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1.
Mol Plant ; 4(6): 947-64, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21486996

ABSTRACT

The non-specific lipid transfer proteins (nsLTPs) are small, basic proteins characterized by a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers. Most nsLTPs are synthesized with an N-terminal signal peptide that localizes the protein to the apoplastic space. The nsLTPs have only been identified in seed plants, where they are encoded by large gene families. We have initiated an analysis of the evolutionary history of the nsLTP family using genomic and EST information from non-seed land plants and green algae to determine: (1) when the nsLTP family arose, (2) how often new nsLTP subfamilies have been created, and (3) how subfamilies differ in their patterns of expansion and loss in different plant lineages. In this study, we searched sequence databases and found that genes and transcripts encoding nsLTPs are abundant in liverworts, mosses, and all other investigated land plants, but not present in any algae. The tertiary structures of representative liverwort and moss nsLTPs were further studied with homology modeling. The results indicate that the nsLTP family has evolved after plants conquered land. Only two of the four major subfamilies of nsLTPs found in flowering plants are present in mosses and liverworts. The additional subfamilies have arisen later, during land plant evolution. In this report, we also introduce a modified nsLTP classification system.


Subject(s)
Carrier Proteins/genetics , Evolution, Molecular , Plant Proteins/genetics , Plants/genetics , Amino Acid Sequence , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Introns/genetics , Molecular Sequence Data , Phylogeny , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Conformation , Sequence Homology, Amino Acid
2.
J Biol Chem ; 284(23): 16037-48, 2009 Jun 05.
Article in English | MEDLINE | ID: mdl-19366703

ABSTRACT

Members of the vascular endothelial growth factor (VEGF) family play a pivotal role in angiogenesis and lymphangiogenesis. They are potential therapeutics to induce blood vessel formation in myocardium and skeletal muscle, when normal blood flow is compromised. Most members of the VEGF/platelet derived growth factor protein superfamily exist as covalently bound antiparallel dimers. However, the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) is predominantly a non-covalent dimer even though the cysteine residues (Cys-44 and Cys-53) forming the intersubunit disulfide bridges in the other members of the VEGF family are also conserved in VEGF-D. Moreover, VEGF-D bears an additional cysteine residue (Cys-25) at the subunit interface. Guided by our model of VEGF-D(DeltaNDeltaC), the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-D(DeltaNDeltaC). The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-D(DeltaNDeltaC). More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-D(DeltaNDeltaC) and increased the dimer to monomer ratio. Specifically, substitutions to hydrophobic amino acids Ile, Leu, and Val, equivalent to those found in other VEGFs, most favorably affected the activity of the recombinant VEGF-D(DeltaNDeltaC). The increased activity of these mutants was mainly due to stabilization of the protein. This study enables us to better understand the structural determinants controlling the biological activity of VEGF-D. The novel variants of VEGF-D(DeltaNDeltaC) described here are potential agents for therapeutic applications, where induction of vascular formation is required.


Subject(s)
Vascular Endothelial Growth Factor D/genetics , Animals , Binding Sites , Blood Flow Velocity , Coronary Circulation , Genetic Variation , Heart/physiology , Homeostasis , Humans , Models, Molecular , Muscle, Skeletal/blood supply , Myocardium/metabolism , Neovascularization, Physiologic , Protein Conformation , Receptors, Vascular Endothelial Growth Factor/physiology , Structure-Activity Relationship , Swine , Vascular Endothelial Growth Factor D/chemistry , Vascular Endothelial Growth Factor D/physiology , Vascular Endothelial Growth Factor D/therapeutic use , Vascular Endothelial Growth Factor Receptor-2/physiology
3.
J Exp Bot ; 59(12): 3485-99, 2008.
Article in English | MEDLINE | ID: mdl-18687588

