Glycan Microarray Research Articles

Natural and Synthetic Sialylated Glycan Microarrays and Their Applications.

This focused chapter serves as a short survey of glycan microarrays that are available with sialylated glycans, including both defined and shotgun arrays, their generation, and their utility in studying differential binding interactions to sialylated compounds, highlighting N-glycolyl (Gc) modified sialylated compounds. A brief discussion of binding interactions by lectins, antibodies, and viruses, and their relevance that have been observed with sialylated glycan microarrays is presented, as well as a discussion of cross-comparisons of array platforms and efforts to centralize and standardize the glycan microarray data.

A Sulfonamide Sialoside Analogue for Targeting Siglec-8 and -F on Immune Cells.

The Siglec family of cell surface receptors have emerged as attractive targets for cell-directed therapies due to their restricted expression on immune cells, endocytic properties, and ability to modulate receptor signaling. Human Siglec-8, for instance, has been identified as a therapeutic target for the treatment of eosinophil and mast cell disorders. A promising strategy to target Siglecs involves the use of liposomal nanoparticles with a multivalent display of Siglec ligands. A key challenge for this approach is the identification of a high affinity ligand for the target Siglec. Here, we report the development of a ligand of Siglec-8 and its closest murine functional orthologue Siglec-F that is capable of targeting liposomes to cells expressing Siglec-8 or -F. A glycan microarray library of synthetic 9-N-sulfonyl sialoside analogues was screened to identify potential lead compounds. The best ligand, 9-N-(2-naphthyl-sulfonyl)-Neu5Acα2-3-[6-O-sulfo]-Galβ1-4GlcNAc (6'-O-sulfo NSANeu5Ac) combined the lead 2-naphthyl sulfonyl C-9 substituent with the preferred sulfated scaffold. The ligand 6'-O-sulfo NSANeu5Ac was conjugated to lipids for display on liposomes to evaluate targeted delivery to cells. Targeted liposomes showed strong in vitro binding/uptake and selectivity to cells expressing Siglec-8 or -F and, when administered to mice, exhibit in vivo targeting to Siglec-F+ eosinophils.

Anti-glycan IgM repertoires in newborn human cord blood.

Natural antibodies are an innate-like subset of serum antibodies involved in host defense, tumor surveillance, homeostasis, and autoimmunity. Defining the natural antibody repertoire is critical for identifying biomarkers, developing vaccines, controlling and preventing autoimmunity, and understanding the development and organization of the immune system. While natural antibodies to protein antigens have been studied in depth, little is known about natural antibodies to carbohydrate antigens. To address this, we profiled IgM from umbilical cord blood and matched maternal sera on a glycan microarray. Since standard methods to detect maternal contamination in cord serum did not have sufficient sensitivity for our study, we developed a highly sensitive microarray-based assay. Using this method, we found that over 50% of the cord samples had unacceptable levels of maternal contamination. For the cord samples with high purity, anti-glycan IgM antibodies were prevalent and recognized a broad range of non-human and human glycans. Using principal component analysis and hierarchical clustering, cord IgM repertoires showed a high degree of similarity with each other but were distinct from maternal IgM repertoires. Our results demonstrate that many anti-glycan antibodies in human serum are natural antibodies and provide new insights into the development of anti-glycan antibody repertoires.

Expedient assembly of Oligo-LacNAcs by a sugar nucleotide regeneration system: Finding the role of tandem LacNAc and sialic acid position towards siglec binding

Sialosides containing (oligo-)N-acetyllactosamine (LacNAc, Galβ(1,4)GlcNAc) as core structure are known to serve as ligands for Siglecs. However, the role of tandem inner epitope for Siglec interaction has never been reported. Herein, we report the effect of internal glycan (by length and type) on the binding affinity and describe a simple and efficient chemo-enzymatic sugar nucleotide regeneration protocol for the preparative-scale synthesis of oligo-LacNAcs by the sequential use of β1,4-galactosyltransferase (β4GalT) and β1,3-N-acetylglucosyl transferase (β3GlcNAcT). Further modification of these oligo-LacNAcs was performed in one-pot enzymatic synthesis to yield sialylated and/or fucosylated analogs. A glycan library of 23 different sialosides containing various LacNAc lengths or Lac core with natural/unnatural sialylation and/or fucosylation was synthesized. These glycans were used to fabricate a glycan microarray that was utilized to screen glycan binding preferences against five different Siglecs (2, 7, 9, 14 and 15).

