Lipid Transfer Proteins

The specificity and the strong binding capacity of the biotin-streptavidin complex makes 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 o C, 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 °C 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.

Membrane contact sites between the endoplasmic reticulum (ER) and mitochondria function as a central
hub for the exchange of phospholipids and calcium. The yeast Endoplasmic Reticulum–Mitochondrion
Encounter Structure (ERMES) complex is composed of five subunits that tether the ER and mitochondria.
Three ERMES subunits (i.e., Mdm12, Mmm1, and Mdm34) contain the synaptotagmin-like mitochondrial
lipid-binding protein (SMP) domain. The SMP domain belongs to the tubular lipid-binding protein
(TULIP) superfamily, which consists of ubiquitous lipid scavenging and transfer proteins. Herein, we
describe the methods for expression and purification of recombinant Mdm12, a bona fide
SMP-containing protein, together with the subsequent identification of its bound phospholipids by
high-performance thin-layer chromatography (HPTLC) and the characterization of its lipid exchange
and transfer functions using lipid displacement and liposome flotation in vitro assays with liposomes as
model biological membranes. These methods can be applied to the study and characterization of novel
lipid-binding and lipid-transfer proteins.

The present study reports, for the first time in literature, the purification and biochemical characterization of a small basic protein from maize seeds similar to plant lipid transfer proteins-2, named mLTP2. The mLTP2 consists of 70 amino acid residues and has an Mr of 7303.83, determined by electrospray ionization mass spectrometry. The primary structure of mLTP2 was determined by automated Edman degradation of the intact protein and peptides obtained from digestions with trypsin and by C-terminal sequencing using carboxypeptidase Y. The mLTP2 exhibits high sequence similarity (51–44% identical positions) with other plant LTP2s previously described.

When plants conquered land, they developed specialized organs, tissues, and cells in order to survive in this new and harsh terrestrial environment. New cell polymers such as the hydrophobic lipid-based polyesters cutin, suberin, and sporopollenin were also developed for protection against water loss, radiation, and other potentially harmful abiotic factors. Cutin and waxes are the main components of the cuticle, which is the waterproof layer covering the epidermis of many aerial organs of land plants. Although the in vivo functions of the group of lipid binding proteins known as lipid transfer proteins (LTPs) are still rather unclear, there is accumulating evidence suggesting a role for LTPs in the transfer and deposition of monomers required for cuticle assembly. In this review, we first present an overview of the data connecting LTPs with cuticle synthesis. Furthermore, we propose liverworts and mosses as attractive model systems for revealing the specific function and activity of LTPs in the biosynthesis and evolution of the plant cuticle.

Eukaryotic cells are characterized by their exquisite compartmentalization resulting from a cornucopia of membrane-bound organelles. Each of these compartments hosts a flurry of biochemical reactions and supports biological functions such as genome storage, membrane protein and lipid biosynthesis/degradation and ATP synthesis, all essential to cellular life. Acting as hubs for the transfer of matter and signals between organelles and throughout the cell, membrane contacts sites (MCSs), sites of close apposition between membranes from different organelles, are essential to cellular homeostasis. One of the now well-acknowledged function of MCSs involves the non-vesicular trafficking of lipids; its characterization answered one long-standing question of eukaryotic cell biology revealing how some organelles receive and distribute their membrane lipids in absence of vesicular trafficking. The endoplasmic reticulum (ER) in synergy with the mitochondria, stands as the nexus for the biosynthesis and distribution of phospholipids (PLs) throughout the cell by contacting nearly all other organelle types. MCSs create and maintain lipid fluxes and gradients essential to the functional asymmetry and polarity of biological membranes throughout the cell. Membrane apposition is mediated by proteinaceous tethers some of which function as lipid transfer proteins (LTPs). We summarize here the current state of mechanistic knowledge of some of the major classes of LTPs and tethers based on the available atomic to near-atomic resolution structures of several "model" MCSs from yeast but also in Metazoans; we describe different models of lipid transfer at MCSs and analyze the determinants of their specificity and directionality. Each of these systems illustrate fundamental principles and mechanisms for the non-vesicular exchange of lipids between eukaryotic membrane-bound organelles essential to a wide range of cellular processes such as at PL biosynthesis and distribution, lipid storage, autophagy and organelle biogenesis.