ABSTRACT

The Arabidopsis thaliana sterol carrier protein-2 (AtSCP2) is a small, basic and peroxisomal protein that in vitro enhances the transfer of lipids between membranes. AtSCP2 and all other plant SCP-2 that have been identified are single-domain polypeptides, whereas in many other eukaryotes SCP-2 domains are expressed in the terminus of multidomain polypeptides. The AtSCP2 transcript is expressed in all analysed tissues and developmental stages, with the highest levels in floral tissues and in maturing seeds. The expression of AtSCP2 is highly correlated with the multifunctional protein-2 (MFP2) involved in beta-oxidation. A. thaliana Atscp2-1 plants deficient in AtSCP2 show altered seed morphology, a delayed germination, and are dependent on an exogenous carbon source to avoid a delayed seedling establishment. Metabolomic investigations revealed 110 variables (putative metabolites) that differed in relative concentration between Atscp2-1 and normal A. thaliana wild-type seedlings. Microarray analysis revealed that many genes whose expression is altered in mutants with a deficiency in the glyoxylate pathway, also have a changed expression level in Atscp2-1.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Carrier Proteins/metabolism , Seedlings/growth & development , Seeds/growth & development , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Germination , Models, Molecular , Mutagenesis, Insertional , Seedlings/chemistry , Seedlings/genetics , Seedlings/metabolism , Seeds/chemistry , Seeds/genetics , Seeds/metabolism
4.
FEBS J ; 275(13): 3421-37, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18537822

ABSTRACT

Arabidopsis thaliana At2g33470 encodes a glycolipid transfer protein (GLTP) that enhances the intervesicular trafficking of glycosphingolipids in vitro. GLTPs have previously been identified in animals and fungi but not in plants. Thus, At2g33470 is the first identified plant GLTP and we have designated it AtGTLP1. AtGLTP1 transferred BODIPY-glucosylceramide at a rate of 0.7 pmol x s(-1), but BODIPY-galactosylceramide and BODIPY-lactosylceramide were transferred slowly, with rates below 0.1 pmol x s(-1). AtGLTP1 did not transfer BODIPY-sphingomyelin, monogalactosyldiacylglycerol or digalactosyldiacylglycerol. The human GLTP transfers BODIPY-glucosylceramide, BODIPY-galactosylceramide and BODIPY-lactosylceramide with rates greater than 0.8 pmol.s(-1). Structural models showed that the residues that are most critical for glycosphingolipid binding in human GLTP are conserved in AtGLTP1, but some of the sugar-binding residues are unique, and this provides an explanation for the distinctly different transfer preferences of AtGLTP1 and human GLTP. The AtGLTP1 variant Arg59Lys/Asn95Leu showed low BODIPY-glucosylceramide transfer activity, indicating that Arg59 and/or Asn95 are important for the specific binding of glucosylceramide to AtGLTP1. We also show that, in A. thaliana, AtGLTP1 together with At1g21360 and At3g21260 constitute a small gene family orthologous to the mammalian GLTPs. However, At1g21360 and At3g21260 did not transfer any of the tested lipids in vitro.


Subject(s)
Arabidopsis/metabolism , Carrier Proteins/chemistry , Carrier Proteins/physiology , Gene Expression Regulation, Plant , Glycosphingolipids/chemistry , Amino Acid Sequence , Antigens, CD/chemistry , Boron Compounds/pharmacology , Escherichia coli/metabolism , Humans , Lactosylceramides/chemistry , Lipids/chemistry , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Sequence Homology, Amino Acid
5.
FEBS J ; 273(24): 5641-55, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17212780

ABSTRACT

Sterol carrier protein-2 (SCP-2) is a small intracellular basic protein domain implicated in peroxisomal beta-oxidation. We extend our knowledge of plant SCP-2 by characterizing SCP-2 from Euphorbia lagascae. This protein consists of 122 amino acids including a PTS1 peroxisomal targeting signal. It has a molecular mass of 13.6 kDa and a pI of 9.5. It shares 67% identity and 84% similarity with SCP-2 from Arabidopsis thaliana. Proteomic analysis revealed that E. lagascae SCP-2 accumulates in the endosperm during seed germination. It showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC). The transfer of BODIPY-PC was almost completely inhibited after addition of phosphatidylinositol, palmitic acid, stearoyl-CoA and vernolic acid, whereas sterols only had a very marginal inhibitory effect. We used protein modelling and site-directed mutagenesis to investigate why the BODIPY-PC transfer mediated by E. lagascae SCP-2 is not sensitive to sterols, whereas the transfer mediated by A. thaliana SCP-2 shows sterol sensitivity. Protein modelling suggested that the ligand-binding cavity of A. thaliana SCP-2 has four methionines (Met12, 14, 15 and 100), which are replaced by leucines (Leu11, 13, 14 and 99) in E. lagascae SCP-2. Changing Leu99 to Met99 was sufficient to convert E. lagascae SCP-2 into a sterol-sensitive BODIPY-PC-transfer protein, and correspondingly, changing Met100 to Leu100 abolished the sterol sensitivity of A. thaliana SCP-2.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Euphorbia/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Sterols/metabolism , Amino Acid Sequence , Amino Acid Substitution , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/metabolism , Biological Transport/genetics , Boron Compounds/chemistry , Carrier Proteins/chemistry , Gene Expression Regulation, Plant , Germination , Models, Biological , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Palmitic Acid/chemistry , Palmitic Acid/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylcholines/pharmacokinetics , Plant Proteins/chemistry , Protein Conformation , Seeds/enzymology , Sequence Alignment , Sterols/chemistry
6.
J Biol Chem ; 279(51): 53544-53, 2004 Dec 17.
Article in English | MEDLINE | ID: mdl-15456765

