Probing N-Glycan Functions in Human Interleukin-17A Based on Chemically Synthesized Homogeneous Glycoforms.

N-Glycosylation represents an essential type of posttranslational modification for proteins. However, deciphering the functions of N-glycosylation remains a challenge due to the lack of analytical and biochemical methods to accurately differentiate the protein glycoforms with various intact glycans. Here we report our synthesis and evaluation of homogeneously glycosylated interleukin-17A (IL-17A), based on a synthetic approach combining solid-phase synthesis of (glyco)peptides, chemoenzymatic glycan modification on segments, and chemical ligations. The obtained homogeneous glycoproteins allow for the demonstration of the stabilizing role of N-glycans during the folding step. A comparison of three IL-17A glycoforms in a normal human dermal fibroblast (NHDF) assay reveals dose-dependent interleukin-6-inducing activities in all cases, wherein the glycoform with sialyl undecasaccharides displays much weaker stimulatory effect than that of the GlcNAc- or GlcNAc(ß1→4)GlcNAc-modified proteins. Further surface plasmon resonance (SPR) and hydrogen/deuterium exchange mass spectroscopic experiments confirm that the evaluated complex type N-glycan impedes the binding between IL-17A and its receptor IL-17RA. This structure-activity relationship study on glycoproteins highlights the viability of applying the de novo approach to probe the roles of N-glycans.

Copper regulates the interactions of antimicrobial piscidin peptides from fish mast cells with formyl peptide receptors and heparin.

Phagocytic cells in fish secrete antimicrobial peptides (AMPs) such as piscidins, glycosaminoglycans such as heparin, and copper ions as first-line immune defenses. Recently, we established that Cu2+ coordination by piscidins 1 (P1) and 3 (P3) enhances their antibacterial activity against membranes and DNA. Interestingly, we noted that physicochemical similarities exist between both piscidins and other AMPs that interact with heparin and induce immune-cell chemotaxis through formyl peptide receptors (FPRs) involved in innate immunity. Thus, we postulated that P1 and P3 interact with heparin and FPRs but that these interactions distinctively depend on Cu2+ Here, we investigate the interactome potentiated by piscidins, heparin, FPR, and Cu2+ Utilizing FPR-transfected cells and neutrophils, we demonstrate that both piscidins exclusively use FPR1 and FPR2 to induce chemotaxis and that Cu2+ reduces their chemotaxis induction. P1 is more effective at activating FPR1 than P3 and other known AMP ligands. Furthermore, the expression of Fpr2 on the surface of neutrophils is down-regulated by both peptides. Copper conjugation of the peptides does not further increase down-regulation, suggesting that the conformational changes induced by the metal translate into reduced FPR efficacy without altering the binding affinity. Using surface plasmon resonance, we show that piscidin-heparin interactions are Cu2+-dependent and reduced at the acidic pH of phagosomes. Although heparin decreases the antimicrobial activity of P3-Cu2+, it does not affect bacterial killing by P1-Cu2+ Copper's effects on modulating the micromolar-range interactions of both piscidins with FPR and heparin suggest that the interactome of these distinct immune agents plays an important role in innate immunity. The interactions between diverse host-defense molecules uncovered here may help inform the design of novel therapeutics to treat immune-related diseases.

A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii.

The increasing incidence of multidrug-resistant Acinetobacter baumannii (MDRAb) infections worldwide has necessitated the development of novel antibiotics. Human defensin 5 (HD5) is an endogenous peptide with a complex architecture and antibacterial activity against MDRAb In the present study, we attempted to simplify the structure of HD5 by removing disulfide bonds. We found that the Cys2-4 bond was most indispensable for HD5 to inactivate MDRAb, although the antibacterial activity of the derivative was significantly attenuated. We then replaced the noncationic and nonhydrophobic residues with electropositive Arg to increase the antibacterial activity of HD5 derivative that contains a Cys2-4 bond, obtaining another derivative termed HD5d5. The in vitro antibacterial assay and irradiation-wound-infection animal experiment both showed that HD5d5 was much more effective than HD5 at eliminating MDRAb Further investigations revealed that HD5d5 efficiently bound to outer membrane lipid A and penetrated membranes, leading to bacterial collapse and peptide translocation. Compared to HD5, more HD5d5 molecules were located in the cytoplasm of MDRAb, and HD5d5 was more efficient at reducing the activities of superoxide dismutase and catalase, causing the accumulation of reactive oxygen species that are detrimental to microbes. In addition, HD5 failed to suppress the pathogenic outer membrane protein A of Acinetobacter baumannii (AbOmpA) at concentrations up to 50 µg/ml, whereas HD5d5 strongly bound to AbOmpA and exhibited a dramatic toxin-neutralizing ability, thus expanding the repertoire of drugs that is available to treat MDRAb infections.