Background. Non-specific Lipid Transfer Proteins (nsLTPs) are widely distributed in the plant kingdom and constitute a superfamily of related proteins. More than 800 different sequences have been characterized so far, but their biological functions remain unclear. It has been clear for years that they present a certain interest for agronomic and nutritional issues. Deciphering their functions means collecting and analyzing a variety of data from gene sequence to protein structure, from cellular localization to the physiological role. As a huge and growing number of new protein sequences are available nowadays, extracting meaningful knowledge from sequence-structure-function relationships calls for the development of new tools and approaches. As nsLTPs show high evolutionary divergence, but a conserved common right-handed superhelix structural fold, and as they are involved in a large number of key roles in plant development and defense, they are a stimulating case study for validati...

Non-specific lipid-transfer proteins (nsLTPs) are major human allergens in many plant species, albeit their role in plant biochemistry is still undefined. They are found in many plant species, either as one or several isoforms according to the species, and usually they are found to concentrate in the outer part of the fruits. In this work, the characterization of tomato nsLTP isoforms was performed on the three main fractions of Piccadilly tomato fruit (peel, pulp and seeds) by using ultracentrifuge devices with molecular cut-off able to retain proteins with molecular weight typical of plant LTPs. The isolated proteins were further analysed by LC-MS, in order to investigate the occurrence and the localization of tomato LTP isoforms. The chromatographic retention times, the molecular masses, the presence of eight cysteine residues in their tertiary structures and the sequence information obtained by MS, although not complete yet, allowed us to identify four different LTP isoforms, not yet reported in the literature, which were found to be concentrated in the seed fractions. None of the molecular masses of these potential LTPs was already present in the UniProtKB/SwissProt database. MALDI imaging experiments confirmed their presence and main localization in seeds, although the actual data hinted at their presence around seeds, rather than exactly in them. These data hint to a complicated scenario concerning LTP proteins in tomato. Figure 5. (A) Full-MS spectra taken directly on tissue (cross in panels D and E) showing lipid-transfer protein masses, compared with full-MS spectra (B) taken in the pulp area (cross in panels D and E). Scanned image (D) and merged MS image (E) of the three analytes clearly outlining their localization near the seeds. M. Bencivenni et al. wileyonlinelibrary.com/journal/jms

"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 produced
by ceramide kinase is an activator of group IVA cytosolic phospholipase
A2a (cPLA2a), the rate-limiting releaser of arachidonic acid
used for pro-inflammatory eicosanoid production3,6–9, which contributes
todisease pathogenesis in asthmaor airwayhyper-responsiveness,
cancer, atherosclerosis and thrombosis.Tomodulate 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 likely10but non-vesicularmechanisms
forC1Psensing, transfer andpresentation remain unexplored11,12.
Moreover, the molecular basis for selective recognition and binding
among signalling lipids with phosphate headgroups, namely C1P,
phosphatidic acid or their lyso-derivatives, remains unclear. Here,
a ubiquitously expressed lipid transfer protein, human GLTPD1,
named here CPTP, is shown to specifically transfer C1P between
membranes. Crystal structures establish C1P binding through a novel
surface-localized, phosphate headgroup recognition centre connected
to an interior hydrophobic pocket that adaptively expands to ensheath
differing-length lipid chains using a cleft-like gating mechanism.
The two-layer, a-helically-dominated ‘sandwich’ topology identifies
CPTP as the prototype for a new glycolipid transfer protein fold13
subfamily.CPTPresides in the cell cytosol but associates with the trans-
Golgi network, nucleus and plasma membrane. RNA interferenceinduced
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
cPLA2a release of arachidonic acid, triggering pro-inflammatory
eicosanoid generation."

Les fruits comestibles renferment de nombreux allergènes qui correspondent essentiellement à des protéines de défense ou protéines PR (pour Pathogenesis Related). Ces protéines, très répandues chez les végétaux, sont des panallergènes. Les principaux allergènes appartiennent aux groupes des PR-2 (1,3β-glucanases), des PR-3 (chitinases de classe I), des PR-4 (transporteurs d'auxine ou germines), des PR-5 (protéines thaumatine-like ou TLP), des PR-10 (protéines Bet v 1-like) et des PR-14 (protéines de transfert des lipides ou LTP). S'y ajoutent des profilines et des pectine estérases. En raison de la présence de ces allergènes dans les pollens, les aeroallergènes sont le plus souvent responsables de la sensibilisation primaire. Ainsi, la plupart des allergies à la pomme dépendent d'une sensibilisation préalable au pollen de bouleau, l'allergène Mal d 1 croisant avec Bet v 1. L'allergie au kiwi ou à la banane sont souvent associées aux allergènes croisants (chitinases, 1,3β-glucanases) du syndrome latex-fruits. La sensibilisation directe par contact ou ingestion (LTP de pêche) est possible et peut donner lieu à des réactions anaphylactiques généralisées, notamment dans les pays du Sud de l'Europe (Espagne). Le diagnostic biologique des allergies aux fruits est souvent délicat en raison du petit nombre d'allergènes recombinants disponibles (rPru p 1, rPru p 3 et rPru p 4 de pêche) et de l'instabilité des extraits de fruits.

