An Antibody-like Polymeric Nanoparticle Removes Intratumoral Galectin-1 to Enhance Antitumor T-Cell Responses in Cancer Immunotherapy.

Antibodies have shown potential to deplete immunosuppressive factors in tumor tissues. However, intrinsic drawbacks, including time-consuming processes in preparation, high cost, and short half-life time, greatly restrict their applications. In this work, we report an antibody-like polymeric nanoparticle (APN) that is capable of specifically capturing and removing galectin-1 in tumor tissues, thereby enhancing the antitumor T-cell responses. The APN is composed of an albumin-polymer hybrid nanoparticle (core) and an acid-responsive PEG shell. The core of the APN contains multiple recognition units and Tuftsin peptides to capture target factors and activate macrophage-mediated phagocytosis, respectively. By employing galactose as recognition units, the APN facilitated the phagocytosis of galectin-1 in tumor tissues, thereby improving the antitumor responses of tumor-infiltrating T cells. Since the recognition units in the APN can be further replaced to capture and remove other peptides/proteins, the APN provides a feasible approach for the development of synthetic nanoformulations to regulate biological systems and treat diseases.

A tandem-repeat galectin-1 from Apostichopus japonicus with broad PAMP recognition pattern and antibacterial activity.

Galectins belong to the family of carbohydrate-binding proteins and play major roles in the immune and inflammatory responses of both vertebrates and invertebrates. In the present study, one novel galectin-1 protein named AjGal-1 was identified from Apostichopus japonicas with an open reading frame of 1179 bp encoding a polypeptide of 392 amino acids. The deduced amino acids sequence of AjGal-1 contained three carbohydrate recognition domains (CRDs) which shared 34-37% identity with that of other galectin proteins from echinodermata, fishes, and birds. In the phylogenetic tree, AjGal-1 was closely clustered with galectins from Mesocentrotus nudus and Paracentrotus lividus. The mRNA transcripts of AjGal-1 were ubiquitously expressed in all the detected tissues, including gut, longitudinal muscle, gonad, coelomocytes, respiratory tree, tentacle and body wall, with the highest expression level in coelomocytes. After Vibrio splendidus stimulation, the mRNA expression levels of AjGal-1 in coelomocytes were significantly increased at 6 and 12 h (P < 0.01) compared with that in control group, and went back to normal level at 72 h. The recombinant protein of AjGal-1 (rAjGal-1) could bind various PAMPs including d-galactose, lipopolysaccharide (LPS), peptidoglycan (PGN) and mannose (Man), and exhibited the highest affinity to d-galactose. Meanwhile, rAjGal-1 could also bind and agglutinate different kinds of microorganisms, including gram-negative bacteria (V. splendidus and Escherichia coli), gram-positive bacteria (Micrococus leteus), and fungi (Pichia pastoris). rAjGal-1 also exhibited anti-microbial activity against V. splendidus and E. coli. All these results suggested that AjGal-1 could function as an important PRR with broad spectrum of microbial recognition and anti-microbial activity against the invading pathogen in A. japonicas.

Isolation of AccGalectin1 from Apis cerana cerana and its functions in development and adverse stress response.

The galectins are a family of lectins that play important roles in development, immunity, and the regulation of cellular responses. Much research has focused on the functions of galectins in mammals, though less in insects. Here, we identified the AccGalectin1 gene in Apis cerana cerana for the first time and explored its functions. The open reading frame of AccGalectin1 is 1449 base pairs and encodes a 482-amino-acid protein. AccGalectin1 expression was high during the transition between developmental stages and was high in the head, thorax, and epidermis compared with its levels in other tissues. In addition, the expression of AccGalectin1 was induced by several adverse stresses, including both abiotic and biotic stresses. A disk diffusion assay of recombinant AccGalectin1 protein revealed possible roles in protecting cells from oxidative stress. Furthermore, the expression levels of multiple oxidative genes (AccCAT, AccTpx1, AccTrx2, etc) were increased after AccGalectin1 was knocked down in Apis cerana cerana using RNA interference. We also observed that the malondialdehyde content in the AccGalectin1-silenced bees was higher than that in the control bees, while the antioxidant enzymatic activities of superoxide dismutase and peroxidase were lower. Considering these results, we suggest that AccGalectin1 may be indispensable for protecting honeybees from biotic and abiotic damage by participating in the oxidative resistance response and the immune response. These results may provide insight into the precise functions of galectins in mammals and other insects.

Studies on immunological and degranulation properties of a galectin-1 purified from goat (Capra hircus) heart.

