Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells.

Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins.

Treatment resistance analysis reveals GLUT-1-mediated glucose uptake as a major target of synthetic rocaglates in cancer cells.

Rocaglates are natural compounds that have been extensively studied for their ability to inhibit translation initiation. Rocaglates represent promising drug candidates for tumor treatment due to their growth-inhibitory effects on neoplastic cells. In contrast to natural rocaglates, synthetic analogues of rocaglates have been less comprehensively characterized, but were also shown to have similar effects on the process of protein translation. Here, we demonstrate an enhanced growth-inhibitory effect of synthetic rocaglates when combined with glucose anti-metabolite 2-deoxy-D-glucose (2DG) in different cancer cell lines. Moreover, we unravel a new aspect in the mechanism of action of synthetic rocaglates involving reduction of glucose uptake mediated by downregulation or abrogation of glucose transporter GLUT-1 expression. Importantly, cells with genetically induced resistance to synthetic rocaglates showed substantially less pronounced treatment effect on glucose metabolism and did not demonstrate GLUT-1 downregulation, pointing at the crucial role of this mechanism for the anti-tumor activity of the synthetic rocaglates. Transcriptome profiling revealed glycolysis as one of the major pathways differentially regulated in sensitive and resistant cells. Analysis of synthetic rocaglate efficacy in a 3D tissue context with a co-culture of tumor and normal cells demonstrated a selective effect on tumor cells and substantiated the mechanistic observations obtained in cancer cell lines. Increased glucose uptake and metabolism is a universal feature across different tumor types. Therefore, targeting this feature by synthetic rocaglates could represent a promising direction for exploitation of rocaglates in novel anti-tumor therapies.

Differential Glycosite Profiling-A Versatile Method to Compare Membrane Glycoproteomes.

Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose-a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.

Imitating evolution's tinkering by protein engineering reveals extension of human galectin-7 activity.

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.

Calorimetric Analysis of the Interplay between Synthetic Tn Antigen-Presenting MUC1 Glycopeptides and Human Macrophage Galactose-Type Lectin.

Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 µM for monoglycosylated peptides to 0.6 µM for triglycosylated peptide. ITC measurements performed in D2O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D2O.

Pro4 prolyl peptide bond isomerization in human galectin-7 modulates the monomer-dimer equilibrum to affect function.

Human galectin-7 (Gal-7; also termed p53-induced gene 1 product) is a multifunctional effector by productive pairing with distinct glycoconjugates and protein counter-receptors in the cytoplasm and nucleus, as well as on the cell surface. Its structural analysis by NMR spectroscopy detected doubling of a set of particular resonances, an indicator of Gal-7 existing in two conformational states in slow exchange on the chemical shift time scale. Structural positioning of this set of amino acids around the P4 residue and loss of this phenomenon in the bioactive P4L mutant indicated cis-trans isomerization at this site. Respective resonance assignments confirmed our proposal of two Gal-7 conformers. Mapping hydrogen bonds and considering van der Waals interactions in molecular dynamics simulations revealed a structural difference for the N-terminal peptide, with the trans-state being more exposed to solvent and more mobile than the cis-state. Affinity for lactose or glycan-inhibitable neuroblastoma cell surface contact formation was not affected, because both conformers associated with an overall increase in order parameters (S2). At low µM concentrations, homodimer dissociation is more favored for the cis-state of the protein than its trans-state. These findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch for Gal-7.

(Phospho)proteomic Profiling of Microsatellite Unstable CRC Cells Reveals Alterations in Nuclear Signaling and Cholesterol Metabolism Caused by Frameshift Mutation of NMD Regulator UPF3A.

DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.

Chicken lens development: complete signature of expression of galectins during embryogenesis and evidence for their complex formation with α-, ß-, δ-, and τ-crystallins, N-CAM, and N-cadherin obtained by affinity chromatography.

The emerging multifunctionality of galectins by specific protein-glycan/protein interactions explains the interest to determine their expression during embryogenesis. Complete network analysis of all seven chicken galectins (CGs) is presented in the course of differentiation of eye lens that originates from a single type of progenitor cell. It answers the questions on levels of expression and individual patterns of distribution. A qualitative difference occurs in the CG-1A/B paralogue pair, underscoring conspicuous divergence. Considering different cell phenotypes, lens fiber and also epithelial cells can both express the same CG, with developmental upregulation for CG-3 and CG-8. Except for expression of the lens-specific CG (C-GRIFIN), no other CG appeared to be controlled by the transcription factors L-Maf and Pax6. Studying presence and nature of binding partners for CGs, we tested labeled galectins in histochemistry and in ligand blotting. Mass spectrometric (glyco)protein identification after affinity chromatography prominently yielded four types of crystallins, N-CAM, and, in the cases of CG-3 and CG-8, N-cadherin. Should such pairing be functional in situ, it may be involved in tightly packing intracellular lens proteins and forming membrane contact as well as in gaining plasticity and stability of adhesion processes. The expression of CGs throughout embryogenesis is postulated to give meaning to spatiotemporal alterations in the local glycome.

