Chemokines and galectins form heterodimers to modulate inflammation

Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin‐1 and galectin‐3, we identified several interacting pairs, such as CXCL12 and galectin‐3. Based on NMR and MD studies of the CXCL12/galectin‐3 heterodimer, we identified contact sites between CXCL12 β‐strand 1 and Gal‐3 F‐face residues. Mutagenesis of galectin‐3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin‐3, but not its mutants, inhibited CXCL12‐induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin‐3 attenuated CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted

Protein lysine-N zeta alkylation and O-phosphorylation mediated by DTT-generated reactive oxygen species

Reactive oxygen species (ROS) play crucial roles in physiology and pathology. In this report, we use NMR spectroscopy and mass spectrometry (MS) to demonstrate that proteins (galectin-1, ubiquitin, RNase, cytochrome c, myoglobin, and lysozyme) under reducing conditions with dithiothreitol (DTT) become alkylated at lysine-N(ζ) groups and O-phosphorylated at serine and threonine residues. These adduction reactions only occur in the presence of monophosphate, potassium, trace metals Fe/Cu, and oxygen, and are promoted by reactive oxygen species (ROS) generated via DTT oxidation. Superoxide mediates the chemistry, because superoxide dismutase inhibits the reaction, and hydroxyl and phosphoryl radicals are also likely involved. While lysine alkylation accounts for most of the adduction, low levels of phosphorylation are also observed at some serine and threonine residues, as determined by western blotting and MS fingerprinting. The adducted alkyl group is found to be a fragment of DTT that forms a Schiff base at lysine N(ζ) groups. Although its exact chemical structure remains unknown, the DTT fragment includes a SH group and a –CHOH–CH(2)– group. Chemical adduction appears to be promoted in the context of a well-folded protein, because some adducted sites in the proteins studied are considerably more reactive than others and the reaction occurs to a lesser extent with shorter, unfolded peptides and not at all with small organic molecules. A structural signature involving clusters of positively charged and other polar groups appears to facilitate the reaction. Overall, our findings demonstrate a novel reaction for DTT-mediated ROS chemistry with proteins

The apical stem–loop of the hepatitis B virus encapsidation signal folds into a stable tri–loop with two underlying pyrimidine bulges

Reverse transcription of hepatitis B virus (HBV) pregenomic RNA is essential for virus replication. In the first step of this process, HBV reverse transcriptase binds to the highly conserved encapsidation signal, epsilon (ε), situated near the 5′ end of the pregenome. ε has been predicted to form a bulged stem–loop with the apical stem capped by a hexa– loop. After the initial binding to this apical stem– loop, the reverse transcriptase synthesizes a 4 nt primer using the bulge as a template. Here we present mutational and structural data from NMR on the apical stem–loop of ε. Application of new isotope-labeling techniques ((13)C/(15)N/(2)H-U-labeling) allowed resolution of many resonance overlaps and an extensive structural data set could be derived. The NMR data show that, instead of the predicted hexa–loop, the apical stem is capped by a stable UGU tri–loop closed by a C-G base pair, followed by a bulged out C. The apical stem contains therefore two unpaired pyrimidines (C1882 and U1889), rather than one as was predicted, spaced by 6 nt. C1882, the 3′ neighbour to the G of the loop-closing C-G base pair, is completely bulged out, while U1889 is at least partially intercalated into the stem. Analysis of 205 of our own HBV sequences and 1026 strains from the literature, covering all genotypes, reveals a high degree of conservation of ε. In particular, the residues essential for this fold are either totally conserved or show rare non-disruptive mutations. These data strongly indicate that this fold is essential for recognition by the reverse transcriptase

(1)H, (13)C, and (15)N backbone and side-chain chemical shift assignments for the 36 proline-containing, full length 29 kDa human chimera-type galectin-3.