Galectins are mammalian lectins characterized by affinity for ß-galactosides and the presence of a conserved carbohydrate recognition domain (CRD). Galectins play crucial role in the causation and progression of deadly human diseases like cancer, neurodegenerative disorders and cardiovascular disorders. Available literature reports relevant roles of galectins in innate as well as adaptive immune responses, along with the modulation of acute inflammatory response. In the current study, we purified the goat heart galectin-1 (GHG-1) and carried out its extensive immunological studies. Immunodiffusion studies revealed that anti-GHG-1 antibodies recognize the GHG-1 more readily as compared to the other galectins, suggesting its preferred utilization in various recognition studies. Antigenic cross-reactivity between galectins isolated from different tissues and species suggest their evolutionary preserved fundamental biological roles. A gradual increase in the lysozyme release was evident when the neutrophils were treated with various neutrophil activating agents. The findings of the present study confirm the increase in lysozyme production under the presence of various neutrophil activators, and thus add new information on GHG-1 induced degranulation.

Examination of the role of galectins in pre-mRNA splicing.

Several lines of evidence have been accumulated to indicate that galectin-1 and galectin-3 are two of the many proteins involved in nuclear splicing of pre-mRNA. First, nuclear extracts, capable of carrying out splicing of pre-mRNA in a cell-free assay, contain both of the galectins. Second, depletion of the galectins from nuclear extracts, using either lactose affinity chromatography or immunoadsorption with antibodies, results in concomitant loss of splicing activity. Third, addition of either galectin-1 or galectin-3 to the galectin-depleted extract reconstitutes the splicing activity. Fourth, the addition of saccharides that bind to galectin-1 and galectin-3 with high affinity (e.g., lactose or thiodigalactoside) to nuclear extract results in inhibition of splicing whereas parallel addition of saccharides that do not bind to the galectins (e.g., cellobiose) fail to yield the same effect. Finally, when a splicing reaction is subjected to immunoprecipitation by antibodies directed against galectin-1, radiolabeled RNA species corresponding to the starting pre-mRNA substrate, the mature mRNA product, and intermediates of the splicing reaction are coprecipitated with the galectin. Similar results were also obtained with antibodies against galectin-3. This chapter describes two key assays used in our studies: one reports on the splicing activity by looking at product formation on a denaturing gel; the other reports on the intermediates of spliceosome assembly using non-denaturing or native gels.

Altered galectin glycosylation: potential factor for the diagnostics and therapeutics of various cardiovascular and neurological disorders.

Galectins are ß-galactoside binding mammalian proteins characterized by the presence of a conserved carbohydrate recognition domain, expressed in almost all taxa of living organisms and involved in broad range of significant biological and physiological functions. Previously, we reported the purification and extensive characterization of galectin-1 from goat (Capra hircus) heart. Interestingly, the purified protein was found to have significant level of glycosylation. This intrigued us to evaluate the involvement of glycosylation in relation to protein's structural and functional integrity in its purified form. In the present study, an extensive comparative physicochemical characterization has been performed between the glycosylated and deglycosylated form of the purified protein. Deglycosylation resulted in an enhanced fluorescence quenching and marked reduction in pH and thermal stability of the purified galectin. Exposure to various biologically active chemicals showed significant differences in the properties and stability profile, causing significant deviations from its regular secondary structure in the deglycosylated form. These results clearly indicated enhanced structural and functional stabilization in the glycosylated galectin. The data revealed herein adds a vital facet demonstrating the significance of galectin expression and glycosylation in causation, progression, and possible therapeutics of associated clinical disorders. Our approach also allowed us to define some key interactions between the purified galectin and carbohydrate ligands that could well serve as an important landmark for designing new drug protocols for various cardiovascular and neurological disorders.

Expression, purification and characterization of galectin-1 in Escherichia coli.

As a member of beta-galactoside-binding proteins family, Galectin-1 (Gal-1) contains a single carbohydrate recognition domain, by means of which it can bind glycans both as a monomer and as a homodimer. Gal-1 is implicated in modulating cell-cell and cell-matrix interactions and may act as an autocrine negative growth factor that regulates cell proliferation. Besides, it can also suppress TH1 and TH17 cells by regulating dendritic cell differentiation or suppress inflammation via IL-10 and IL-27. In the present study, Gal-1 monomer and concatemer (Gal-1②), which can resemble Gal-1 homodimer, were expressed in Escherichia coli and their bioactivities were analyzed. The results of this indicate that both Gal-1 and Gal-1② were expressed in E. coli in soluble forms with a purity of over 95% after purifying with ion-exchange chromatography. Clearly, both Gal-1 and Gal-1② can effectively promote erythrocyte agglutination in hemagglutinating activity assays and inhibit Jurkat cell proliferation in MTT assays. All these data demonstrate that bacterially-expressed Gal-1 and Gal-1② have activities similar to those of wild type human Gal-1 whereas the bioactivity of concatemer Gal-1② was stronger than those of the bacterially-expressed and wild type human Gal.

