Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers.

The interaction of multi-LacNAc (Galß1-4GlcNAc)-containing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.

Lactose-Modified Chitosan Gold(III)-PEGylated Complex-Bioconjugates: From Synthesis to Interaction with Targeted Galectin-1 Protein.

Galectins (Gal) are a family of glycan-binding proteins characterized by their affinity for ß-galactosides. Galectin-1 (Gal-1), a dimeric lectin with two galactoside-binding sites, regulates cancer progression and immune responses. Coordination chemistry has been engaged to develop versatile multivalent neoglycoconjugates for binding Gal-1. In this study we report a fast and original method to synthesize hybrid gold nanoparticles in which a hydrochloride lactose-modified chitosan, named CTL, is mixed with dicarboxylic acid-terminated polyethylene glycol (PEG), leading to shell-like hybrid polymer-sugar-metal nanoparticles (CTL-PEG-AuNPs). The aim of this paper is to preliminarily study the interaction of the CTL-PEG-AuNPs with a target protein, namely, Gal-1, under specific conditions. The molecular interaction has been measured by Transmission Electron Microscopy (TEM), UV-vis, and Raman Spectroscopy on a large range of Gal-1 concentrations (from 0 to 10-12 M). We observed that the interaction was strongly dependent on the Gal-1 concentration at the surface of the gold nanoparticles.

Synergistic targeting of alphavbeta3 integrin and galectin-1 with heteromultivalent paramagnetic liposomes for combined MR imaging and treatment of angiogenesis.

Effective and specific targeting of nanoparticles is of paramount importance in the fields of targeted therapeutics and diagnostics. In the current study, we investigated the targeting efficacy of nanoparticles that were functionalized with two angiogenesis-specific targeting ligands, an alpha(v)beta(3) integrin-specific and a galectin-1-specific peptide. We show in vitro, using optical techniques and MRI, that the dual-targeting approach produces synergistic targeting effects, causing a dramatically elevated uptake of nanoparticles as compared to single ligand targeting.

Galectin-1 protein modified gold (III)-PEGylated complex-nanoparticles: Proof of concept of alternative probe in colorimetric glucose detection.

Galectins (Gal) are a family of dimeric lectins, composed by two galactoside-binding sites implicated in the regulation of cancer progression and immune responses. In this study, we report for the first time the synthesis and the physical-chemical characterization of galectin-1-complex-gold COOH-terminated polyethlenglicole (PEG)-coated NPs (Gal-1 IN PEG-AuNPs) and their ability to recognize glucose in an aqueous solution with a concentration varying from 10 mM to 100 pM. The chemical protocol consistsof three steps: (i) complexation between galectin-1Gal-1 and tetrachloroauric acid (HAuCl4) to form gold-protein grains; (ii) staking process of COOH-terminated polyethlenglicole molecules (PEG) onto Gal-1-Au complex and (iii) reduction of hybrid metal ions to obtain a colloidal stable solution. During the complexation, the spectral signatures related to the Gal-1 orientation on the gold surface have been found to change due to its protonation state. The effective glucose monitoring was detected by UV-vis, Raman spectroscopy and Transmission Electron Microscopy (TEM). Overall, we observed that the interaction is strongly dependent on the Gal-1 conformation at the surface of gold nanoparticles.

Multivalent interactions between lectins and supramolecular complexes: Galectin-1 and self-assembled pseudopolyrotaxanes.

Supramolecular chemistry has been employed to develop flexible and adaptable multivalent neoglycoconjugates for binding galectin-1 (Gal-1). Gal-1, a dimeric lectin with two galactoside-binding sites, regulates cancer progression and immune responses. Self-assembled pseudopolyrotaxanes consisting of lactoside-displaying cyclodextrin (LCD) "beads" threaded onto polyviologen "strings" display mobile ligands as a result of cyclodextrin rotation about, and limited translation along, the polymer chain. The pseudopolyrotaxanes rapidly and efficiently precipitate Gal-1 and provide valency-corrected enhancements of up to 30-fold compared to native lactose and 20-fold over free LCD in a T-cell agglutination assay. A supramolecular statistical effect was observed, wherein the efficacy of Gal-1 inhibition correlates with the number of ligands connected to each other solely through mechanical and noncovalent interactions. Such flexible and adaptable self-assembled pseudopolyrotaxanes show promise for the study of multivalent interactions and targeting of therapeutically relevant lectins.

The role of Galectin-1 in the interaction between chondrocytes and a lactose-modified chitosan.

