Glycomimetic Peptides as Therapeutic Tools.

The entry of peptides into glycobiology has led to the development of a unique class of therapeutic tools. Although numerous and well-known peptides are active as endocrine regulatory factors that bind to specific receptors, and peptides have been used extensively as epitopes for vaccine production, the use of peptides that mimic sugars as ligands of lectin-type receptors has opened a unique approach to modulate activity of immune cells. Ground-breaking work that initiated the use of peptides as tools for therapy identified sugar mimetics by screening phage display libraries. The peptides that have been discovered show significant potential as high-avidity, therapeutic tools when synthesized as multivalent structures. Advantages of peptides over sugars as drugs for immune modulation will be illustrated in this review.

Resolution of Eczema with Multivalent Peptides.

The integrity of the skin is an important aspect of QOL. Whether caused by genetic deficiencies or environmental insults, disruption of the surface barrier allows irritants and allergens to penetrate the skin, which initiates inflammatory responses by immune cells that often lead to life-long allergies. In this study, eczema was induced on depilated mouse skin with topical lipopolysaccharide or a mixture of Staphylococcal enterotoxin B and an extract of house dust mites, which resulted in thickening of the epidermis, epidermal disruption, and abundant neutrophils in the dermis. Within 14 days of topical treatment with 1 µM svL4, a tetravalent peptide, neutrophils were absent, and the epidermis had returned to a normal morphology. The sequence of svL4 contains glutamine residues that serve as a cross-linking substrate for transglutaminase 2, which gains access to the skin surface where the epidermis becomes disrupted. In contrast, topical application of 1 µM svH1C, a peptide mimetic of sialic acid that lacks glutamine residues, or 1 µM dexamethasone was ineffective in restoring normal epidermal morphology. The data suggest that svL4 would be a powerful treatment for resolving severe eczema.

The Discovery and Function of Filaggrin.

Keratohyalin granules were discovered in the mid-19th century in cells that terminally differentiate to form the outer, cornified layer of the epidermis. The first indications of the composition of these structures emerged in the 1960s from a histochemical stain for histidine, followed by radioautographic evidence of a high incidence of histidine incorporation into newly synthesized proteins in cells containing the granules. Research during the next three decades revealed the structure and function of a major protein in these granules, which was initially called the 'histidine-rich protein'. Steinert and Dale named the protein 'filaggrin' in 1981 because of its ability to aggregate keratin intermediate filaments. The human gene for the precursor, 'profilaggrin,' was reported in 1991 to encode 10, 11 or 12 nearly identical repeats. Remarkably, the mouse and rat genes encode up to 20 repeats. The lifetime of filaggrin is the time required for keratinocytes in the granular layer to move into the inner cornified layer. During this transition, filaggrin facilitates the collapse of corneocytes into 'building blocks' that become an impermeable surface barrier. The subsequent degradation of filaggrin is as remarkable as its synthesis, and the end-products aid in maintaining moisture in the cornified layer. It was apparent that ichthyosis vulgaris and atopic dermatitis were associated with the absence of this protein. McLean's team in 2006 identified the cause of these diseases by discovering loss-of-function mutations in the profilaggrin gene, which led to dysfunction of the surface barrier. This story illustrates the complexity in maintaining a healthy, functional epidermis.

Stories From the Dendritic Cell Guardhouse.

Phagocytic cells [dendritic cells (DCs), macrophages, monocytes, neutrophils, and mast cells] utilize C-type (Ca2+-dependent) lectin-like (CLEC) receptors to identify and internalize pathogens or danger signals. As monitors of environmental imbalances, CLEC receptors are particularly important in the function of DCs. Activation of the immune system requires, in sequence, presentation of antigen to the T cell receptor (TCR) by DCs, interaction of co-stimulatory factors such as CD40/80/86 on DCs with CD40L and CD28 on T cells, and production of IL-12 and/or IFN-α/ß to amplify T cell differentiation and expansion. Without this sequence of events within an inflammatory environment, or in a different order, antigen-specific T cells become unresponsive, are deleted or become regulatory T cells. Thus, the mode by which CLEC receptors on DCs are engaged can either elicit activation of T cells to achieve an immune response or induce tolerance. This minireview illustrates these aspects with Dectin-1, DEC205, the mannose receptor and CLEC10A as examples.

An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides.

BACKGROUND: Receptors 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. METHODS: A 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. RESULTS: The 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. CONCLUSION: sv6D 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.

A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D.

We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 µM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.

Monocyte galactose/N-acetylgalactosamine-specific C-type lectin receptor stimulant immunotherapy of an experimental glioma. Part 1: stimulatory effects on blood monocytes and monocyte-derived cells of the brain.