Chemoenzymatic Synthesis of DSGb5 and Sialylated Globo-series Glycans.

Sialic-acid-binding, immunoglobulin-type lectin-7 (Siglec-7) is present on the surface of natural killer cells. Siglec-7 shows preference for disialylated glycans, including α(2,8)-α(2,3)-disialic acids or internally branched α(2,6)-NeuAc, such as disialosylglobopentaose (DSGb5). Herein, DSGb5 was synthesized by a one-pot multiple enzyme method from Gb5 by α2,3-sialylation (with PmST1) followed by α2,6-sialylation (with Psp2,6ST) in 23 % overall yield. DSGb5 was also chemoenzymatically synthesized. The protection of the nonreducing-end galactose of Gb5 as 3,4-O-acetonide, 3,4-O-benzylidene, and 4,6-O-benzylidene derivatives provided DSGb5 in overall yields of 26 %, 12 %, and 19 %, respectively. Gb3, Gb4, and Gb5 were enzymatically sialylated to afford a range of globo-glycans. Surprisingly, DSGb5 shows a low affinity for Siglec-7 in a glycan microarray binding affinity assay. Among the synthesized globo-series glycans, α6α3DSGb4 shows the highest binding affinity for Siglec-7.

Chemoenzymatic Synthesis of DSGb5 and Sialylated Globo‐series Glycans

AbstractSialic‐acid‐binding, immunoglobulin‐type lectin‐7 (Siglec‐7) is present on the surface of natural killer cells. Siglec‐7 shows preference for disialylated glycans, including α(2,8)‐α(2,3)‐disialic acids or internally branched α(2,6)‐NeuAc, such as disialosylglobopentaose (DSGb5). Herein, DSGb5 was synthesized by a one‐pot multiple enzyme method from Gb5 by α2,3‐sialylation (with PmST1) followed by α2,6‐sialylation (with Psp2,6ST) in 23 % overall yield. DSGb5 was also chemoenzymatically synthesized. The protection of the nonreducing‐end galactose of Gb5 as 3,4‐O‐acetonide, 3,4‐O‐benzylidene, and 4,6‐O‐benzylidene derivatives provided DSGb5 in overall yields of 26 %, 12 %, and 19 %, respectively. Gb3, Gb4, and Gb5 were enzymatically sialylated to afford a range of globo‐glycans. Surprisingly, DSGb5 shows a low affinity for Siglec‐7 in a glycan microarray binding affinity assay. Among the synthesized globo‐series glycans, α6α3DSGb4 shows the highest binding affinity for Siglec‐7.

Practical considerations for printing high-density glycan microarrays to study weak carbohydrate-protein interactions

Interactions of carbohydrates and proteins are essential for many biological processes and glycan microarrays have emerged as powerful tools to rapidly assess these carbohydrate-protein interactions. Diverse platforms to immobilize glycans on glass slides for subsequent probing of the specificities of glycan-binding proteins (GBPs) have evolved. It has been suggested that high local glycan density on microarrays is crucial for detecting low-affinity interactions. To determine the influence of printing efficacy on GBP binding, we compared N-hydroxyl succinimide (NHS)-ester activated glass slides from three different manufacturers and evaluated two different printing buffers. Large differences in binding efficacies of Concanavalin A, peanut agglutinin, and Ricinus communis agglutinin 120 were observed. On some slides, low affinity interactions were missed altogether. Addition of polyethylenglycol (PEG) 400 to the printing buffer significantly enhanced the sensitivity of the binding assays. After monitoring printing efficacy over prolonged printing times, substantial effects resulting from progressing hydrolysis of the NHS-esters during the printing run on one type of slides were found. Printing efficiency of glycans strongly depends on the type of NHS-ester activated slides, the printing buffer, and the printing time. We provide practical advice for selecting the right printing conditions for particular applications.

Next-Generation Glycan Microarray Enabled by DNA-Coded Glycan Library and Next-Generation Sequencing Technology.