SILAC-Based Quantification of TGFBR2-Regulated Protein Expression in Extracellular Vesicles of Microsatellite Unstable Colorectal Cancers.

Microsatellite unstable (MSI) colorectal cancers (CRCs) are characterized by mutational inactivation of Transforming Growth Factor Beta Receptor Type 2 (TGFBR2). TGFBR2-deficient CRCs present altered target gene and protein expression. Such cellular alterations modulate the content of CRC-derived extracellular vesicles (EVs). EVs function as couriers of proteins, nucleic acids, and lipids in intercellular communication. At a qualitative level, we have previously shown that TGFBR2 deficiency causes overall alterations in the EV protein content. To deepen the basic understanding of altered protein dynamics, this work aimed to determine TGFBR2-dependent EV protein signatures in a quantitative manner. Using a stable isotope labeling with amino acids in cell culture (SILAC) approach for mass spectrometry-based quantification, 48 TGFBR2-regulated proteins were identified in MSI CRC-derived EVs. Overall, TGFBR2 deficiency caused upregulation of several EV proteins related to the extracellular matrix and nucleosome as well as downregulation of proteasome-associated proteins. The present study emphasizes the general overlap of proteins between EVs and their parental CRC cells but also highlights the impact of TGFBR2 deficiency on EV protein composition. From a clinical perspective, TGFBR2-regulated quantitative differences of protein expression in EVs might nominate novel biomarkers for liquid biopsy-based MSI typing in the future.

TGFBR2­dependent alterations of microRNA profiles in extracellular vesicles and parental colorectal cancer cells.

In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor ß receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF­ß signaling. This pathway can alter the expression of coding and non­coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post­transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116­TGFBR2 MSI cell line model enables the doxycycline (dox)­inducible reconstituted expression of TGFBR2 in an isogenic background (­dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2­dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2­dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2­deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR­381­3p in TGFBR2­deficient MSI tumor cells and their secreted EVs.

Deciphering the galectin-12 protein interactome reveals a major impact of galectin-12 on glutamine anaplerosis in colon cancer cells.

Galectins are ß-galactoside binding proteins which possess a variety of functions including modulation of apoptosis, growth and differentiation. Hence, alterations in the expression profile have been associated with loss of cellular homeostasis contributing to tumor growth and progression. Though galectin-12 is significantly downregulated in several tumor entities, including colon cancer, its impact on cellular homeostasis as well as galectin-12 specific binding partners have not been identified so far. We therefore established an experimental strategy which is based on reversible cross-link immunoprecipitation to capture the galectin-12 protein interactome in colon cancer cells. By applying this approach, we identified 10 novel candidates of galectin-12 interacting proteins including the neutral amino acid exchanger SLC1A5. Remarkably, we uncovered that binding of galectin-12 to SLC1A5 significantly reduced glutamine uptake in our model cell line. Consequently, utilization of glutamine carbon for biomass synthesis was profoundly affected, suggesting galectin-12 as a novel inhibitor of glutamine anaplerosis in colon cancer cells. More detailed analysis revealed that colon cancer cells can counteract galectin-12 mediated glutamine deprivation by induction of compensatory mechanisms which facilitate adaption to low-glutamine conditions and thus survival.

Design-functionality relationships for adhesion/growth-regulatory galectins.

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.

Chicken GRIFIN: binding partners, developmental course of localization and activation of its lens-specific gene expression by L-Maf/Pax6.

Tissue lectins appear to be involved in a broad range of physiological processes, as reflected for the members of the family of galectins by referring to them as adhesion/growth-regulatory effectors. In order to clarify the significance of galectin presence, key challenges are to define their binding partners and the profile of localization. Having identified the chicken galectin-related interfiber protein (C-GRIFIN) as lens-specific protein present in the main body of adult lens, we here report its interaction with lens proteins in ligand blotting. The assumption for pairing with α-, ß- and δ-crystallins was ascertained by mass spectrometric detection of their presence in eluted fractions obtained by affinity chromatography. Biochemical and immunohistochemical monitoring revealed protein presence from about 3-day-old embryos onwards, mostly in the cytoplasm of elongated posterior cells, later in secondary lens fiber cells. On the level of gene expression, its promoter was activated by transcription factor L-Maf alone and together with Pax6 like a crystallin gene, substantiating C-GRIFIN's status as lens-specific galectin. Using this combined strategy for counterreceptor and expression profiling by bio- and histochemical methods including light, electron and fluorescence microscopy, respective monitoring in lens development can now be taken to the level of the complete galectin family.