OBJECTIVES: Immunotherapy with immunostimulants is an attractive therapy against gliomas. C-type lectin receptors specific for galactose/N-acetylgalactosamine (GCLR) regulate cellular differentiation, recognition, and trafficking of monocyte-derived cells. A peptide mimetic of GCLR ligands (GCLRP) was used to activate blood monocytes and populations of myeloid-derived cells against a murine glioblastoma. METHODS: The ability of GCLRP to stimulate phagocytosis by human microglia and monocyte-derived cells of the brain (MDCB) isolated from a human glioblastoma was initially assessed in vitro. Induction of activation markers on blood monocytes was assayed by flow cytometry after administration of GCLRP to naive mice. C57BL/6 mice underwent stereotactic intracranial implantation of GL261 glioma cells and were randomized for tumor size by magnetic resonance imaging, which was also used to assess increase in tumor size. Brain tumor tissues were analyzed using flow cytometry, histology, and enzyme-linked immunosorbent assay with respect to tumor, peritumoral area, and contralateral hemisphere regions. RESULTS: GCLRP exhibited strong stimulatory effect on MDCBs and blood monocytes in vitro and in vivo. GCLRP was associated with an increased percentage of precursors of dendritic cells in the blood (P = 0.003), which differentiated into patrolling macrophages in tumoral (P = 0.001) and peritumoral areas (P = 0.04), rather than into dendritic cells, as in control animals. Treatment with GCLRP did not result in a significant change in survival of mice bearing a tumor. CONCLUSIONS: In vitro and in vivo activation of monocytes was achieved by administration of GCLR to mice. GCLRP-activated blood monocytes were recruited to the brain and exhibited specific phenotypes corresponding with tumor region (glioma, peritumoral zone, and contralateral glioma-free hemisphere). GCLRP treatment alone was associated with increased glioma mass as the result of the infiltration of phagocytic cells. Regional specificity for MDCB may have significant tumor treatment implications.

Monocyte galactose/N-acetylgalactosamine-specific C-type lectin receptor stimulant immunotherapy of an experimental glioma. Part II: combination with external radiation improves survival.

BACKGROUND: A peptide mimetic of a ligand for the galactose/N-acetylgalactosamine-specific C-type lectin receptors (GCLR) exhibited monocyte-stimulating activity, but did not extend survival when applied alone against a syngeneic murine malignant glioma. In this study, the combined effect of GCLRP with radiation was investigated. METHODS: C57BL/6 mice underwent stereotactic intracranial implantation of GL261 glioma cells. Animals were grouped based on randomized tumor size by magnetic resonance imaging on day seven. One group that received cranial radiation (4 Gy on days seven and nine) only were compared with animals treated with radiation and GCLRP (4 Gy on days seven and nine combined with subcutaneous injection of 1 nmol/g on alternative days beginning on day seven). Magnetic resonance imaging was used to assess tumor growth and correlated with survival rate. Blood and brain tissues were analyzed with regard to tumor and contralateral hemisphere using fluorescence-activated cell sorting analysis, histology, and enzyme-linked immunosorbent assay. RESULTS: GCLRP activated peripheral monocytes and was associated with increased blood precursors of dendritic cells. Mean survival increased (P < 0.001) and tumor size was smaller (P < 0.02) in the GCLRP + radiation group compared to the radiation-only group. Accumulation of dendritic cells in both the tumoral hemisphere (P < 0.005) and contralateral tumor-free hemisphere (P < 0.01) was associated with treatment. CONCLUSION: Specific populations of monocyte-derived brain cells develop critical relationships with malignant gliomas. The biological effect of GCLRP in combination with radiation may be more successful because of the damage incurred by tumor cells by radiation and the enhanced or preserved presentation of tumor cell antigens by GCLRP-activated immune cells. Monocyte-derived brain cells may be important targets for creating effective immunological modalities such as employing the receptor system described in this study.

Peptide sugar mimetics prevent HIV type 1 replication in peripheral blood mononuclear cells in the presence of HIV-positive antiserum.