Mammalian PITPβ (phosphatidylinositol transfer protein β) is a 272-amino-acid polypeptide capable of transferring PtdIns, PtdCho and SM (sphingomyelin) between membrane bilayers. It has been reported that Ser262 present in the C-terminus of PITPβ is constitutively phosphorylated and determines Golgi localization. We provide evidence for the expression of an sp (splice) variant of PITPβ (PITPβ-sp2) where the C-terminal 15 amino acids of PITPβ-sp1 are replaced by an alternative C-terminus of 16 amino acids. PITPβ-sp1 is the product of the first 11 exons, whereas PITPβ-sp2 is a product of the first 10 exons followed by the twelfth exon – exon 11 being ‘skipped’. Both splice variants are capable of PtdIns and PtdCho transfer, with PITPβ-sp2 being unable to transport SM. PITPβ is ubiquitously expressed, with the highest amounts of PITPβ found in HL60 cells and in rat liver; HL60 cells express only PITPβ-sp1, whereas rat liver expresses both sp variants in similar amounts. In both cell ty...

Mammalian PITPβ (phosphatidylinositol transfer protein β) is a 272-amino-acid polypeptide capable of transferring PtdIns, PtdCho and SM (sphingomyelin) between membrane bilayers. It has been reported that Ser 262 present in the C-terminus of PITPβ is constitutively phosphorylated and determines Golgi localization. We provide evidence for the expression of an sp (splice) variant of PITPβ (PITPβ-sp2) where the C-terminal 15 amino acids of PITPβ-sp1 are replaced by an alternative C-terminus of 16 amino acids. PITPβ-sp1 is the product of the first 11 exons, whereas PITPβ-sp2 is a product of the first 10 exons followed by the twelfth exon-exon 11 being 'skipped'. Both splice variants are capable of PtdIns and PtdCho transfer, with PITPβ-sp2 being unable to transport SM. PITPβ is ubiquitously expressed, with the highest amounts of PITPβ found in HL60 cells and in rat liver; HL60 cells express only PITPβ-sp1, whereas rat liver expresses both sp variants in similar amounts. In both cell types, PITPβ-sp1 is constitutively phosphorylated and both the PtdIns and PtdCho forms of PITPβ-sp1 are present. In contrast, PITPβ-sp2 lacks the constitutively phosphorylated Ser 262 (replaced with glutamine). Nonetheless, both PITPβ variants localize to the Golgi and, moreover, dephosphorylation of Ser 262 of PITPβ-sp1 does not affect its Golgi localization. The presence of PITPβ sp variants adds an extra level of proteome complexity and, in rat liver, the single gene for PITPβ gives rise to seven distinct protein species that can be resolved on the basis of their charge differences.

Mammalian PITPβ (phosphatidylinositol transfer protein β) is a 272-amino-acid polypeptide capable of transferring PtdIns, PtdCho and SM (sphingomyelin) between membrane bilayers. It has been reported that Ser 262 present in the C-terminus of PITPβ is constitutively phosphorylated and determines Golgi localization. We provide evidence for the expression of an sp (splice) variant of PITPβ (PITPβ-sp2) where the C-terminal 15 amino acids of PITPβ-sp1 are replaced by an alternative C-terminus of 16 amino acids. PITPβ-sp1 is the product of the first 11 exons, whereas PITPβ-sp2 is a product of the first 10 exons followed by the twelfth exon-exon 11 being 'skipped'. Both splice variants are capable of PtdIns and PtdCho transfer, with PITPβ-sp2 being unable to transport SM. PITPβ is ubiquitously expressed, with the highest amounts of PITPβ found in HL60 cells and in rat liver; HL60 cells express only PITPβ-sp1, whereas rat liver expresses both sp variants in similar amounts. In both cell types, PITPβ-sp1 is constitutively phosphorylated and both the PtdIns and PtdCho forms of PITPβ-sp1 are present. In contrast, PITPβ-sp2 lacks the constitutively phosphorylated Ser 262 (replaced with glutamine). Nonetheless, both PITPβ variants localize to the Golgi and, moreover, dephosphorylation of Ser 262 of PITPβ-sp1 does not affect its Golgi localization. The presence of PITPβ sp variants adds an extra level of proteome complexity and, in rat liver, the single gene for PITPβ gives rise to seven distinct protein species that can be resolved on the basis of their charge differences.