Detection of malignancy-associated phosphoproteome changes in human colorectal cancer induced by cell surface binding of growth-inhibitory galectin-4.

Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.

In-depth characterization of the neuroinflammatory reaction induced by peripheral surgery in an animal model.

Delirium is a common complication seen after surgery and anesthesia, in particular in older patients. Although the etiology of postoperative delirium is only incompletely understood, various lines of evidence suggest that proinflammatory signaling from the peripheral site of inflammation to central nervous system results in a decrease of cerebral acetylcholine (ACh) levels thereby inducing neuroinflammation. To corroborate this theory, we applied an animal model for characterization of the neuroinflammatory response after partial hepatectomy (HPx). In this model, the surgery-induced decrease in cerebral ACh levels can be prevented by intraoperative application of physostigmine. Thus, ACh-associated changes in the expression and secretion of inflammation-related compounds can be assessed by comparing the results obtained after surgery, in physostigmine-treated and untreated controls. This way we were able to show that the decrease of cerebral ACh triggers increased secretion of IL-1ß, IL-6, TNFα, MIP-2 (CCL3), RANTES, MCP1, IFNgamma, and IP-10. A gene array covering the expression of 370 inflammation-related genes indicated 13 candidates that are induced upon cerebral ACh decrease after HPx. Quantification of the changes in the expression of these candidates by the comparative CT method revealed a significant increase (> 1.5-fold) in the expression of IL-1ß, IL-6, TNFα, MIP2, RANTES, MCP1, TLR2, TLR4, HMGB1, TNFSF6, TNFSF12, IL1R1 and ILR6. Thus, our results suggest that peripheral surgery induces a reduction of cerebral ACh levels which trigger a complex neuroinflammatory response. From a clinical point of view, manipulating cerebral ACh levels by procholinergic drugs such as physostigmine could become an option to therapeutically target this kind of neuroinflammation.

Distinct Activities of Glycolytic Enzymes Identify Chronic Lymphocytic Leukemia Patients with a more Aggressive Course and Resistance to Chemo-Immunotherapy.

A higher capacity to grow under hypoxic conditions can lead to a more aggressive behavior of tumor cells. Determining tumor activity under hypoxia may identify chronic lymphocytic leukemia (CLL) with aggressive clinical course and predict response to chemo-immunotherapy (CIT). A metabolic score was generated by determining pyruvate kinase and lactate dehydrogenase, key enzymes of glycolysis, ex vivo in primary CLL samples under normoxic and hypoxic conditions. This score was further correlated with clinical endpoints and response to CIT in 96 CLL patients. 45 patients were classified as metabolic high risk (HR), 51 as low risk (LR). Treatment-free survival (TFS) was significantly shorter in HR patients (median 394 vs 723 days, p = .021). 15 HR patients and 14 LR patients received CIT after sample acquisition. HR patients had a significantly shorter progression-free survival after treatment compared to LR patients (median 216 days vs not reached, p = .008). Multivariate analysis evaluating age, IGHV, TP53 deletion or mutation and 11q22-23 deletion besides the capacity of tumor cells to grow under severe hypoxic conditions identified the metabolic profile as the strongest independent risk factor for shorter TFS (hazard ratio 2.37, p = .011). The metabolic risk can provide prognostic and predictive information complementary to genetic biomarkers and identify patients who might benefit from alternative treatment approaches.

Glycan Chains of Gangliosides: Functional Ligands for Tissue Lectins (Siglecs/Galectins).