Synthetic green fluorescent protein chromophore analogues with a positive charge at the phenyl-like group.

Synthetic green fluorescent protein (GFP) chromophore analogues with a positive charge at the phenyl-like group have the highly electrophilic amidine carbon, smaller LUMO-HOMO energy gap, red-shifted electronic absorptions and fluorescent emissions, and accelerated E-Z thermoisomerization rates. They are water-labile and their hydrolysis results in ring-opening of the imidazolinone moiety with a half life around 25-37 h in D2O at 25 °C.

Synthetic Modification within the "RPRL" Region of Apelin Peptides: Impact on Cardiovascular Activity and Stability to Neprilysin and Plasma Degradation.

Apelin is an important mammalian peptide hormone with a range of physiological roles, especially in the cardiovascular system. The apelinergic system is a promising target for treatment of disease, but this remains to be realized due to rapid proteolysis of apelin-derived peptides by proteases, including neprilysin (NEP). The synthetic analogues modified within the NEP degradation site ("RPRL" motif) showed improved in vitro proteolytic stability while maintaining receptor-binding affinities, with three candidate peptides retaining full cardiovascular activities for potential therapeutic application. Many such analogues proved physiologically inactive even with relatively conservative modifications, highlighting the importance of this region for full agonist activity of this peptide hormone.

Synthesis, characterization and systematic comparison of FITC-labelled GnRH-I, -II and -III analogues on various tumour cells.

Targeted tumour therapy is the focus of recent cancer research. Gonadotropin-releasing hormone (GnRH) analogues are able to deliver anticancer agents selectively into tumour cells, which highly express GnRH receptors. However, the effectiveness of different analogues as targeting moiety in drug delivery systems is rarely compared, and the investigated types of cancer are also limited. Therefore, we prepared selectively labelled, fluorescent derivatives of GnRH-I, -II and -III analogues, which were successfully used for drug targeting. In this manuscript, we investigated these analogues' solubility, stability and passive membrane permeability and compared their cellular uptake by various cancer cells. We found that these labelled GnRH conjugates provide great detectability, without undesired cytotoxicity and passive membrane permeability. The introduced experiments with these conjugates proved their reliable tracking, quantification and comparison. Cellular uptake efficiency was studied on human breast, colon, pancreas and prostate cancer cells (MCF-7, HT-29, BxPC-3, LNCaP) and on dog kidney cells (Madin-Darby canine kidney). Each of the three conjugates was taken up by GnRH-I receptor-expressing cells, but the different cells preferred different analogues. Furthermore, we demonstrated for the first time the high cell surface expression of GnRH-I receptors and the effective cellular uptake of GnRH analogues on human pharynx tumour (Detroit-562) cells. In summary, our presented results detail that the introduced conjugates could be innovative tools for the examination of the GnRH-based drug delivery systems on various cells and offer novel information about these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Semisynthesis of biologically active glycoforms of the human cytokine interleukin†6.

Human interleukin 6 (IL-6) is a potent cytokine with immunomodulatory properties. As the influence of N-glycosylation on the in vivo activities of IL-6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL-6 was established by sequential native chemical ligation. The four cysteines of IL-6 are convenient for ligations and require only the short synthetic glycopeptide 43-48. The Cys-peptide 49-183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1-42 was accessible by the simultaneous cleavage of two inteins, leading to the 1-42 thioester with the native N-terminus. Ligation and refolding studies showed that the inherently labile Asp-Pro bond 139-140 was detrimental for the sequential C- to N-terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full-length IL-6 glycoproteins, which showed full bioactivity after efficient refolding and purification.

Gly25-Ser26 amyloid ß-protein structural isomorphs produce distinct Aß42 conformational dynamics and assembly characteristics.