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.

Galectin-1 is an interactive protein of selenoprotein M in the brain.

Selenium, an essential trace element for human health, mainly exerts its biological function through selenoproteins. Selenoprotein M (SelM) is one of the highly expressed selenoproteins in the brain, but its biological effect and molecular mechanism remain unclear. Thus, the interactive protein of SelM was investigated in this paper to guide further study. In order to avoid protein translational stop, the selenocysteine-encoding UGA inside the open reading frame of SelM was site-directly changed to the cysteine-encoding UGC to generate the SelM' mutant. Meanwhile, its N terminal transmembrane signal peptide was also cut off. This truncated SelM' was used to screen a human fetal brain cDNA library by the yeast two-hybrid system. A new interactive protein of SelM' was found to be galectin-1 (Gal-1). This protein-protein interaction was further verified by the results of fluorescence resonance energy transfer techniques, glutathione S-transferase pull-down and co-immunoprecipitation assays. As Gal-1 plays important roles in preventing neurodegeneration and promoting neuroprotection in the brain, the interaction between SelM' and Gal-1 displays a new direction for studying the biological function of SelM in the human brain.

Isolation of galectin-1 from human platelets: its interaction with actin.

Galectins are a family of animal lectins defined by their ß-galactoside-binding specificity and a consensus sequence in their carbohydrate-recognition domain. Galectin-1 (Gal-1) is expressed as a non-covalently linked homodimer present in a variety of tissues. Here we describe its isolation from human platelets by a procedure involving ionic exchange chromatography and affinity chromatography on lactose-agarose. Platelet Gal-1 co-purifies with actin, forming an actin-Gal-1 complex which does no dissociate even after treatment with sodium dodecyl sulfate. The presence of both proteins was confirmed by Western blot and by trypsin digestion followed by mass spectrometry identification. By hemagglutination assays we studied the response of recombinant Gal-1/actin, mixed and pre-incubated in different proportions, and then tested against neuraminidase treated rabbit red blood cells. The complex formation was confirmed by confocal microscopy, showing that both proteins co-localised in resting platelets as well as in thrombin-activated ones. These results suggest that endogenous Gal-1 forms an intracellular complex with monomeric actin and that, after platelet activation, Gal-1 could play a role in the polymerization-depolymerization process of actin, which concludes in platelet aggregation.

Antiproliferative effects of galectin-1 from Rana catesbeiana eggs on human leukemia cells and its binding proteins in human cells.

Galectin-1 from American bullfrog, RCG1, was isolated to high purity, and its growth inhibitory properties against human cells were examined. The results demonstrated that highly purified RCG1 induced large cell aggregates and revealed cell-type-specific growth inhibition. It significantly inhibited all human leukemia cell lines tested such as HL-60, U937, and K562 cells but did not inhibit human colon cancer cell line, Colo 201, or mouse mammary tumor cell line FM3A cells. Although most of the galectin-induced growth inhibitions are known to be apoptic, RCG1 induced growth arrest and neither apoptosis nor necrosis. RCG1-mediated growth inhibition was specifically suppressed by the corresponding sugar, lactose, but not by sucrose or even the structurally similar sugar, melibiose. Several studies have reported that galectin-mediated biological functions were modulated by charge modification. Since the high purity of RCG1 was demonstrated but a moderate degree of growth inhibition occurred, it is possible protein charge modification was examined by isoelectric focusing, and it was found to be highly heterogeneous in charge. RCG1 binding proteins in human cells were analyzed by lectin blotting using biotinylated RCG1, and lectin blotting revealed that in human cell extracts the specific proteins at molecular weight 37 and 50 kDa possessed the responsive features of RCG1 binding and lactose competition.

Purification, characterization, sequencing and biological chemistry of galectin-1 purified from Capra hircus (goat) heart.

A soluble ß-galactoside binding 14.5 kDa lectin was purified from the heart of Capra hircus. Its metal independent nature, preferential affinity for ß-D-lactose and 90-94% homology with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. The secondary structures of the deduced amino acid sequences were generally conserved with previously reported Gal-1. Exposure of the purified protein to varying temperature and pH, oxidant, thiol blocking reagents, denaturants and detergents resulted in significant changes in UV (ultraviolet), fluorescence, CD (circular dichroism) and FTIR (fourier transform infra red) spectra, thus strongly emphasizing the vitality of regular secondary structure of galectins for maintaining their active conformation. Bioinformatics studies corroborated the results obtained in wet lab. Our findings based on physico-chemical properties, oxidative inactivation and structural analysis of the goat heart galectin-1 suggests significant implications in potential biological and clinical applications.