Molecular signals on the cell surface are responsible for adhesion and communication. Of relevance in this respect, their chemical properties endow carbohydrates with the capacity to store a maximum of information in a minimum of space. One way to present glycans on the cell surface is their covalent conjugation to a ceramide anchor. Among the resulting glycosphingolipids, gangliosides are special due to the presence of at least one sialic acid in the glycan chains. Their spatial accessibility and the dynamic regulation of their profile are factors that argue in favor of a role of glycans of gangliosides as ligands (counterreceptors) for carbohydrate-binding proteins (lectins). Indeed, as discovered first for a bacterial toxin, tissue lectins bind gangliosides and mediate contact formation (trans) and signaling (cis). While siglecs have a preference for higher sialylated glycans, certain galectins also target the monosialylated pentasaccharide of ganglioside GM1. Enzymatic interconversion of ganglioside glycans by sialidase action, relevant for neuroblastoma cell differentiation and growth control in vitro, for axonogenesis and axon regeneration, as well as for proper communication between effector and regulatory T cells, changes lectin-binding affinity profoundly. The GD1a-to-GM1 "editing" is recognized by such lectins, for example, myelin-associated glycoprotein (siglec-4) losing affinity and galectin-1 gaining reactivity, and then translated into postbinding signaling. Orchestrations of loss/gain of affinity, of ganglioside/lectin expression, and of lectin presence in a network offer ample opportunities for fine-tuning. Thus glycans of gangliosides such as GD1a and GM1 are functional counterreceptors by a pairing with tissue lectins, an emerging aspect of ganglioside and lectin functionality.

Adhesion/growth-regulatory galectins tested in combination: evidence for formation of hybrids as heterodimers.

The delineation of the physiological significance of protein (lectin)-glycan recognition and the structural analysis of individual lectins have directed our attention to studying them in combination. In this report, we tested the hypothesis of hybrid formation by using binary mixtures of homodimeric galectin-1 and -7 as well as a proteolytically truncated version of chimera-type galectin-3. Initial supportive evidence is provided by affinity chromatography using resin-presented galectin-7. Intriguingly, the extent of cell binding by cross-linking of surface counter-receptor increased significantly for monomeric galectin-3 form by the presence of galectin-1 or -7. Pulsed-field gradient NMR (nuclear magnetic resonance) diffusion measurements on these galectin mixtures indicated formation of heterodimers as opposed to larger oligomers. 15N-1H heteronuclear single quantum coherence NMR spectroscopy and molecular dynamics simulations allowed us to delineate how different galectins interact in the heterodimer. The possibility of domain exchange between galectins introduces a new concept for understanding the spectrum of their functionality, particularly when these effector molecules are spatially and temporally co-expressed as found in vivo.

Reaction of a Programmable Glycan Presentation of Glycodendrimersomes and Cells with Engineered Human Lectins To Show the Sugar Functionality of the Cell Surface.

Chemical and biological tools are harnessed to investigate the impact of spatial factors for functional pairing of human lectins with counterreceptors. The homodimeric adhesion/growth-regulatory galectin-1 and a set of covalently linked homo-oligomers from di- to tetramers serve as proof-of-principle test cases. Glycodendrimersomes provide a versatile and sensitive diagnostic platform to reveal thresholds for ligand density and protein concentration in aggregation assays (trans-activity), irrespective of linker length between lectin domains. Monitoring the affinity of cell binding and ensuing tumor growth inhibition reveal the linker length to be a bidirectional switch for cis-activity. The discovery that two aspects of lectin functionality (trans- versus cis-activity) respond non-uniformly to a structural change underscores the power of combining synthetic and biological tools to advance understanding of the sugar functionality of the cell surface.

Studying the Structural Significance of Galectin Design by Playing a Modular Puzzle: Homodimer Generation from Human Tandem-Repeat-Type (Heterodimeric) Galectin-8 by Domain Shuffling.

Tissue lectins are emerging (patho)physiological effectors with broad significance. The capacity of adhesion/growth-regulatory galectins to form functional complexes with distinct cellular glycoconjugates is based on molecular selection of matching partners. Engineering of variants by changing the topological display of carbohydrate recognition domains (CRDs) provides tools to understand the inherent specificity of the functional pairing. We here illustrate its practical implementation in the case of human tandem-repeat-type galectin-8 (Gal-8). It is termed Gal-8 (NC) due to presence of two different CRDs at the N- and C-terminal positions. Gal-8N exhibits exceptionally high affinity for 3'-sialylated/sulfated ß-galactosides. This protein is turned into a new homodimer, i.e., Gal-8 (NN), by engineering. The product maintained activity for lactose-inhibitable binding of glycans and glycoproteins. Preferential association with 3'-sialylated/sulfated (and 6-sulfated) ß-galactosides was seen by glycan-array analysis when compared to the wild-type protein, which also strongly bound to ABH-type epitopes. Agglutination of erythrocytes documented functional bivalency. This result substantiates the potential for comparative functional studies between the variant and natural Gal-8 (NC)/Gal-8N.