Interactions of glycans with proteins, cells, and microorganisms play important roles in cell-cell adhesion and host-pathogen interaction. Glycan microarray technology, in which multiple glycan structures are immobilized on a single glass slide and interrogated with glycan-binding proteins (GBPs), has become an indispensable tool in the study of protein-glycan interactions. Despite its great success, the current format of the glycan microarray requires expensive, specialized instrumentation and labor-intensive assay and image processing procedures, which limit automation and possibilities for high-throughput analyses. Furthermore, the current microarray is not suitable for assaying interaction with intact cells due to their large size compared to the two-dimensional microarray surface. To address these limitations, we developed the next-generation glycan microarray (NGGM) based on artificial DNA coding of glycan structures. In this novel approach, a glycan library is presented as a mixture of glycans and glycoconjugates, each of which is coded with a unique oligonucleotide sequence (code). The glycan mixture is interrogated by GBPs followed by the separation of unbound coded glycans. The DNA sequences that identify individual bound glycans are quantitatively sequenced (decoded) by powerful next-generation sequencing (NGS) technology, and copied numbers of the DNA codes represent relative binding specificities of corresponding glycan structures to GBPs. We demonstrate that NGGM generates glycan-GBP binding data that are consistent with that generated in a slide-based glycan microarray. More importantly, the solution phase binding assay is directly applicable to identifying glycan binding to intact cells, which is often challenging using glass slide-based glycan microarrays.

Tracking polysaccharides during white winemaking using glycan microarrays reveals glycoprotein-rich sediments.

Winemaking results in a significant amount of sediments that are formed in the tanks, the vats and in the bottles before and after fermentation. Little is known about the biochemical composition of these sediments apart from the fact that they are assumed to be derived in large part from the grape matrix. Glycan microarray technology offers a relatively rapid means to track the polysaccharides from their origin in the grape material and throughout the various steps in the winemaking process. In this study Comprehensive Microarray Polymer Profiling (CoMPP) was used to investigate the glycan-rich composition of particularly white grapes during winemaking and then investigate the effects of recombinant and commercial enzyme formulations on wine sediment compositions. The gross lees or sediments produced in the absence of enzymes were found to be composed of an abundance of homogalacturonans, rhamnogalacturonans, arabinans and galactans in addition to an abundance of extensins and arabinogalactan proteins. The addition of enzymes was shown to strip off the homogalacturonan and much of the rhamnogalacturonan with its side chains revealing a sediment layer composed almost exclusively of extensins and arabinogalactan proteins. The effect of winemaking techniques was shown to have an effect on the glycan-rich wine sediment compositions and holds implications for the management of gross lees in a winery environment.

Maskless Photochemical Printing of Multiplexed Glycan Microarrays for High-Throughput Binding Studies.

Spatially encoded glycan microarrays promise to rapidly accelerate our understanding of glycan binding in myriad biological processes, which could lead to new therapeutics and previously unknown drug targets. Here, we bring together a digital micromirror device, microfluidic introduction of inks, and advanced surface photochemistry to produce multiplexed glycan microarrays with reduced feature diameters, an increased number of features per array, and precise control of glycan density at each feature. The versatility of this platform was validated by printing two distinct glycan microarrays where, in the first, different glycans were immobilized to create a multiplexed array and, in another, the density of a single glycan was varied systematically to explore the effect of surface presentation on lectin-glycan binding. For lectin binding studies on these miniaturized microarrays, a microfluidic incubation chip was developed that channels multiple different protein solutions over the array. Using the multiplexed array, binding between eight lectin solutions and five different glycosides was determined, such that a single array can interrogate the binding between 40 lectin-glycan combinations. The incubation chip was then used on the array with varied glycan density to study the effects of glycan density on lectin binding. These results show that this novel printer could rapidly advance our understanding of critical unresolved questions in glycobiology, while simultaneously increasing the throughput and reducing the cost of these experiments.

Active cancer vaccine targeting carbohydrates for immunotherapy

Abstract Transformed cells present cell surface neoantigens, including aberrantly expressed carbohydrates. Targeting tumor-associated carbohydrate antigens could be useful for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a non-human carbohydrate consumed in diet and accumulates on human cancer cells, leading to expression of neoantigens. Passive immunotherapy with anti-Neu5Gc antibodies in mice inhibits tumor growth. Here we describe an active cancer vaccination immunotherapy strategy to target Neu5Gc-positive tumors. We generated glyconanoparticles from engineered red blood cells to form nano-ghosts (NGs) that either express (NGpos) or lack expression (NGneg) of Neu5Gc-glycoconjugates. We then show that optimized immunization of Neu5Gc-deficient Cmah-KO mice with NGpos glyconanoparticles induce a robust, diverse and long-lasting anti- Neu5Gc IgG immune response. Anti-Neu5Gc IgG antibodies also migrated into Neu5Gc-positive tumors and inhibited tumor growth in-vivo. Using glycan microarrays we also show that the kinetics and quality of the immune responses influence the efficacy of the vaccine. Hence, immunotherapy targeting Neu5Gc-carbohydrate neoantigens is promising.