Galectin-1 and HIV-1 Infection.

Initial binding of human immunodeficiency virus-1 (HIV-1) to its susceptible CD4(+) cells is the limiting step for the establishment of infection as the avidity of viral envelope gp120 for CD4 is not high and the number of viral envelope spikes on the surface is found to be low compared to highly infectious viruses. Several host factors, such as C-type lectins, are listed as being able to enforce or facilitate the crucial interaction of HIV-1 to the susceptible cell. Recent works suggest that a host soluble beta-galactoside-binding lectin, galectin-1, also facilitates both virion binding and the infection of target cells in a manner dependent on lactose but not mannose, suggesting that this soluble galectin can be considered as a host factor that influences HIV-1 pathogenesis. In this chapter, we describe methods used to investigate the potential role of the galectin family in HIV-1-mediated disease progression.

Purification, characterization, structural analysis and protein chemistry of a buffalo heart galectin-1.

A soluble ß-galactoside-binding lectin was purified by gel filtration chromatography from Bubalus bubalis heart. Its metal-independent nature, molecular weight of 14.5 kDa, preferential affinity for ß-D-lactose, and 87-92% identity with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. Stokes radii determination using gel filtration under reducing and non-reducing conditions revealed its homo-dimeric nature, further confirming its Gal-1 nomenclature. The purified lectin was found to be the most stable mammalian heart galectin purified till date, suggesting its preferential use in various recognition studies. Treatment of the purified lectin with oxidizing agent, thiol blocking reagents, denaturants, and detergents resulted in significant changes in UV-VIS, fluorescence, CD and FTIR spectra, which strongly emphasized the important aspect of regular secondary structure of galectins for the maintenance of their active conformation. Reduction of the activity of the purified lectin after oxidation by H2O2, with remarkable fluorescence quenching, may suggest potential role for galectin-1 in free radical-induced, oxidative stress-mediated cardiovascular disorders. The predictions of bioinformatics studies were found to be in accordance with the results obtained in wet lab.

Glycosylation of purified buffalo heart galectin-1 plays crucial role in maintaining its structural and functional integrity.

A buffalo heart galectin-1 purified by gel filtration chromatography revealed the presence of 3.55% carbohydrate content, thus it is the first mammalian heart galectin found to be glycosylated in nature and emphasizes the need to perform deglycosylation studies. Physicochemical comparative analysis between the properties of the native and deglycosylated proteins was carried out to understand the significance of glycosylation. The deglycosylated protein exhibited lesser thermal and pH stability compared to the native galectin. When exposed to thiol blocking reagents, denaturants, and detergents, remarkable differences were observed in the properties of the native and deglycosylated protein. Compared to the native glycosylated protein, the deglycosylated galectin showed enhanced fluorescence quenching when exposed to various agents. CD and FTIR analysis showed that deglycosylation of the purified galectin and its exposure to different chemicals resulted in significant deviations from regular secondary structure of the protein, thus emphasizing the significance of glycosylation for maintaining the active conformation of the protein. The remarkable differences observed in the properties of the native and deglycosylated galectin add an important dimension to the significance of protein glycosylation and its associated biological and clinical relevance.

Proteomic investigation of glioblastoma cell lines treated with wild-type p53 and cytotoxic chemotherapy demonstrates an association between galectin-1 and p53 expression.

Global protein analysis of treated and untreated glioblastoma cell lines was performed. Proteomic analysis revealed the identity of proteins that were significantly modulated by the treatment with wild-type TP53 and the cytotoxic chemotherapy SN38. In particular, galectin-1 was found to be negatively regulated by transfection with TP53 and further down-regulated by SN38. Expression level changes were confirmed by Western blot. Subsequent analysis of several high-grade glioma cell lines demonstrated very high levels of galectin-1, regardless if the cell lines contained mutant or wild-type TP53. High expression of galectin-1 in a human orthotopic murine tumor model was also detected by immunohistochemistry and revealed a consistent pattern of preferential expression in peripheral or leading tumor edges. Further examination of galectin-1 expression through microarray analysis in tumor materials from patients confirmed galectin-1 as a valuable biomarker and possible therapeutic target. These results demonstrate the utility of using proteomic approaches to interrogate and identify potential useful targets for cancer therapy by evaluating specific tumor responses, either positive or negative, to various therapies.

