, to mature SP-C results in slower aggregation kinetics and altered amyloid fibril formation, and reduces the surface activity of phospholipid-bound SP-C. These data suggest that the propeptide part of SP-Ci prevents unfolding by locking the N-terminal part of the helix, and that acidic pH results in structural disordering of the region that is proteolytically cleaved to generate SP-C.     

Expression and membrane localization of MCT isoforms along the length of the human intestine

Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells. monocarboxylate transporter; short-chain fatty acids; absorption; short-chain fatty acid transport; mammalian colon     

Characterization of a novel amphiregulin-related molecule in 12-O-tetradecanoylphorbol-13-acetate-treated breast cancer cells

Amphiregulin (AR) can be induced at the mRNA level by 17-β-estradiol (E₂) or the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This study compares the effects of TPA and E₂ on the regulation of processing of AR isoforms and on subcellular localization in human MCF-7 breast cancer cells. AR was localized in the nucleus of MCF-7 cells after E₂ treatment, whereas it was predominantly secreted after TPA treatment. AR isoforms of 28, 18, and 10 kDa and an additional species of approximately 55–60 kDa were detected in the cellular conditioned media after TPA stimulation. Expression of this unusual AR isoform was inhibited by protein kinase C (PKC) inhibitors such as bryostatin or H-7. The biochemical properties of this isoform are consistent with it being an N-linked glycosylated form of the AR precursor that contains unprocessed mannose residues. The size of this large isoform is reduced to approximately 40 kDa after treating the TPA-induced MCF-7 cells with tunicamycin or treating the conditioned media of such cells with N-glycosidase F or with endoglycosidase H. Moreover, this isoform is able to bind several lectins with specificity for mannose residues. The 55–60 kDa glycosylated AR isoform, like lower Mr AR isoforms, is able to bind to heparin and to stimulate the growth of MCF-10A cells by interacting with the EGF receptor. These data suggest that TPA activation of PKC may be involved in post-translational modifications of AR, such as glycosylation, and in alteration of its subcellular routing to predominantly a secretory pathway. © 1996 Wiley-Liss, Inc.     

Palmitoylation and processing of the lipopeptide surfactant protein C

Pulmonary surfactant, a mixture of lipids and proteins, reduces the surface tension at the air-water interface of the lung alveoli by forming a surface active film. This way, it prevents alveoli from collapsing and facilitates the work of breathing. Surfactant protein C (SP-C) plays an important role in this surfactant function. SP-C is expressed as a proprotein (proSP-C), which becomes posttranslationally modified with palmitate and undergoes several rounds of proteolytical cleavage. This results in the formation of mature SP-C, which is stored in the lamellar bodies (LB) and finally secreted into the alveolar space. Recently, new insights into the sorting, processing and palmitoylation of proSP-C have been obtained by mutagenesis studies. Moreover, reports on the association of development of lung disease with SP-C deficiency have led to new insights into the importance of SP-C for proper surfactant homeostasis. In addition, new information has become available on the role of the palmitoyl chains of SP-C in surface activity. This review summarizes these recent developments in the processing and function of SP-C, with particular emphasis on the signals for and role of palmitoylation of SP-C.     

Structural characterization of recombinant human interferon-gammas derived from two different mammalian cells

Recombinant human interferon-gammas (rHuIFN-gamma s) were obtained from two different mammalian cells (mouse C127 cells and Chinese hamster ovary, CHO, cells) cultured in a microcarrier culture system. Both rHuIFN-gamma s were purified using sequential chromatographies for their comparison of structural properties. The peptide maps of HuIFN-gamma s digested with V8 protease and Western blot analysis demonstrated that C127 cells yielded mainly about 25kDa component and CHO cells produced about 25kDa and about 20kDa components. By the identification of glycosylated peptides, it was suggested that 20kDa and 25kDa components are glycosylated at one and at two sites, respectively. C-terminal amino acid sequence analysis indicated that both rHuIFN-gamma s consisted of at least six different species lacking 2 to 16 amino acid residues from C-terminus, so that C-termini of both rHuIFN-gamma s were slightly different from each other. Amino acid sequence and composition analyses of N-terminal peptides demonstrated that N-termini of both rHuIFN-gamma s were blocked and were supposed to be identical with that of natural HuIFN-gamma. These results suggested that different molecular heterogeneities of rHuIFN-gamma s resulted from the difference of post-translational modifications of host cells.     

Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells

Pulmonary surfactant is a lipoprotein complex that functions to reduce surface tension at the air liquid interface in the alveolus of the mature lung. In late gestation glycogen-laden type II cells shift their metabolic program toward the synthesis of surfactant, of which phosphatidylcholine (PC) is by far the most abundant lipid. To investigate the cellular site of surfactant PC synthesis in these cells we determined the subcellular localization of two key enzymes for PC biosynthesis, fatty acid synthase (FAS) and CTP:phosphocholine cytidylyltransferase-alpha (CCT-alpha), and compared their localization with that of surfactant storage organelles, the lamellar bodies (LBs), and surfactant proteins (SPs) in fetal mouse lung. Ultrastructural analysis showed that immature and mature LBs were present within the glycogen pools of fetal type II cells. Multivesicular bodies were noted only in the cytoplasm. Immunogold electron microscopy (EM) revealed that the glycogen pools were the prominent cellular sites for FAS and CCT-alpha. Energy-filtering EM demonstrated that CCT-alpha bound to phosphorus-rich (phospholipid) structures in the glycogen. SP-B and SP-C, but not SP-A, localized predominantly to the glycogen stores. Collectively, these data suggest that the glycogen stores in fetal type II cells are a cellular site for surfactant PC synthesis and LB formation/maturation consistent with the idea that the glycogen is a unique substrate for surfactant lipids.     

Lack of palmitoylation redirects p59Hck from the plasma membrane to p61Hck-positive lysosomes

Hck, a protein-tyrosine kinase of phagocytes, is the unique member of the Src family expressed under two alternatively translated isoforms differing in their N-terminal site of acylation: p61(Hck) has an additional 21-amino acid sequence comprising a single myristoylation motif, whereas p59(Hck) N terminus has myristoylation and palmitoylation sites. To identify the molecular determinants involved in the targeting of each isoform, they were fused to GFP and expressed in HeLa and CHO cells. p61(Hck) was associated with lysosomal vesicles, whereas p59(Hck) was found at the plasma membrane and to a low extent associated with lysosomes. Their unique N-terminal domains were sufficient to target GFP to the corresponding intracellular compartments. Mutation of the palmitoylation site of p59(Hck) redirected this isoform to lysosomes, indicating that the palmitoylation state governs the association of p59(Hck) with the plasma membrane or with lysosomes. In addition, both isoforms and the nonpalmitoylated p59(Hck) mutant were found on the Golgi apparatus, suggesting a role of this organelle in the subcellular sorting of Hck isoforms. Regarding their subcellular localizations, we propose that bi-acylated p59(Hck) might transduce plasma membrane receptor signals, whereas p61(Hck) and the nonpalmitoylated p59(Hck) might control the biogenesis of phagolysosomes, two functions yet proposed for Hck in phagocytes.     

Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C

Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4--7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.     

Characterization and binding properties of human fetal lung fatty acid-binding proteins

When delipidated Mr>10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.Abbreviations AO anthroyloxy - 12-AS 12-(9-anthroyloxy)stearic acid - FABP fatty acid-binding protein - NBD-PE [N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine - Pal-CoA palmitoyl coenzyme A - PITC phenylisothiocyanate - PBS phosphate-buffered saline - PtdCho phosphatidylcholine - SUV small unilamellar vesicle - Tris tris(hydroxymethyl) amino methane