Differential Recognition of Diet-Derived Neu5Gc-Neoantigens on Glycan Microarrays by Carbohydrate-Specific Pooled Human IgG and IgA Antibodies.

Sialic acids (Sias) cover vertebrate cell surface glycans. N-Acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc) are common Sia in mammals. Humans cannot synthesize Neu5Gc but accumulate it on cells through red-meat rich diets, generating numerous immunogenic Neu5Gc-neoantigens. Consequently, humans have diverse anti-Neu5Gc antibodies affecting xenotransplantation, cancer, atherosclerosis, and infertility. Anti-Neu5Gc antibodies circulate as IgG, IgM, and IgA isotypes; however, repertoires of the different isotypes in a large population have not been studied yet. Here, we used glycan microarrays to investigate anti-Neu5Gc IgGs and IgAs in intravenous immunoglobulin (IVIG) or pooled human IgA, respectively. Binding patterns on microarrays fabricated with Neu5Gc- and Neu5Ac-glycans, together with inhibition assays, revealed that different IVIG preparations have highly specific anti-Neu5Gc IgG reactivity with closely related repertoires, while IgAs show cross-reactivity against several Neu5Ac-glycans. Such different anti-Neu5Gc IgG/IgA repertoires in individuals could possibly mediate distinctive effects on human diseases.

A Tumor-Selective Monoclonal Antibody from Immunization with a Tumor-Associated Mucin Glycopeptide

We have previously studied the generation of immune responses after vaccination with tumor-associated carbohydrate antigen (TACA)-containing glycopeptides from the tandem repeat (TR) sequence of MUC4, an aberrantly expressed mucin in pancreatic adenocarcinomas. A specific lead antigen from that study containing the Thomsen-Friedenreich TACA disaccharide facilitated the pursuit of a monoclonal antibody to this synthetic hapten. Initial evaluation of polyclonal antiserum resulting from immunization with a KLH conjugate of this glycopeptide into rabbits showed high titer antibodies by ELISA assays, and selective immunoreactivity with MUC4+ cells by western blot and flow cytometry techniques. Glycan microarray analysis showed an intriguing binding pattern where the antiserum showed near complete specificity for MUC4 TR glycopeptides and peptides, relative to all components on the array. Tissue staining also showed distinct tumor specificity to pancreatic tumor tissue in relation to normal pancreatic tissue, with a preference for more aggressive tumor foci. Based on this data, we produced a monoclonal antibody whose binding and reactivity profile was similar to that of the polyclonal serum, with the added benefit of being more specific for the N-terminal glycosylated peptide domain. This epitope represents a novel immunogen to potentially develop diagnostic antibodies or immunotherapies against various MUC4-positive cancers.

Species‐specific differences in heparin‐induced modulation of IL‐12 family cytokines

