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BBA - Biomembranes (v.1715, #2)

Editorial Board (pp. ii).

Temperature dependence of the Langmuir monolayer packing of mycolic acids from Mycobacterium tuberculosis by Masumi Villeneuve; Mizuo Kawai; Hideki Kanashima; Motoko Watanabe; David E. Minnikin; Hiroo Nakahara (pp. 71-80).

Phase diagrams of the Langmuir monolayer of dicyclopropyl alpha mycolic acid (α-MA), cyclopropyl methoxy mycolic acid (MeO-MA), and cyclopropyl ketomycolic acids (Keto-MA) from Mycobacterium tuberculosis were obtained by thermodynamic analysis of the surface pressure ( π) vs. average molecular area ( A) isotherms at temperatures in the range of 10–46 °C. The Langmuir monolayers of MAs were shown to exhibit various phases depending on the temperature ( T) and the π values. In the Langmuir monolayer of Keto-MA, the carbonyl group in the meromycolate chain apparently touches the water surface to give the molecule a W-shape in all the temperatures and surface pressures studied. Keto-MA formed a rigid solid condensed film, with four hydrocarbon chains packing together, not observed in the others. In contrast, the monolayer films of α-and MeO-MAs having no such highly hydrophilic intra-chain groups in the meromycolate chain were mostly in liquid condensed phase. This novel insight into the packing of mycolic acids opens up new avenues for the study of the role of mycolic acids in the mycobacterial cell envelopes and pathogenic processes.

Keywords: Mycolic acid; Mycobacterium tuberculosis; Langmuir monolayer; Thermodynamics; Phase diagram; Fatty acid conformation

Structure and orientation study of fusion peptide FP23 of gp41 from HIV-1 alone or inserted into various lipid membrane models (mono-, bi- and multibi-layers) by FT-IR spectroscopies and Brewster angle microscopy by Sabine Castano; Bernard Desbat (pp. 81-95).

In the present work, we study the structure and the orientation of the 23 N-terminal peptide of the HIV-1 gp 41 protein (AVGIGALFLGFLGAAGSTMGARS) called FP23. The behaviour of FP23 was investigated alone at the air/water interface and inserted into various lipid model systems: in monolayer or multibilayers of a DOPC/cholesterol/DOPE/DOPG (6/5/3/2) and in a DMPC bilayer. PMIRRAS and polarized ATR spectroscopy coupled with Brewster angle microscopy and spectral simulations were used to precisely determine the structure and the orientation of the peptide in its environment as well as the lipid perturbations induced by the FP23 insertion. The infra-red results show the structural polymorphism of the FP23 and its ability to transit quasi irreversibly from an α-helix to antiparallel β-sheets. At the air/water interface, the transition is induced by compression of the peptide alone and is modulated by compression and lipid to peptide ratio (Ri) when FP23 is inserted into a lipid monolayer. In multibilayers and in a single bilayer, there is coexistence in quasi equal proportions of α-helix and antiparallel β-sheets of FP23 at low peptide content (Ri=100, 200) while antiparallel β-sheets are predominant at high FP23 concentration (Ri=50). In (multi)bilayer systems, evaluation of dichroic ratios and sprectral simulations show that both the α-helix and the antiparallel β-sheets are tilted at diluted FP23 concentrations (tilt angle of α-helix with respect to the normal of the interface=36.5±3.0° for FP23 in multibilayers of DOPC/Chol/DOPE/DOPG at Ri=200 and 39.0±5.0° in a single bilayer of DMPC at Ri=100 and tilt angle of the β-sheets=36.0±2.0° for the β-sheets in multibilayers and 30.0±2.0° in the lipid bilayer). In parallel, the FP23 induces an increase of the lipid chain disorder which shows both by an increase of the methylene stretching frequencies and an increase of the average C–C–C angle of the acyl chains. At high FP23 content (Ri=50), the antiparallel β-sheets induce a complete disorganization of the lipid chains in (multi)bilayers.

Keywords: Abbreviations; DMPC; dimirystoyl-phosphatidylcholine; DOPC; dioleyl-phosphatidylcholine; DOPG; dioleyl-phosphatidylglycerol; DOPE; dioleyl-phosphatidylethanolamine; PMIRRAS; polarization modulation infra-red reflection absorption spectroscopy; ATR; attenuated total reflexion spectroscopy; BAM; Brewster angle microscopyFusion peptide FP23; Lipid-bound structure; Peptide orientation; PMIRRAS; ATR spectroscopy; Brewster angle microscopy

Rose Bengal located within liposome do not affect the activity of inside-out oriented Na,K-ATPase by H�rica de Lima Santos; Carolina Fortes Rigos; Antonio Cl�udio Tedesco; Pietro Ciancaglini (pp. 96-103).