9 p.Galectin-3 has a unique trimodular design consisting of the canonical lectin domain, a collagen-like tandem-repeat section and an N-terminal peptide with two sites for Ser phosphorylation. Structural characterization of the full-length protein with its non-lectin part (115 of 250 residues) will help understand the multifunctionality of this potent cellular effector. Here, we report 1H, 13C, and 15N chemical shift assignments as determined by heteronuclear NMR spectroscopy.This work was generously supported by a research grant from the National Cancer Institute (CA-096090) to KHM; EC funding (GlycoHIT, contract no. 260600; GLYCOPHARM, contract no. 217297) to JJB & HJG, and European Research Council funding (ERC AdG °249929) to CW. NMR instrumentation was provided with funds from the National Science Foundation (BIR-961477), the University of Minnesota Medical School, and the Minnesota Medical Foundation.Peer reviewe

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with N-15-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

11 p.Galectin-3 (Gal-3) is a multifunctional effector acting extracellularly after non-classical secretion, in the cytoplasm and the nucleus. Its modular display of a carbohydrate recognition domain (CRD) attached to a tail of collagen-like repeats (nine in the human protein) and an N-terminal peptide is unique in this lectin family and not yet fully explored, as is the physiological significance of serine and tyrosine phosphorylation. Using a series of nine synthetic (phospho)peptides and the 15N-labeled CRD of human Gal-3 as well as measuring chemical shift perturbations in mixtures, potential for peptide reactivity was revealed in Gal-3’s backface. Tyrosine phosphorylation reduced the affinity, while serine phosphorylation of the N-terminal peptide was essential. Controls with sequence scrambling underscored inherent specificity. These results detect capacity for distinct sites of intramolecular recognition in Gal-3, adjustable by phosphorylation, and thus prompt analysis using the full-length protein.We gratefully acknowledge the financial support by MINECO of Spain (Grant CTQ2012-32025) and Comunidad de Madrid (MHit project) as well as the EC-funded BM1003 and CM1102 COST actions,the GlycoHIT program (contract No. 260600) and the GLYCOPHARM ITN project. M.A.B. acknowledges a FPI Ph.D. fellowship from the Spanish Ministry of Economy and Competitiveness.Peer reviewe

Hybrid ligands with calixarene and thiodigalactoside groups:galectin binding and cytotoxicity

Galectins have diverse functions and are involved in many biological processes because of their complex intra- and extracellular activities. Selective and potent inhibitors for galectins will be valuable tools to investigate the biological functions of these proteins. Therefore, we describe here the synthesis of galectin inhibitors with a potential "chelate effect". These compounds are designed to bind to two different binding sites on galectins simultaneously. In this paper a series of asymmetric "hybrid" compounds are prepared, which combine two galectin ligands (1) a substituted thiodigalactoside derivative and (2) an antagonist calixarene-based therapeutic agent. NMR spectroscopy was used to evaluate the interactions of these compounds with Galectin-1 and -3. In addition, cellular experiments were conducted to compare the cytotoxic effects of the hybrids with those of a calixarene derivative. While only the thiodigalactoside part of the hybrids showed strong binding, the calixarene part was responsible for observed cytoxoxicity effects, suggesting that the calixarene moiety may also be addressing a non-galectin target

Biodegradable Poly(ester) Urethane Acrylate Resins for Digital Light Processing: From Polymer Synthesis to 3D Printed Tissue Engineering Constructs

Digital light processing (DLP) is one of the most accurate and fastest additive manufacturingtechnologies to produce a variety of products, from patient-customized biomedical implants toconsumer goods; however, DLP’s use in tissue engineering is limited largely due to a lack ofbiodegradable resins. Herein, a library of biodegradable urethane acrylate-capped poly(esters)(with variations in molecular weight) is investigated as the basis for a DLP printable ink fortissue engineering. The synthesized oligomers show good printability in a DLP resin, capableof creating complex structures with mechanical properties matching those of medium-softtissues (1–3 MPa). While fabricated films from different molecular weight resins showed fewdifferences in surface topology, wettability, and protein adsorption, the adhesion and metabolicactivity of L929 and human dermal fibroblasts (HDFs) were significantly different: resins fromhigher molecular weight oligomers provided greater cell adhesion and metabolic activity. Theseprintable and biodegradable resins show the importance of oligomer molecular weight onscaffold properties, and facilitate the printing of elastomeric customizable scaffolds for a varietyof tissue engineering applications.</div