Purification and some properties of galectin-1 derived from water buffalo (Bubalus bubalis) brain.

An increasing number of galectins have been found in various animal species, the most abundant of which is galectin-1. The purpose of the present study was to purify and characterize galectin-1 from buffalo brain. We purified the galectin using a combination of ammonium sulphate fractionation and affinity chromatography and the homogeneity was determined by both native polyacrylamide gel electrophoresis (PAGE) and denaturing SDS-PAGE. The molecular weight of the galectin as determined by SDS-PAGE under reducing conditions and by gel filtration column under native conditions was 13.8 and 24.5 kDa, respectively, suggesting a dimeric form of galectin. The most potent inhibitor of the galectin activity was lactose, giving complete inhibition of hemagglutination at 0.8 mM. Galectin showed higher specificity towards human blood group A. Free thiol groups were estimated at a molar ratio of 2.9. The effects of alkylating reagents (iodoacetate and iodoacetamide) on saccharide binding of the galectin were studied. Both alkylating reagents significantly inactivated the activity of the galectin within 20 min. The temperature and pH stability of the galectin were determined. Our findings based on physico-chemical properties, carbohydrate and blood group specificities of the galectin may have future implications in biological and clinical applications.

Crosslinking of low-affinity glycoprotein ligands to galectin LEC-1 using a photoactivatable sulfhydryl reagent.

The N-terminal lectin domain (Nh) of the tandem repeat-type nematode galectin LEC-1 has a lower affinity for sugars than the C-terminal lectin domain. To confirm that LEC-1 forms a complex with N-acetyllactosamine-containing glycoproteins, we used several mutants of LEC-1 in which a unique cysteine residue was introduced into the Nh domain and examined their binding to bovine asialofetuin with a photoactivatable sulfhydryl crosslinking reagent. A crosslinked product was formed with the Q38C mutant, strongly suggesting the low-affinity interaction of Nh with the glycoprotein could be detected with this system.

Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3.

We have shown that galectin-1 and galectin-3 are functionally redundant splicing factors. Now we provide evidence that both galectins are directly associated with spliceosomes by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific antisera. Both galectin antisera co-precipitated splicing substrate, splicing intermediates and products in active spliceosomes. Protein factors co-precipitated by the galectin antisera included the Sm core polypeptides of snRNPs, hnRNP C1/C2 and Slu7. Early spliceosomal complexes were also immunoprecipitated by these antisera. When splicing reactions were sequentially immunoprecipitated with galectin antisera, we found that galectin-1 containing spliceosomes did not contain galectin-3 and vice versa, providing an explanation for the functional redundancy of nuclear galectins in splicing. The association of galectins with spliceosomes was (i) not due to a direct interaction of galectins with the splicing substrate and (ii) easily disrupted by ionic conditions that had only a minimal effect on snRNP association. Finally, addition of excess amino terminal domain of galectin-3 inhibited incorporation of galectin-1 into splicing complexes, explaining the dominant-negative effect of the amino domain on splicing activity. We conclude that galectins are directly associated with splicing complexes throughout the splicing pathway in a mutually exclusive manner and they bind a common splicing partner through weak protein-protein interactions.

IgA1 is the premier serum glycoprotein recognized by human galectin-1 since T antigen (Galbeta1-->3GalNAc-) is far superior to non-repeating N-acetyl lactosamine as ligand.

Human heart galectin-1 (HHL) was separated by high pressure liquid chromatography from endogenous glycoproteins co-purified with it during affinity chromatography. These glycoproteins offered excellent ligands for HHL binding and were rich in T antigen (Galbeta1-->3 GalNAc-) of O-linked oligosaccharides. In enzyme linked lectin assay and hemagglutination inhibition assay, human IgA1, bovine fetuin and other O-glycosylated T antigen-bearing glycoproteins bound to the lectin efficiently in contrast to single N-acetyl lactosamine (LacNAc)-bearing N-linked oligosaccharides released from them and to IgG which is not O-glycosylated. HHL binding to IgA1 and fetuin was unaffected by removal of their N-linked oligosaccharides by alpha-mannosidase. When immobilized, O-glycosylated serum proteins but not IgG could capture HHL from its solutions. Desialylated or polymeric IgA1 was better inhibitor than monomeric IgA1. The findings suggest a possible role for galectin-1 in anchoring of microbial and cancer cells known to be rich in T antigen, in high serum IgA1 turn over and in tissue sequestering of IgA1 immune complexes especially after their microbial desialylation in IgA nephropathy and other immune complex-mediated disorders.