DPPC:DPPE–proteoliposomes (in which the enzyme is inside-out oriented) and DLOPC:DLOPE–proteoliposomes (in which the enzyme is only 40% inside-out oriented) is an excellent model for studying the selective effect of the reactive oxygen species, produced by the photo-activation of Rose Bengal. Both proteoliposomes used, when submitted to photo-irradiation with laser using 1200 mJ/cm² energy dose, in the absence of the Rose Bengal, did not shown any effect in the ATPase activity and in the integrity of its systems. Also, no effect was observed using 50 μM of Rose Bengal encapsulated in the interior of the DPPC:DPPE–proteoliposome system. But, when we use 50 μM of Rose Bengal, present only in the extravesicular environment, and photo-irradiation with a laser dose of 200 mJ/cm², it results in the loss of 40–50% of the ATPase activity, with damage of the DPPC:DPPE–proteoliposome integrity. Using a dose of 400 mJ/cm² the ATPase activity was totality lost. Consequently, these effects could be correlated with direct damage in the peptide structure. The photo-irradiation of the system constituted by DLOPC:DLOPE–proteoliposome in the presence of Rose Bengal, encapsulated only in the interior compartment or in the extra-liposomal environments, revealed a gradual decrease of the ATPase activity, maintaining it at 30% after a dose of 1200 mJ/cm² and losing total ATPase activity at 800 mJ/cm², respectively, with the loss of integrity of this vesicular system in both conditions studied. The generated singlet oxygen could attack the double linkage present in the fatty acid structure of the lipid instead of the amino acid in the protein structure and, in a second step, result in an indirect inactivation of the enzyme activity. In summary, these results indicated that singlet oxygen species produced by photo-oxidation of Rose Bengal using laser light could act in protein and lipid structure depending on its proportion or distribution.

Keywords: Rose Bengal; Na,K-ATPase; DPPC:DPPE–Liposome; ATPase activity

Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1 by Ruben J. Boado; Jian Yi Li; Chun Chu; Fumio Ogoshi; Petra Wise; William M. Pardridge (pp. 104-110).

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood–brain barrier (BBB). Owing to the high affinity (low K_m) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low K_m of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys¹⁶⁰ of LAT1 and Cys¹¹⁰ of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl₂ with a K_i of 0.56±0.11 μM. The inhibitory effect of HgCl₂ for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl₂ for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys⁸⁸ and Cys⁴³⁹, altered amino acid transport. The V_max was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys⁴³⁹ residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.

Keywords: Phenylketonuria; PKU; Brain amino acid transport; LAT1; Site-directed mutagenesis

Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules by Sonja M.K. Alanko; Katrin K. Halling; Stina Maunula; J. Peter Slotte; Bodil Ramstedt (pp. 111-121).

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.

Keywords: Cholesterol; Cholestatrienol; Sterol displacement; Quenching; Spin-labeled phosphatidylcholine; Ceramide

Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity by Patrick Garidel; Michael Rappolt; Andra B. Schromm; J�rg Howe; Karl Lohner; J�rg Andr�; Michel H.J. Koch; Klaus Brandenburg (pp. 122-131).

The physicochemical properties and biological activities of rough mutant lipopolysaccharides Re (LPS Re) as preformed divalent cation (Mg²⁺, Ca²⁺, Ba²⁺) salt form or as natural or triethylamine (Ten⁺)-salt form under the influence of externally added divalent cations were investigated using complementary methods: Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopic (FT-IR) measurements for the β↔α gel to liquid crystalline phase behaviour of the acyl chains of LPS, synchrotron radiation X-ray diffraction studies for their aggregate structures, electron density calculations of the LPS bilayer systems, and LPS-induced cytokine (interleukin-6) production in human mononuclear cells. The divalent cation salt forms of LPS exhibit considerable changes in physicochemical parameters such as acyl chain mobility and aggregate structures as compared to the natural or monovalent cation salt forms. Concomitantly, the biological activity was much lower in particular for the Ca²⁺- and Ba²⁺-salt forms. This decrease in activity results mainly from the conversion of the unilamellar/cubic aggregate structure of LPS into a multilamellar one. The reduced activity also clearly correlates with the higher order – lower mobility – of the lipid A acyl chains. Both effects can be understood by an impediment of the interactions of LPS with binding proteins such as lipopolysaccharide-binding protein (LBP) and CD14 due to the action of the divalent cations.

Keywords: Cytokine; DSC; Electron density; Lipopolysaccharide; X-ray scattering

Characterization of CLCA protein expressed in ductal cells of rat salivary glands by Jun Yamazaki; Kazuhiko Okamura; Kazunari Ishibashi; Kenji Kitamura (pp. 132-144).

A molecular entity for Ca²⁺-dependent Cl⁻ transport has not been well characterized in salivary cells. Here, we identify a rat CLCA homologue (rCLCA1) using a polymerase chain reaction (PCR)-based strategy. The full length of the isoform was 3.3 kb, and the predicted open reading frame encoded a 903-amino acid protein. Immunoblotting using a specific anti-rCLCA antibody recognizing near the amino-terminus showed the expression of N-glycosylated 120- and 86-kDa proteins in the membrane fraction of rCLCA1-transfected HEK293 cells. Reverse transcription-PCR results showed mRNA expressions in rat submandibular gland (SMG), ileum, and lung. Intense immunostaining was detected in the striated ducts, but not in the acinar cells, of SMG. Immunoblot for the membrane fraction of SMG revealed the existence of 137- and 90-kDa protein species. N-glycosidase F reduced the size of these bands toward those of the deglycosylated forms in the transfected HEK293 cells. A marked ionomycin-induced Cl⁻ conductance was observed in the transfected cells. The current was Ca²⁺-dependent and sensitive to niflumic acid and DIDS. rCLCA1 proteins are probably responsible for modulation of Ca²⁺-dependent Cl⁻ transport in salivary ductal cells, where the 137- and 90-kDa proteins may be modified posttranslationally in a manner similar to those in the heterologous expression system.

Keywords: Ca; ²⁺; -activated Cl; ⁻; channel; CLCA; N-glycosylation; Epithelium; Submandibular gland; Ileum

Biomembranes Cumulative Contents (pp. 145-145).

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BBA - Biomembranes (v.1715, #2)