IL‐12 family cytokines play a central role in the immune system by connecting the activities of innate and adaptive immunity. IL‐12 family members, IL‐12 and IL‐23, share a common p40 subunit that contains a prominent heparin binding domain. We have previously exploited this heparin binding segment for heparin affinity‐based purification of recombinant IL‐12. We also demonstrated that exogenous heparin can enhance IL‐12‐mediated interferon‐gamma (IFN‐g) production. However, the interaction between heparin and IL‐12/IL‐23 at the molecular level and the mechanisms by which heparin modulates the biological functions of these cytokines have not been explored. In addition, little is known about the species dependence of IL‐12 and IL‐23 in the context of sulfated glycosaminoglycans. Using isothermal titration calorimetry (ITC), we found that heparin can bind at micromolar concentrations to both human IL‐12 (hIL‐12) (Kd=9.8±0.2 mM) and murine IL‐12 (mIL‐12) (Kd=45.2±1.1 mM). ITC studies also showed that only heparin molecules longer that 8 saccharide units are able to bind to IL‐12. We confirmed the interaction of heparan sulfate (HS)‐derived compounds and hIL‐12/mIL‐12 via a novel, high throughput glycan microarray. Results from the microarray demonstrated that only HS‐derived molecules with 3 sulfate groups per disaccharide can bind to hIL‐12 and mIL‐12. In bioactivity studies, heparin was found to enhance hIL‐12 and human IL‐23 (hIL‐23) in human cells. On the other hand, though heparin did not increase the activity of mIL‐2 in 2D6 murine lymphocytes or C57BL/6 splenocytes, heparin enhanced the activity of mouse IL‐23 (mIL‐23) in C57BL/6 splenocytes. In cross‐species studies, heparin enhanced mIL‐12 activity in human NK‐9MI cells (1.55‐fold) whereas hIL‐12 was inactive in murine 2D6 cells and remained inactive in the presence of heparin. Interestingly, heparin was found to enhance mIL‐23 in human HEK Blue™ IL‐23 cells and hIL‐23 bioactivities in C57BL/6 splenocytes. Based on these and other data, we developed a working model in which heparin serves as a co‐receptor by stabilizing the hIL‐12/hIL‐12 receptor complex. We hypothesize that the mIL‐12/mIL‐12 receptor complex is sufficiently stable so that it does not benefit from heparin involvement. On the other hand, the interactions of hIL‐12/hIL‐12R, hIL‐23/hIL‐23R, and mIL‐23/mIL‐23R may be less stable so as to benefit from the addition of heparin. Ongoing ITC studies will evaluate the thermodynamics of the hIL‐12/hIL‐12R, mIL‐12/mIL‐12R, hIL‐23/hIL‐23R, and mIL‐23/mIL‐23R complexes in the presence and absence of heparin. Differences in binding affinity may help explain the species‐specific nature of heparin‐enhanced activity of the IL‐12 family cytokines.Support or Funding InformationThis work was supported by funding from the National Institutes of Health, National Cancer Institute (R01CA172631), the Arkansas Biosciences Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ABO Tolerance Following Treatment of Infant Mice with A-Expressing MHC-Identical Erythrocytes

Purpose ABO-incompatible heart transplantation (ABOi HTx) is safe in young children and increases donor access. Post-ABOi HTx, B cell tolerance develops to donor ABO blood group antigen(s) by mechanisms not fully defined. To study ABO tolerance, we developed A-transgenic mice (A-Tg) that express A-antigen on vascular endothelium, erythrocytes (RBCs) and lymphocytes; we demonstrated A-antigen-specific tolerance induced by HTx into 4 wk-old, MHC-identical, wild-type (WT) mice. Intentional induction of tolerance may allow subsequent ABOi HTx. Recently, we showed ABO tolerance could be intentionally induced in WT mice after ip injection of infant mice with intact A-Tg blood cells (BCs). Herein, we sought to determine specific A-Tg cell type(s) capable of inducing tolerance. Methods WT BALB/c (BALB) mice at 7 days of age were left untreated or injected ip (weekly × 3) with either unfractionated BCs, erythrocytes (RBCs), peripheral blood mononuclear cells (PBMCs), or splenocytes from A-Tg BALB mice (see Table). Two weeks later, all mice were injected ip (weekly × 5) with human A-RBC (A-antigen challenge) in an attempt to elicit anti-A antibody (Ab) production. Serum anti-A and third-party (non-A anti-human) Ab were assessed by hemagglutination or ABH glycan microarray. Results In response to challenge with A-antigen, high levels of anti-A Ab were produced both in untreated mice and in mice previously treated with A-Tg PBMCs or splenocytes (Table). In contrast, anti-A Ab remained undetectable/very low in mice treated as infants with A-Tg BCs or RBCs. Third-party Ab responses were high for all groups. Conclusion A-Tg RBCs induced robust A-antigen-specific tolerance in infant mice, whereas A-Tg lymphocytes (PBMCs, splenocytes) did not. Future studies will explore mechanisms of A-Tg RBC tolerance induction with the goal to design synthetic ABH-multivalent polyethylene glycol (PEG) glycoconjugates for intentional ABO tolerance to allow subsequent ABOi HTx safely.

GLAD: GLycan Array Dashboard, a visual analytics tool for glycan microarrays.

Traditional glycan microarray data is typically presented as excel files with limited visualization and interactivity. Thus, comparisons and analysis of glycan array data have been difficult, and there is need for a tool to facilitate data mining of glycan array data. GLAD (GLycan Array Dashboard) is a web-based tool to visualize, analyze, present and mine glycan microarray data. GLAD allows users to input multiple data files to create comparisons. GLAD extends the capability of the microarray data to produce more comparative visualizations in the form of grouped bar charts, heatmaps, calendar heatmaps, force graphs and correlation maps in order to analyze broad sets of samples. Additionally, it allows users to filter, sort and normalize the data and view glycan structures in an interactive manner, to facilitate faster visual data mining. GLAD is freely available for use on the Web at https://glycotoolkit.com/Tools/GLAD/ with all major modern browsers (Edge, Firefox, Chrome, Safari). Full documentation and video tutorials for GLAD can be found on https://glycotoolkit.com/GLAD.

