Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs.
The Arabidopsis CAO gene encodes a 52-kDa protein with predicted localization in the plastid compartment. Here, we report that CAO is an intrinsic Rieske iron-sulfur protein of the plastidenvelope inner and thylakoid membranes. Activity measurements revealed that CAO catalyzes chlorophyllide a to chlorophyllide b conversion in vitro and that the enzyme was only slightly active with protochlorophyllide a, the nonreduced precursor of chlorophyllide a. Protein import and organelle fractionation studies identified CAO to be distinct from Ptc52 in the substrate-dependent transport pathway of NADPH:protochlorophyllide oxidoreductase A but instead to be part of a separate translocon complex. This complex was involved in the regulated import and stabilization of the chlorophyllide b-binding light-harvesting proteins Lhcb1 (LHCII) and Lhcb4 (CP29) in chloroplasts. Together, our results provide insights into the plastid subcompartmentalization and evolution of chlorophyll precursor biosynthesis in relation to protein import in higher plants.chlorophyll biosynthesis ͉ light-harvesting proteins ͉ protein import
BackgroundReceptors specific for the sugar N-acetylgalactosamine (GalNAc) include the human type II, C-type lectin receptor macrophage galactose-type lectin/C-type lectin receptor family member 10A (MGL/CLEC10A/CD301) that is expressed prominently by human peripheral immature dendritic cells, dendritic cells in the skin, alternatively-activated (M2a) macrophages, and to lesser extents by several other types of tissues. CLEC10A is an endocytic receptor on antigen-presenting cells and has been proposed to play an important role in maturation of dendritic cells and initiation of an immune response. In this study, we asked whether a peptide that binds in the GalNAc-binding site of CLEC10A would serve as an effective tool to activate an immune response against ovarian cancer.MethodsA 12-mer sequence emerged from a screen of a phage display library with a GalNAc-specific lectin. The peptide, designated svL4, and a shorter peptide consisting of the C-terminal 6 amino acids, designated sv6D, were synthesized as tetravalent structures based on a tri-lysine core. In silico and in vitro binding assays were developed to evaluate binding of the peptides to GalNAc-specific receptors. Endotoxin-negative peptide solutions were administered by subcutaneous injection and biological activity of the peptides was determined by secretion of cytokines and the response of peritoneal immune cells in mice. Anti-cancer activity was studied in a murine model of ovarian cancer.ResultsThe peptides bound to recombinant human CLEC10A with high avidity, with half-maximal binding in the low nanomolar range. Binding to the receptor was Ca2+-dependent. Subcutaneous injection of low doses of peptides into mice on alternate days resulted in several-fold expansion of populations of mature immune cells within the peritoneal cavity. Peptide sv6D effectively suppressed development of ascites in a murine ovarian cancer model as a monotherapy and in combination with the chemotherapeutic drug paclitaxel or the immunotherapeutic antibody against the receptor PD-1. Toxicity, including antigenicity and release of cytotoxic levels of cytokines, was not observed.Conclusionsv6D is a functional ligand for CLEC10A and induces maturation of immune cells in the peritoneal cavity. The peptide caused a highly significant extension of survival of mice with implanted ovarian cancer cells with a favorable toxicity and non-antigenic profile.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0339-5) contains supplementary material, which is available to authorized users.
The motif Glu-X-X-His/Asn-X-Arg is conserved in the first and third membrane-spanning domains of all lightharvesting chlorophyll a/b-and a/c-binding proteins in chloroplasts. Molecular modeling of synthetic peptides containing the sequence Glu-Ile-Val-His-Ser-Arg, a motif found in the apoprotein of the major light-harvesting complex in plants, generated a loop structure formed by intrapeptide, electrostatic attraction between Glu and Arg. His, Asn, and charge-compensated Glu-Arg pairs are known ligands of the magnesium atom in chlorophyll. The prediction that this structure should bind two molecules of chlorophyll was confirmed experimentally with an assay based on fluorescence resonance energy transfer between peptides and chlorophyll a. Motifs with both potential ligands bound approximately two times the amount of chlorophyll as one in which His was replaced by Ala. These results support the conclusion that formation of this intermediate, within membranes of the envelope, is a crucial step in assembly of lightharvesting complexes and a mechanism that regulates import of the apoproteins into the chloroplast.
BackgroundThe physico-chemical properties of chlorophylls b and c have been known for decades. Yet the mechanisms by which these secondary chlorophylls support assembly and accumulation of light-harvesting complexes in vivo have not been resolved.PresentationBiosynthetic modifications that introduce electronegative groups on the periphery of the chlorophyll molecule withdraw electrons from the pyrrole nitrogens and thus reduce their basicity. Consequently, the tendency of the central Mg to form coordination bonds with electron pairs in exogenous ligands, a reflection of its Lewis acid properties, is increased. Our hypothesis states that the stronger coordination bonds between the Mg atom in chlorophyll b and chlorophyll c and amino acid sidechain ligands in chlorophyll a/b- and a/c-binding apoproteins, respectively, enhance their import into the chloroplast and assembly of light-harvesting complexes.TestingSeveral apoproteins of light-harvesting complexes, in particular, the major protein Lhcb1, are not detectable in leaves of chlorophyll b-less plants. A direct test of the hypothesis – with positive selection – is expression, in mutant plants that synthesize only chlorophyll a, of forms of Lhcb1 in which weak ligands are replaced with stronger Lewis bases.ImplicationsThe mechanistic explanation for the effects of deficiencies in chlorophyll b or c points to the need for further research on manipulation of coordination bonds between these chlorophylls and chlorophyll-binding proteins. Understanding these interactions will possibly lead to engineering plants to expand their light-harvesting antenna and ultimately their productivity.
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