Background. Non-specific Lipid Transfer Proteins (nsLTPs) are widely distributed in the plant kingdom and constitute a superfamily of related proteins. More than 800 different sequences have been characterized so far, but their biological functions remain unclear. It has been clear for years that they present a certain interest for agronomic and nutritional issues. Deciphering their functions means collecting and analyzing a variety of data from gene sequence to protein structure, from cellular localization to the physiological role. As a huge and growing number of new protein sequences are available nowadays, extracting meaningful knowledge from sequence-structure-function relationships calls for the development of new tools and approaches. As nsLTPs show high evolutionary divergence, but a conserved common right-handed superhelix structural fold, and as they are involved in a large number of key roles in plant development and defense, they are a stimulating case study for validati...

Recognition of lipid antigens by T lymphocytes is well established. Lipids are recognized by T cells when presented in association with CD1 antigen-presenting molecules. Both microbial and self lipids stimulate specific T lymphocytes, thus participating in immune reactions during infections and autoimmune diseases. The immune system uses a variety of strategies to solubilise lipid antigens, to facilitate their internalization, processing, and loading on CD1 molecules. Recent studies in the field of lipid antigen presentation have revealed new mechanisms which allow the immune system to sense lipids as stimulatory antigens.

Th e aim of this study was to evaluate the eff ect of a low saturated fat, low cholesterol diet on plasma lipopoproteins, pre β-high density lipoprotein (HDL) formation, lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activities as well as on the ability of plasma to stimulate cellular cholesterol effl ux. Twelve male type 1 diabetic patients with plasma cholesterol >5.0 mmol/L were studied while consuming their usual diet and after 6 weeks of a low fat, low cholesterol diet. Pre β-HDL formation was measured using crossed immuno-electrophoresis. Plasma LCAT, CETP and PLTP activities were assayed by exogenous substrate methods. Th e ability of plasma to promote cellular cholesterol effl ux out of Fu5AH rat hepatoma cells and out of human skin fi broblasts was also determined. Saturated fat intake was lowered (P = 0.001), due to replacement with carbohydrates, while mono-and polyunsaturated fat intake remained unchanged. Cholesterol intake decreased as well (P = 0.003). Th e changes in plasma total cholesterol, very low and low density lipoprotein (VLDL+LDL) cholesterol, HDL cholesterol, HDL phospholipids, apolipoprotein (apo) A-I, plasma LCAT activity and PLTP activity were not signifi cant. Plasma CETP activity (P = 0.008) and pre β-HDL formation (P = 0.008) decreased. Th e ability of plasma to promote cholesterol effl ux out of fi broblasts and Fu5AH cells remained unchanged. Reduction in dietary saturated fat and cholesterol intake does not adversely aff ect cellular cholesterol effl ux to plasma from type 1 diabetic patients, despite a drop in pre β-HDL formation.

Phospholipid transfer protein (PLTP) exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP) in the circulation. LA-PLTP is associated with apoA-I while the HA-PLTP complex is enriched with apoE. To study the interaction of PLTP with apolipoproteins, we carried out surface plasmon resonance analyses. These demonstrated a concentration-dependent binding of recombinant human PLTP, which represents an active PLTP form, and LA-PLTP to apoE, apoA-I, and apoA-IV within a nanomolar K D range. To study whether LA-PLTP can be transformed into an active form, we incubated it in the presence of proteoliposomes containing apoE, apoA-I or apoA-IV. The apoE proteoliposomes induced a concentration-dependent activation of LA-PLTP. ApoA-IV proteoliposomes also activated LA-PLTP in a concentration-dependent manner, whereas apoA-I proteoliposomes had no such effect. These observations suggest that PLTP is capable of interacting with apoE, apoA-I, and apoA-IV, and that these interactions regulate PLTP-activity levels in plasma.