One of the earliest events in amyloid ß-protein (Aß) self-association is nucleation of Aß monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aß42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create Aß42 from 26-O-acyliso-Aß42 (iAß42) through a pH jump from 3 to 7.4. We also synthesized Nα-acetyl-Ser26-iAß42 (Ac-iAß42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aß42 itself at neutral pH. Data from experiments monitoring increases in ß-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy-mass spectrometry) were quite consistent. A rank order of Ac-iAß42>iAß42>Aß42 was observed. Photochemically cross-linked iAß42 displayed an oligomer distribution with a prominent dimer band that was not present with Aß42. These dimers also were observed selectively in iAß42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAß42 and Aß42 appear to be due to the conversion of iAß42 into "pure" Aß42 monomer, a nascent form of Aß42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aß42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aß42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aß oligomers and higher-order assemblies.

Construction and characterization of novel hirulog variants with antithrombin and antiplatelet activities.

The RGD sequence was used to design potent hirudin isoform 3 mimetic peptides with both antithrombin activity and antiplatelet aggregation activity. The RGD and proline were inserted between the catalytic active binding domain (D-Phe-Pro-Arg-Pro) on the N-terminus and the anion-binding exosite binding domain (QGDFEPIPEDAYDE) on the Cterminus. Thrombin titration assay and ATP-induced platelet aggregation test revealed that the peptide with the linker RGDWP or RGDGP possessed potent antithrombin and antiplatelet activities, while other peptides without the Pro residue in the linker only showed antithrombin activity. Similar results were obtained in the RGD-containing hirulog-1 variants. Our study indicates that the inserted Pro residue facilitates the exposure of RGD and the binding of the peptide to glycoprotein IIb/IIIa (GPIIb/IIIa). The strategy of combining the RGD sequence and the Pro residue may be used for future designs of bifunctional antithrombotic agents.

Lipid sorting by ceramide structure from plasma membrane to ER for the cholera toxin receptor ganglioside GM1.

The glycosphingolipid GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane lipid can specify trafficking in these pathways, we synthesized GM1 isoforms with alternate ceramide domains and imaged their trafficking in live cells. Only GM1 with unsaturated acyl chains sorted efficiently from PM to TGN and ER. Toxin binding, which effectively crosslinks GM1 lipids, was dispensable, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. The results implicate a protein-dependent mechanism of lipid sorting by ceramide structure and provide a molecular explanation for the diversity and specificity of retrograde trafficking by CT in host cells.

The role of G-domain orientation and nucleotide state on the Ras isoform-specific membrane interaction.

Ras proteins are proto-oncogenes that function as molecular switches linking extracellular stimuli with an overlapping but distinctive range of biological outcomes. Although modulatable interactions between the membrane and the Ras C-terminal hypervariable region (HVR) harbouring the membrane anchor motifs enable signalling specificity to be determined by their location, it is becoming clear that the spatial orientation of different Ras proteins is also crucial for their functions. To reveal the orientation of the G-domain at membranes, we conducted an extensive study on different Ras isoforms anchored to model raft membranes. The results show that the G-domain mediates the Ras-membrane interaction by inducing different sets of preferred orientations in the active and inactive states with largely parallel orientation relative to the membrane of most of the helices. The distinct locations of the different isoforms, exposing them to different effectors and regulators, coupled with different G-domain-membrane orientation, suggests synergy between this type of recognition motif and the specificity conferred by the HVR, thereby validating the concept of isoform specificity in Ras.

Natural and synthetic chiral isoforms of crustacean hyperglycemic hormone from the crayfish Astacus leptodactylus: hyperglycemic activity and hemolymphatic clearance.

In the crayfish Astacus leptodactylus, as in several crustacean species, the crustacean hyperglycemic hormone is present as two isoforms differing by the chirality of the third residue, a phenylalanine. In the present work, isoforms synthesized full length by solid-phase peptide synthesis have been purified, refolded, the location of the disulfide bridges has been checked, their immunoreactivity against different antibodies have been analyzed and their hyperglycemic activity tested, to ensure the identity of the synthetic peptides with their natural homologs. Different parameters of the hyperglycemic activity of both isoforms were studied. In addition to a difference in the kinetics of hyperglycemia, already known from other studies, it was observed that the dose-response was different depending on the season where experiments were performed, the response being stronger in spring than in autumn, especially for the d-Phe containing isoform. A dosage method based on sandwich enzyme linked immunosorbent assay (ELISA) has been developed to measure hemolymphatic levels of the isoforms after spiking of the animals with one isoform or the other. It was found that hemolymphatic clearance was identical for both isoforms, indicating that their differential effect is not linked to their different lifetime in the hemolymph but may rather rely on other mechanisms such as their binding to different target tissues.

Antitumor effects and cell selectivity of temporin-1CEa, an antimicrobial peptide from the skin secretions of the Chinese brown frog (Rana chensinensis).

Many antimicrobial peptides from amphibian exhibit additional anticancer properties due to a similar mechanism of action at both bacterial and cancer cells. We have previously reported the cDNA sequence of the antimicrobial peptide temporin-1CEa precursor cloned from the Chinese brown frog Rana chensinensis. In this study, we purified, synthesized and structurally characterized temporin-1CEa from the skin secretions of R. chensinensis. The cytotoxicity and cell selectivity of temporin-1CEa were further examined on twelve human carcinoma cell lines and on normal human umbilical vein smooth muscle cells (HUVSMCs). Our results indicated that temporin-1CEa has the amino acid sequence of FVDLKKIANIINSIF-NH(2), and exhibits 50-56% identity with temporin family peptides from other frog species. The CD spectra for temporin-1CEa adopted a well-defined α-helical structure in 50% TFE/water solution. The results of MTT assay showed that temporin-1CEa exhibits cytotoxicity to all tested cancer cell lines in a concentration-dependent manner, being MCF-7 cells the most sensitive. Moreover, temporin-1CEa had lower hemolytic effect to human erythrocytes and had no significant cytotoxicity to normal HUVSMCs at concentrations showed potent antitumor activity. In summary, temporin-1CEa, an amphiphilic α-helical cationic peptide, may represent a novel anticancer agent for breast cancer therapy, considering its cancer cell selectivity and relatively lower cytotoxicity to normal cells.

Synthesis and crystallographic analysis of new sulfonamides incorporating NO-donating moieties with potent antiglaucoma action.

Several aromatic/heterocyclic sulfonamide scaffolds have been used to synthesize compounds incorporating NO-donating moieties of the nitrate ester type, which have been investigated for the inhibition of five physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms: hCA I (offtarget), II, IV and XII (antiglaucoma targets) and IX (antitumor target). Some of the new compounds showed effective in vitro inhibition of the target isoforms involved in glaucoma, and the X-ray crystal structure of one of them revealed factors associated with the marked inhibitory activity. In an animal model of ocular hypertension, one of the new compounds was twice more effective than dorzolamide in reducing elevated intraocular pressure characteristic of this disease, anticipating their potential for the treatment of glaucoma.

Anticonvulsant 4-aminobenzenesulfonamide derivatives with branched-alkylamide moieties: X-ray crystallography and inhibition studies of human carbonic anhydrase isoforms I, II, VII, and XIV.

Aromatic amides comprising branched aliphatic carboxylic acids and 4-aminobenzenesulfonamide were evaluated for their inhibition of carbonic anhydrase (CA) isoforms. Of the most anticonvulsant-active compounds (2, 4, 13, 16, and 17), only 13, 16, and 17 were potent inhibitors of CAs VII and XIV. Compounds 9, 14, and 19 inhibited CA II, while 10 and 12 inhibited all isoforms. Structural studies suggest that differences in the active sites' hydrophobicity modulate the affinity of the inhibitors.

Synthesis and immune response of non-native isomers of vascular endothelial growth factor.

Native proteins often lack immunogenicity and thus limit vaccine and mAb development. We described here a unique method to enhance the immunogenicity of native proteins. This is achieved by creating non-native isomers of disulfide proteins (X-isomers) using the method of disulfide scrambling. X-isomers have the potential to be developed as vaccines and effective immunogens, as they are capable of breaking the immune tolerance and eliciting antibodies that cross-react with the native protein. In this report, we describe production of X-isomers of vascular endothelial growth factor (X-VEGF). The aim is to develop X-VEGF for cancer immunotherapy targeting reduction of VEGF. The production of mouse X-VEGF is achieved by expressing the short version of VEGF (1-110) commonly shared by all VEGF isoforms, with two Cys --> Ala mutations at Cys(51) and Cys(60) to generate R-VEGF(110) (R stands for fully reduced). R-VEGF(110) was then allowed to undergo oxidative folding in the absence of denaturant to form N-VEGF(110) (N stands for native) or in the presence of denaturant to generate five fractions of X-VEGF(110) isomers. While N-VEGF(110) exhibits only marginal immunogenicity in mice, all five fractions of X-VEGF(110) isomers were shown to elicit high titers of antibodies that cross-react with N-VEGF(110). In sera of immunized mice, the amounts of anti-N-VEGF antibodies elicited by X-VEGF(110) isomers range from 54 to 186 mug/mL, which are compatible with or greater than the concentration required for effective therapy using anti-VEGF MAbs. The underlying mechanism of enhanced immunogenicity of X-VEGF(110) is investigated and elaborated. These data suggest that X-VEGF(110) isomers are potential compounds in developing active immunotherapy for treatment of VEGFR bearing tumors and the wet form of age-related macular degeneration.