Evidences for the involvement of the Galectin-1 in the interaction of pig chondrocytes with a lactose-modified chitosan, namely Chitlac, are reported. The Chitlac glycopolymer has been shown to promote pig chondrocyte aggregation and to induce extracellular matrix production. Highly pure Galectin-1 was obtained from pig spleen by affinity chromatography and its identity was determined by ion spray mass spectrometry analysis of tryptic peptide fragments obtained after in-gel digestion. The complete sequence of pig Galectin-1 CDS was obtained by screening a pig EST database using human Galectin-1 sequence as template. The Galectin-1 cDNA was cloned into a pGEX-4T-1 expression vector and the recombinant protein was purified, characterized and used to produce a rabbit anti-serum. Recombinant Galectin-1 interacts in a dose-dependent manner with Chitlac as determined with ELISA assay. Expression level of galectin-1 gene, quantified by real-time PCR, was significantly higher in chondrocytes cultivated on Chitlac. In the same way, the presence of Chitlac stimulates secretion of Galectin-1 in culture medium that, by immunohistochemical analysis, revealed to be clustered on the surface of Chitlac-induced aggregates. These data indicate the role of Galectin-1 as a bridging agent between Chitlac and chondrocyte aggregates.

The effect of galectin 1 on 3T3 cell proliferation on chitosan membranes.

Galectin-1 (GAL1), a beta-galactoside-binding protein, functions in cell adhesion, development, and growth regulation. A number of studies suggest that GAL1 play an important role in enhancing cell adhesion to extracellular matrix and inducing cell proliferation. Chitosan is a derivative of chitin extracted from lobsters, crabs and shrimps' exoskeletons. In clinical medicine, chitosan membrane had been used as a semi-permeable biological dressing. Although chitosan membranes show no cytotoxicity, some cell types (e.g. 3T3 cells) fail to attach and proliferate on their surface. In these studies, we show that over-expression of GAL1 does not enhance 3T3 cell proliferation on chitosan membranes. However, coating the chitosan membrane with recombinant GAL1 proteins significantly expedites 3T3 cells proliferation. The enhanced cell growth was inhibited by thiodigalactoside (TDG, a potent inhibitor of beta-galactoside binding) and GAL1 monoclonal antibodies, suggesting GAL1's specific effect on the proliferation of 3T3 cells upon chitosan membranes. Moreover, immunoblotting detected a markedly suppressed tyrosine phosphorylation in several proteins on 3T3 cell growths upon GAL1-coated chitosan membrane. Pretreating the cells with sodium fluoride (NaF, a phosphatase inhibitor) inhibits the attachment and proliferation of 3T3 cells. These findings support a proposed role for altered levels of protein phosphorylation in GAL1-mediated cell attachment and proliferation on chitosan membranes.

Isolation and characterization of chimeric human Fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow.

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Effect of recombinant galectin-1 on the growth of immortal rat chondrocyte on chitosan-coated PLGA scaffold.

The effect of galectin-1 (GAL1) on the growth of immortal rat chondrocyte (IRC) on chitosan-modified PLGA scaffold is investigated. The experimental results showed that water absorption ratio of chitosan-modified PLGA scaffold was 70% higher than that of PLGA alone after immersion in ddH(2)O for 2 weeks, indicating that chitosan-modification significantly enhances the hydrophilicity of PLGA. The experimental results also showed that GALl efficiently and spontaneously coats the chitosan-PLGA scaffold surface to promote adhesion and growth of immortal rat chondrocyte (IRC). To investigate the effect of endogenous GAL1, the full-length GAL1 cDNAs were cloned and constructed into pcDNA3.1 vectors to generate a plasmid expressed in IRC (IRC-GAL1). The results showed that IRC-GAL1 growth was significantly higher than that of IRC on chitosan-PLGA scaffold. The GAL1-potentiated IRC growth on chitosan-PLGA scaffold was dose-dependently inhibited by TDG (specific inhibitor of GAL1 binding). These results strongly suggest that GAL1 is critical for enhancing IRC cell adhesion and growth on chitosan-PLGA scaffold. Moreover, GAL1-coating or expression tends to promote IRC cell-cell aggregation on chitosan-PLGA scaffold and significantly enhances IRC migration. These results suggest that GAL1 probably could induce tissue differentiation and facilitates cartilage reconstruction. In conclusion, the experimental results suggest that both GAL1 and chitosan are important for enhancing IRC cell adhesion and growth on PLGA scaffold, and GAL1 is a potential biomaterial for tissue engineering.