ABSTRACT

This is the first report describing the cloning and characterization of sterol carrier protein-2 (SCP-2) from plants. Arabidopsis thaliana SCP-2 (AtSCP-2) consists of 123 amino acids with a molecular mass of 13.6 kDa. AtSCP-2 shows 35% identity and 56% similarity to the human SCP-2-like domain present in the human D-bifunctional protein (DBP) and 30% identity and 54% similarity to the human SCP-2 encoded by SCP-X. The presented structural models of apo-AtSCP-2 and the ligand-bound conformation of AtSCP-2 reveal remarkable similarity with two of the structurally known SCP-2s, the SCP-2-like domain of human DBP and the rabbit SCP-2, correspondingly. The AtSCP-2 models in both forms have a similar hydrophobic ligand-binding tunnel, which is extremely suitable for lipid binding. AtSCP-2 showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC) from donor membranes to acceptor membranes. The transfer of BODIPY-PC was almost completely inhibited after addition of 1-palmitoyl 2-oleoyl phosphatidylcholine or ergosterol. Dimyristoyl phosphatidic acid, stigmasterol, steryl glucoside, and cholesterol showed a moderate to marginal ability to lower the BODIPY-PC transfer rate, and the single chain palmitic acid and stearoyl-coenzyme A did not affect transfer at all. Expression analysis showed that AtSCP-2 mRNA is accumulating in most plant tissues. Plasmids carrying fusion genes between green fluorescent protein and AtSCP-2 were transformed with particle bombardment to onion epidermal cells. The results from analyzing the transformants indicate that AtSCP-2 is localized to peroxisomes.


Subject(s)
Arabidopsis/metabolism , Carrier Proteins/biosynthesis , Carrier Proteins/chemistry , Agar/chemistry , Amino Acid Sequence , Animals , Antigens, Plant , Binding, Competitive , Boron Compounds/pharmacology , Carrier Proteins/metabolism , Cholesterol/pharmacology , Cloning, Molecular , Computational Biology , Electrophoresis, Polyacrylamide Gel , Ergosterol/pharmacology , Escherichia coli/metabolism , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/pharmacology , Glucosides/pharmacology , Glutathione Transferase/metabolism , Glycerophospholipids/pharmacology , Green Fluorescent Proteins/metabolism , Humans , Lipids/chemistry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Onions , Palmitic Acid/pharmacology , Peroxisomes/metabolism , Phosphatidylcholines/pharmacology , Phylogeny , Plant Proteins/chemistry , Plant Proteins/metabolism , Plasmids/metabolism , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA/metabolism , Rabbits , Recombinant Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sterols/chemistry , Stigmasterol/pharmacology , Time Factors
7.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 7): 1288-90, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12832789

ABSTRACT

Human vascular adhesion protein-1 (VAP-1) is a membrane-bound multifunctional glycoprotein with both adhesive and enzymatic properties. The protein belongs to the copper-containing amine oxidase (CAO) family, which use 2,4,5-trihydroxyphenylalanine quinone as a cofactor. Here, the crystallization and preliminary X-ray analysis of a mammalian CAO, human VAP-1, is reported. The protein was expressed in Chinese hamster ovary cells as a full-length form with an N-terminal transmembrane region and multiple glycosylation sites. Hexagonal crystals with unit-cell parameters a = b = 225.9, c = 218.7 A, alpha = beta = 90, gamma = 120 degrees were obtained using the vapour-diffusion method. Data from three different crystals were collected at 100 K using synchrotron radiation and were processed to 3.2 A resolution with 95.9% completeness and an R(merge) of 19.6%.


Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Cell Adhesion Molecules/chemistry , Amine Oxidase (Copper-Containing)/isolation & purification , Cell Adhesion Molecules/isolation & purification , Cloning, Molecular , Crystallization , Glycosylation , Humans , Recombinant Proteins , X-Ray Diffraction
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