Microbe-focused glycan array screening platform

Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.

History and future of shotgun glycomics.

Glycans in polysaccharides and glycoconjugates of the hydrophilic exterior of all animal cells participate in signal transduction, cellular adhesion, intercellular signaling, and sites for binding of pathogens largely through protein-glycan interactions. Microarrays of defined glycans have been used to study the binding specificities of biologically relevant glycan-binding proteins (GBP), but such arrays are limited by their lack of diversity or relevance to the GBP being investigated. Shotgun glycan microarrays are made up of structurally undefined glycans that were released from natural sources, labeled with bifunctional reagents so that they can be monitored during their purification using multidimensional chromatographic procedures, stored as a tagged glycan library (TGL) and subsequently printed onto microarrays at equal molar concentrations. The shotgun glycan microarray is then interrogated with a biologically relevant GBP and the corresponding glycan ligands can be retrieved from the TGL for detailed structural analysis and further functional analysis. Shotgun glycomics extended the defined glycan microarray to a discovery platform that supports functional glycomic analyses and may provide a useful process for ultimately defining the human glycome.

Deconstructing Wine Grape Cell Walls with Enzymes During Winemaking: New Insights from Glycan Microarray Technology.

Enzyme-aid maceration is carried out in most modern winemaking industries with a range of positive impacts on wine production. However, inconsistencies in enzyme efficiency are an issue complicated by unclear targets (limited information available on berry cell wall architecture of different cultivars) and the complex wine environment (i.e., fermenting must). Recent studies have been performed to develop a clearer picture of grape cell wall structures, maceration effects, and interactions between important wine compounds and grape-derived polysaccharides. This review highlights critically important recent studies on grape berry cell wall changes during ripening, the importance of enzymes during maceration (skin contact phase) and deconstruction processes that occur during alcoholic fermentation. The novelty of the Comprehensive Microarray Polymer Profiling (CoMPP) technique using cell wall probes (e.g., antibodies) as a method for following cell wall derived polymers during different biological and biotechnological processes is discussed. Recent studies, using CoMPP together with classical analytical methods, confirmed the developmental pattern of berry cell wall changes (at the polymer level) during grape ripening. This innovative technique were also used to track enzyme-assisted depectination of grape skins during wine fermentation and determine how this influence the release of wine favourable compounds. Furthermore, polysaccharides (e.g., arabinogalactan proteins) present in the final wine could be identified. Overall, CoMPP provides a much more enriched series of datasets compared to traditional approaches. Novel insights and future studies investigating grape cell wall and polyphenol interactions, and the tailoring of enzyme cocktails for consistent, effective and “customized” winemaking is advanced and discussed.

Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies: Implications for the molecular recognition of its ligands

The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal GalNAc residues present in the tumor-associated Tn antigen (αGalNAc-Ser/Thr) and its sialylated form, the sialyl-Tn antigen. However, human MGL has multiple splice variants, and whether these variants have distinct ligand-binding properties is unknown. Here, using glycan microarrays, we compared the binding properties of the short MGL 6C (MGLshort) and the long MGL 6B (MGLlong) splice variants, as well as of a histidine-to-threonine mutant (MGLshort H259T). Although the MGLshort and MGLlong variants displayed similar binding properties on the glycan array, the MGLshort H259T mutant failed to interact with the sialyl-Tn epitope. As the MGLshort H259T variant could still bind a single GalNAc monosaccharide on this array, we next investigated its binding characteristics to Tn-containing glycopeptides derived from the MGL ligands mucin 1 (MUC1), MUC2, and CD45. Strikingly, in the glycopeptide microarray, the MGLshort H259T variant lost high-affinity binding toward Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGLshort H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in WT MGLshort, His259 mediates H bonds directly or engages the Tn-glycopeptide backbone through water molecules. These bonds were lost in MGLshort H259T, thus explaining its lower binding affinity. Together, our results suggest that MGL not only connects to the Tn carbohydrate epitope, but also engages the underlying peptide via a secondary binding pocket within the MGL carbohydrate recognition domain containing the His259 residue.