Cells of the immune system express a number of receptors that bind carbohydrate ligands. We questioned whether peptide mimetics of these ligands will activate phagocytic cells and thereby enhance an antiviral response. Short peptide sequences were identified by computational modeling of docking to glycan-specific lectins, selected as receptor analogs, and incorporated into quadravalent structures by peptide synthesis. A peptide with the sequence HPSLK bound to several lectins specific for monosaccharides and to lectins specific for Neu5Ac-Gal-containing complex glycans, whereas a longer sequence, NPSHPLSG, bound only lectins specific for the more complex glycans. In cultures of peripheral blood mononuclear cells (PBMCs) these peptides stimulated phagocytosis of opsonized microspheres. The peptides inhibited replication of HIV-1 in PBMC cultures by 20-80% at concentrations between 1 nM and 1 muM but inhibited replication 100% in the presence of diluted HIV-positive antiserum that alone inhibited replication by 30%. HPSLK caused about 50% loss of viability of cells at 1 mM, a concentration 10(6)-fold higher than an effective inhibitory concentration, but no toxicity was observed with NPSHPLSG. These results demonstrated that peptidomimetics of glycan ligands of cellular receptors are effective in activating phagocytosis, which may be a factor in providing complete inhibition of HIV-1 replication in vitro.

A biologically active peptide mimetic of N-acetylgalactosamine/galactose.

BACKGROUND: Glycosylated proteins and lipids are important regulatory factors whose functions can be altered by addition or removal of sugars to the glycan structure. The glycans are recognized by sugar-binding lectins that serve as receptors on the surface of many cells and facilitate initiation of an intracellular signal that changes the properties of the cells. We identified a peptide that mimics the ligand of an N-acetylgalactosamine (GalNAc)-specific lectin and asked whether the peptide would express specific biological activity. FINDINGS: A 12-mer phage display library was screened with a GalNAc-specific lectin to identify an amino acid sequence that binds to the lectin. Phage particles that were eluted from the lectin with free GalNAc were considered to have been bound to a GalNAc-binding site. Peptides were synthesized with the selected sequence as a quadravalent structure to facilitate receptor crosslinking. Treatment of human peripheral blood mononuclear cells for 24 h with the peptide stimulated secretion of interleukin-8 (IL-8) but not of IL-1beta, IL-6, IL-10, or tumor necrosis factor-alpha (TNF-alpha). The secretion of IL-21 was stimulated as strongly with the peptide as with interferon-gamma. CONCLUSION: The data indicate that the quadravalent peptide has biological activity with a degree of specificity. These effects occurred at concentrations in the nanomolar range, in contrast to free sugars that generally bind to proteins in the micro- to millimolar range.

The small CAB-like proteins of Synechocystis sp. PCC 6803 bind chlorophyll. In vitro pigment reconstitution studies on one-helix light-harvesting-like proteins.

The large family of light-harvesting-like proteins contains members with one to four membrane spanning helices with significant homology to the chlorophyll a/b-binding antenna proteins of plants. From structural as well as evolutionary perspective, it is likely that the members of this family bind chlorophylls and carotenoids. However, undisputable evidence is still lacking. The cyanobacterial small CAB-like proteins (SCPs) are one-helix proteins with compelling similarity to the first and third transmembrane helix of LHCII (LHCIIb) including the chlorophyll-binding motifs. They have been proposed to act as chlorophyll-carrier proteins. Here, we analyze the in vivo absorption spectra of single scp deletion mutants in Synechocystis sp. PCC 6803 and compare the in vitro pigment binding ability of the SCP pairs ScpC/D and ScpB/E with the one of LHCII and a synthetic peptide containing the chlorophyll-binding motif (Eggink LL, Hoober JK (2000) J Biol Chem 275:9087-9090). We demonstrate that deletion of scpB alters the pigmentation in the cyanobacterial cell. Furthermore, we are able to show that chlorophylls and carotenoids interact in vitro with the pairs of ScpC/D and ScpB/E, demonstrated by fluorescence resonance energy transfer and circular dichroism.

Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts.

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.

A role for chlorophyllide a oxygenase in the regulated import and stabilization of light-harvesting chlorophyll a/b proteins.

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 plastid-envelope 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.

Influence of structure on binding of chlorophylls to peptide ligands.

Four classes of chlorophyll (Chl), a, b, c, and d, are involved in photosynthesis within cyanobacteria, algae, and plants. These classes have different evolutionary origins, chemical properties, and biological functions. Our results demonstrate that peptide-bound ligands provided by the imidazole group of histidine and the charge-compensated glutamate-arginine ion pair readily form coordination bonds with Chls a and d but do not interact significantly with Chls b and c. These ligands are apparently not sufficiently strong Lewis bases to displace strongly coordinated water from Chls b and c. These differences determine specificity of binding of Chls in light-harvesting complexes and play an important role in assembly of stable Chl-protein complexes, which has had a profound impact on the evolution of photosynthetic organisms.

Synthesis of chlorophyll b: localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit.

BACKGROUND: Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly. RESULTS: Fluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy. However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane. The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center. Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center. A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042. Fragmentation of the protein after incorporation of 125I- identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species. The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction. CONCLUSION: CAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C. reinhardtii cells. Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development. Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis.