Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins.

The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-ß (IFN-ß), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.

Synthesis of [11C]interleukin 8 using a cell-free translation system and L-[11C]methionine.

Positron emission tomography (PET), which requires a compound labeled with a positron emitter radioisotope as an imaging probe, is one of the most useful and valuable imaging modalities in molecular imaging. It has several advantages over other imaging modalities, particularly in sensitive and quantitative investigations of molecular functions and processes in vivo. Recent advances in biopharmaceuticals development have increased interest in practical methods for proteins and peptides labeling with positron emitter radioisotope for PET molecular imaging. Here, we propose a novel approach for preparing positron emitter-labeled proteins and peptides based on biochemical synthesis using a reconstituted cell-free translation system. In this study, [(11)C]interleukin 8 (IL-8; MW 9.2 kDa) was successfully synthesized by the cell-free system in combination with l-[(11)C]methionine. The in vitro biochemical reaction proceeded smoothly and gave maximum radioactivity of [(11)C]IL-8 at 20 min with a radiochemical yield of 63%. Purification of [(11)C]IL-8 was achieved by conventional cation exchange and ultrafiltration methods, resulting in enough amount of radioactivity with excellent radiochemical purity (>95%) for small-animal imaging. This study clearly demonstrates that cell-free protein production system combined with positron emitter-labeled amino acid holds great promise as a novel approach to prepare radiolabeled proteins and peptides for PET imaging.

Monomeric and dimeric CXCL8 are both essential for in vivo neutrophil recruitment.

Rapid mobilization of neutrophils from vasculature to the site of bacterial/viral infections and tissue injury is a critical step in successful resolution of inflammation. The chemokine CXCL8 plays a central role in recruiting neutrophils. A characteristic feature of CXCL8 is its ability to reversibly exist as both monomers and dimers, but whether both forms exist in vivo, and if so, the relevance of each form for in vivo function is not known. In this study, using a 'trapped' non-associating monomer and a non-dissociating dimer, we show that (i) wild type (WT) CXCL8 exists as both monomers and dimers, (ii) the in vivo recruitment profiles of the monomer, dimer, and WT are distinctly different, and (iii) the dimer is essential for initial robust recruitment and the WT is most active for sustained recruitment. Using a microfluidic device, we also observe that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient, and the gradients created by different CXCL8 variants elicit different neutrophil migratory responses. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and glycosaminoglycans (GAGs) on endothelial cells. On the basis of our data, we propose that dynamic equilibrium between CXCL8 monomers and dimers and their differential binding to CXCR2 and GAGs mediates and regulates in vivo neutrophil recruitment. Our finding that both CXCL8 monomer and dimer are functional in vivo is novel, and indicates that the CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease.

Evaluación de la citotoxicidad de la aflatoxina y la fumonisina en células intestinales de porcino / Evaluation of the cytotoxicity of AFB1, FB1 and AFB1/FB1 in intestinal cell

La coexistencia de la aflatoxina (AFB) y la fumonisina (FB) es ampliamenteconocida en muchas partes del mundo; sin embargo existen pocos estudiosque describan el efecto sinérgico de ambas micotoxinas in vivo o in vitro. Elobjetivo de este trabajo fue evaluar la citotoxicidad y el efecto de AFB y FBsobre la morfología, la capacidad proliferativa celular y la síntesis deinterleucina 8 (IL-8) en una línea celular de epitelio intestinal porcino (IPEC-1).Respecto a la morfología celular, ésta se vio afectada únicamente en lasconcentraciones más altas de AFB (50 μM) y FB (500 μM). Sin embargo laproliferación celular, el daño celular y la síntesis de IL-8 se vieron afectadascon la combinación AFB/FB (1,3/3,7; 2/3,7 y 5/10 μM, respectivamente), alcompararlas con el efecto individual de estas micotoxinas a las mismasconcentraciones (p < 0,05). Nuestros datos indican que la combinaciónAFB/FB en concentraciones bajas muestra un efecto sinérgico, alterando lamorfofisiología de las células utilizadas, lo que puede implicar, in vivo, laentrada de otras toxinas o agentes biológicos al estar alterada la barreraintestinal, impactando negativamente en la salud humana o animal(AU)

The coexistence of the aflatoxin (AFB) and fumonisin (FB) has been widelydocumented in many parts of the world. However, few studies describing thesynergy effect of both mycotoxins in vivo and/or in vitro are available. Theobjective of this study consisted on evaluating the effect of AFB and FB onthe morphology, the capacity of cellular proliferation, cytotoxicity andinterleukina-8 (IL-8) synthesis in a porcine intestinal epithelial cell line (IPEC-1).Concerning to the cellular morphology it was only affected in theconcentrations higher of AFB (50 μM) and FB (500 μM). However, the cellularproliferation, the cellular damage and synthesis of IL-8 they were affectedwhen present in combination the AFB/FB (1.3/3.7; 2/3.7 and 5/10 μMrespectively) with that showed by the individual effect of similar concentrationsof these mycotoxins (p < 0.05). Our data indicate that the combination ofAFB/FB in low concentrations showed a synergy effect, altering the cellularmorfophisiology, which can imply in vivo the entrance of other toxins orbiological agents for alteration of the intestinal barrier impacting negatively inthe human or animal health(AU)

Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8.

Human interleukin 8 (hIL-8), a neutrophil-activating and chemotactic cytokine, is known to play an important role in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. This cytokine belongs to the family of CXC chemokines, mediating the response through binding to the seven-transmembrane helical G protein-coupled receptors CXCR1 and CXCR2. For the first time, we employed the expressed protein ligation (EPL) strategy to chemokine synthesis and subsequent modification. The ligation site was chosen with respect to the position of four cysteine residues within the hIL-8 sequence. Ligation with synthetic peptides that carry cysteine at their N-termini resulted in full-length hIL-8 and the specifically carboxyfluorescein-labelled analogue [K69(CF)]hIL-8(1-77). [K69(CF)]hIL-8(1-77) was fully active as shown by inhibition of cAMP production. Furthermore, this analogue was used to study receptor internalisation in human promyelotic HL60 cells that express CXCR1 and CXCR2 receptors. Binding and quenching studies were performed on HL60 membranes and suggest that the C-terminus of IL-8 is accessible to solvent in the receptor-bound state. Thus, we introduce here a powerful approach that allows the site-specific incorporation of chemical modifications into the sequence of chemokines, which opens new avenues for studying IL-8 function.

Enantiospecific synthesis of the (9S,18R)-diastereomer of the leukocyte adhesion inhibitor cyclamenol A.

Cyclamenol A is one of the very few non-carbohydrate and non-peptide natural products that inhibit leukocyte adhesion to endothelial cells. We report on the first enantioselective total synthesis of the (9S, 18R)-diastereomer of this macrocyclic polyene lactam. Key elements of the synthesis are i) the synthesis of the required chiral building blocks by employing readily accessible building blocks from the chiral pool, that is, (S)-malic acid and (R)-hydroxyisobutyric acid, ii) assembly of a linear polyene precursor by means of Wittig and Horner olefination reactions as key C-C bond-forming transformations, iii) ring closure by means of a vanadium-mediated pinacolisation reaction and iv) conversion of the generated cis-diol into a (Z)-olefin to complete the entire polyene system of the natural product. Attempts to close the macrocyclic ring by a macrolactamisation, a double Stille coupling or direct olefination in a McMurry reaction failed. Crucial to the successful completion of the synthesis was the correct orchestration of the final steps. It was necessary to first deprotect the intermediate formed after macrocycle formation and to generate the sensitive heptaene system in the last step by means of a Corey-Hopkins sequence.

Synthesis of proteins by native chemical ligation.

A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chemical ligation was demonstrated by the one-step preparation of a cytokine containing multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-containing protein molecule. Native chemical ligation is an important step toward the general application of chemistry to proteins.

Interleukin-8 processing by neutrophil elastase, cathepsin G and proteinase-3.

Activated neutrophils secrete two forms of IL-8 with 77 and 72 amino acids, IL-8(77) and IL-8(72), along with proteinases that could process these cytokines. Significant conversion of IL-8(77) to more potent, N-terminally truncated forms was observed upon incubation with neutrophil granule lysates and purified proteinase-3. IL-8(72) was considerably more resistant to proteolytic processing than IL-8(77). The present observations indicate that neutrophil proteinases released in inflamed tissues convert IL-8 to more active forms and therefore tend to conserve or enhance, rather than decrease IL-8 activity.

A synthetic peptide which specifically inhibits heat-treated interleukin-8 binding and chemotaxis for neutrophils.

Interleukin-8 (IL-8) is a peptide which is secreted by stimulated human monocytes and which is chemotactic for human neutrophils. We synthesized three overlapping peptides spanning the amino-terminal region of the IL-8 sequence. None of the peptides retained the chemotactic activity of the native molecule. One of the peptides, IL-8(3-25), inhibited the neutrophil chemotactic activity of recombinant IL-8 (rIL-8) which had been preheated to 40 degrees C but did not reduce neutrophil chemokinesis, or the chemotactic activity of unheated rIL-8, FMLP, C5a or LTB4. Interleukin-8 exhibited similar binding kinetics and chemotaxis for neutrophils regardless of whether it had been pretreated at 40 degrees C. In addition, IL-8(3-25) was also able to decrease the binding of preheated IL-8 to neutrophils. IL-8(3-25), which can self-associate, binds directly to receptors on the neutrophil. The data suggest that heat-treated, but not untreated, IL-8 causes the IL-8(3-25) multimers to disaggregate, allowing the monomeric peptide to directly bind to the IL-8 receptor and thus inhibiting IL-8/receptor binding.

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide.

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)