Diversity in penaeidin antimicrobial peptide form and function.

Penaeidins are a diverse family of two-domain antimicrobial peptides expressed in shrimp. Variation in penaeidin sequence results in functional diversity, which was discovered using synthetic reproductions of native penaeidins. An isoform of penaeidin class 3 from Litopenaeus setiferus (Litset Pen3-4) was synthesized using native ligation and compared directly with the synthetic penaeidin class 4 known to be expressed in the same organism. New antimicrobial activity data are included in this review that emphasize differences in effectiveness that are apparent from a direct comparison of two classes. A novel approach to intact penaeidin analysis is presented in the form of Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry, which has implications for the identification of individual penaeidin isoforms without chemical modification or enzymatic cleavage. The new information included in this review helps gather the perspective on relevance of penaeidin diversity to antimicrobial function, the use of synthetic peptides as tools to evaluate specific immune functions and the application of high mass resolution, top-down sequencing methods to the intact analysis of individual penaeidin isoforms.

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit.

The potassium channel accessory subunit KChIP2 associates with Kv4.2 channels in the cardiac myocyte and is involved in the regulation of the transient outward current (I(to)) during the early phase of repolarization of the action potential. As a first step to biophysically probe the mechanism of KChIP2, we have chemically synthesized its minimal isoform, KChIP2d, using Boc chemistry solid phase peptide synthesis in conjunction with native chemical ligation. The synthetic KChIP2d protein is primarily alpha-helical as predicted and becomes more structured upon binding calcium as assessed by (1)H-NMR and CD spectroscopy. Synthetic KChIP2d is in a monomer-dimer equilibrium in solution, and there is evidence for two monomer binding sites on an N-terminal peptide of Kv4.2. Planned future studies include the incorporation of fluorescent and spin labeled probes in KChIP2d to yield structural information in parallel with electrophysiologic studies to elucidate KChIP2d's mechanism of action.

Evidence for noncooperative metal binding to the alpha domain of human metallothionein.

In the present study, we investigated the metal-binding reactivity of the isolated alpha domain of human metallothionein isoform 1a, with specific emphasis on resolving the debate concerning the cooperative nature of the metal-binding mechanism. The metallation reaction of the metal-free alpha domain with Cd2+ was unequivocally shown to proceed by a noncooperative mechanism at physiologic pH by CD and UV absorption spectroscopy and ESI MS. The data clearly show the presence of intermediate partially metallated metallothionein species under limiting Cd2+ conditions. Titration with four molar equivalents of Cd2+ was required for the formation of the Cd4alpha species in 100% abundance. The implications of a noncooperative metal-binding mechanism are that the partially metallated and metal-free species are stable intermediates, and thus may have a potential role in the currently undefined function of metallothionein.

"Click peptides"--chemical biology-oriented synthesis of Alzheimer's disease-related amyloid beta peptide (abeta) analogues based on the "O-acyl isopeptide method".

A clear understanding of the pathological mechanism of amyloid beta peptide (Abeta) 1-42, a currently unexplained process, would be of great significance for the discovery of novel drug targets for Alzheimer's disease (AD) therapy. To date, though, the elucidation of these Abeta1-42 dynamic events has been a difficult issue because of uncontrolled polymerization, which also poses a significant obstacle in establishing experimental systems with which to clarify the pathological function of Abeta1-42. We have recently developed chemical biology-oriented pH- or phototriggered "click peptide" isoform precursors of Abeta1-42, based on the "O-acyl isopeptide method", in which a native amide bond at a hydroxyamino acid residue, such as Ser, is isomerized to an ester bond, the target peptide subsequently being generated by an O-N intramolecular acyl migration reaction. These click peptide precursors did not exhibit any self-assembling character under physiological conditions, thanks to the presence of the one single ester bond, and were able to undergo migration to give the target Abeta1-42 in a quick and easy, one-way (so-called "click")conversion reaction. The use of click peptides could be a useful strategy to investigate the biological functions of Abeta1-42 in AD through inducible activation of Abeta1-42 self-assembly.