Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays

Carbohydrate post-translational modifications on proteins are important determinants of protein function in both normal and disease biology. We have developed a method to allow the efficient, multiplexed study of glycans on individual proteins from complex mixtures, using antibody microarray capture of multiple proteins followed by detection with lectins or glycan-binding antibodies. Chemical derivatization of the glycans on the spotted antibodies prevented lectin binding to those glycans. Multiple lectins could be used as detection probes, each targeting different glycan groups, to build up lectin binding profiles of captured proteins. By profiling both protein and glycan variation in multiple samples using parallel sandwich and glycan-detection assays, we found cancer-associated glycan alteration on the proteins MUC1 and CEA in the serum of pancreatic cancer patients. Antibody arrays for glycan detection are highly effective for profiling variation in specific glycans on multiple proteins and should be useful in diverse areas of glycobiology research.     

Quantifiable fluorescent glycan microarrays

A glycan microarray was developed by using 2,6-diaminopyridine (DAP) as a fluorescent linker and printing of the glycan-DAP conjugates (GDAPs) on epoxy-activated glass slides. Importantly, all coupled GDAPs showed a detectable level of concentration-dependent GDAP fluorescence under blue laser excitation (495 nm) that can be used for both grid location and on-slide quantification. A glycan array including a large number of GDAP’s derived from natural and commercially available free glycans was constructed and glycan interactions with various plant lectins were investigated. In addition, binding parameters of lectins to glycans were obtained by varying both the amount of GDAPs on the array and the lectin concentration in analyses. These data demonstrate the general utility of GDAP microarrays for functional glycomic analyses and for determining binding parameters of glycan binding proteins (GBPs).     

应用凝集素芯片检测肝癌细胞膜表面糖链变化

利用凝集素糖链特异亲和原理构建对细胞膜表面糖链进行即时检测的凝集素芯片体系,检测肝癌发生过程中细胞膜糖链的变化.从H22细胞系、正常小鼠和肝癌模型鼠肝组织中提取细胞进行荧光标记,激光扫描仪检测凝集素位点捕获的细胞,根据凝集素特异亲和性确定细胞膜表面糖表达谱,显微镜下观察捕获细胞的形态.对凝集素芯片捕获细胞的最佳条件进行探讨,用甘露糖抑制试验、流式细胞仪和不同血型红细胞验证了凝集素捕获细胞的特异性.结果显示:正常和肝癌小鼠肝细胞膜表面糖链存在较大差异,正常组只有PSA、DSL、STL、NPL凝集素位点捕获到细胞,实验组只有LTL和DBA位点没有捕获到细胞,提示小鼠肝癌组织细胞膜表面糖链显著增加,细胞膜上唾液酸、乙酰葡萄糖、乙酰半乳糖、甘露糖和半乳糖糖链表达增加,这些糖链及其相关糖蛋白可能在肝癌的发生和发展中起一定作用.该凝集素芯片有较好的稳定性和特异性,可以对细胞膜表面糖链进行动态、即时、通量的检测,为研究细胞膜表面聚糖在细胞发育和癌变等过程中的变化提供了一个技术平台.     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.     

One-step biotinylation procedure for carbohydrates to study carbohydrate-protein interactions

Protein-carbohydrate interactions play crucial roles in numerous biological processes. To study these interactions, we developed a simple and fast procedure for the biotinylation of carbohydrates based on reductive amination. The method allows complete and stable biotinylation of small quantities of oligosaccharides and includes a rapid and simple procedure to remove excess labeling reagent. After biotinylation, the structural and biological integrity of the glycans was intact as determined by HPLC, mass spectrometry, and a plant lectin assay. By using the human C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin), we demonstrate that the biotinylated glycans can be used in a glycan array to determine binding specificities of lectins. Moreover, we show that fluorescent beads coated with selected biotinylated glycans bind to DC-SIGN-expressing dendritic cells in vitro. Finally, by using biotinylated high-mannose N-glycans, we could visualize DC-SIGN-expressing cells in lymph node tissue. The availability of easy biotinylation methods for oligosaccharides such as those described here greatly facilitates the functional analysis of lectins. In addition, the biotinylated glycans will be great tools for investigating functional lectin receptors in situ.     

Use of a library of mutated Maackia amurensis hemagglutinin for profiling the cell lineage and differentiation

Thirty-five variant lectins were prepared by mutations of two amino acids within the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH). Each lectin showed unique carbohydrate specificity according to their bindings to soluble polyacrylamide with various mono- and oligosaccharides and to glycophorin A. The relative intensity of the bindings of carcinoma, myeloid, fibroblastic, and melanoma cells to immobilized MAH variant lectins was examined. Each cell line showed distinct profiles regarding the number of cells bound to wild-type and 35 MAH variants and the differences and the similarities in these binding profiles were quantitatively documented by the cluster analysis. The cell lines were classified into several groups and these groups surprisingly corresponded to the lineage of the cells. These results indicated that a library of mutated MAH is useful as a tool for the profiling of various cells based on the variations of the surface glycans.     

Lectin microarrays identify cell-specific and functionally significant cell surface glycan markers

Glycosylation is among the most complex posttranslational modifications with an extremely high level of diversity that has made it refractory to high-throughput analyses. Despite its resistance to high-throughput techniques, glycosylation is important in many critical cellular processes that necessitate a productive approach to their analysis. To facilitate studies in glycosylation, we developed a high-throughput lectin microarray for defining mammalian cell surface glycan signatures. Using the lectin microarray we established a binary analysis of cell binding and hierarchical organization of 24 mammalian cell lines. The array was also used to document changes in cell surface glycosylation during cell development and differentiation of primary murine immune system cells. To establish the biological and clinical importance of glycan signatures, the lectin microarray was applied in two systems. First, we analyzed the cell surface glycan signatures and were able to predict mannose-dependent tropism using a model pathogen. Second, we used the glycan signatures to identify novel lectin biomarkers for cancer stem-like cells in a murine model. Thus, lectin microarrays are an effective tool for analyzing diverse cell processes including cell development and differentiation, cell-cell communication, pathogen-host recognition, and cell surface biomarker identification.     

Fluorescent labeling agents change binding profiles of glycan-binding proteins

Interactions of glycan-binding proteins (GBPs) with glycans are essential in cell adhesion, bacterial/viral infection, and cellular signaling pathways. Experimental characterization of these interactions based on glycan microarrays typically involves (1) labeling GBPs directly with fluorescent reagents before incubation with the microarrays, or (2) labeling GBPs with biotin before the incubation and detecting the captured GBPs after the incubation using fluorescently labeled streptavidin, or (3) detecting the captured GBPs after the incubation using fluorescently labeled antibodies raised against the GBPs. The fluorescent signal is mostly measured ex situ after excess fluorescent materials are washed off. In this study, by using a label-free optical scanner for glycan microarray detection, we measured binding curves of 7 plant lectins to 24 glycans: four β1-4-linked galactosides, three β1-3-linked galactosides, one β-linked galactoside, one α-linked N-acetylgalactosaminide, eight α2-3-linked sialosides, and seven α2-6-linked sialosides. From association and dissociation constants deduced by global-fitting the binding curves, we found that (1) labeling lectins directly with fluorescent agents change binding profiles of lectins, in some cases by orders of magnitude; (2) those lectin-glycan binding reactions characterized with large dissociation rates, though biologically relevant, are easily missed or deemed insignificant in ex situ fluorescence-based assays as most captured lectins are washed off before detection. This study highlights the importance of label-free real-time detection of protein-ligand interactions and the potential pitfall in interpreting fluorescence-based assays for characterization of protein-glycan interactions.     

Natural glycan microarrays

Glycan microarrays are emerging as increasingly used screening tools with a high potential for unraveling protein–carbohydrate interactions: probing hundreds or even thousands of glycans in parallel, they provide the researcher with a vast amount of data in a short time-frame, while using relatively small amounts of analytes. Natural glycan microarrays focus on the glycans’ repertoire of natural sources, including both well-defined structures as well as still-unknown ones. This article compares different natural glycan microarray strategies. Glycan probes may comprise oligosaccharides from glycoproteins as well as glycolipids and polysaccharides. Oligosaccharides may be purified from scarce biological samples that are of particular relevance for the carbohydrate-binding protein to be studied. We give an overview of strategies for glycan isolation, derivatization, fractionation, immobilization and structural characterization. Detection methods such as fluorescence analysis and surface plasmon resonance are summarized. The importance of glycan density and multivalency is discussed. Furthermore, some applications of natural glycan microarrays for studying lectin and antibody binding are presented.     

Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines

Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan-binding lectins. The screening of breast cancer MDA-MB435 cells, cervical cancer HeLa cells and colon cancer Caco-2, HCT116 and HCT116-FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4-deoxy-4-guanidino-Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell-lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan-lectin interactions that are exhibited by immortalised cell lines.     

A novel strategy for mammalian cell surface glycome profiling using lectin microarray

The glycome represents the total set of glycans expressed in a cell. The glycome has been assumed to vary between cell types, stages of development and differentiation, and during malignant transformation. Analysis of the glycome provides a basis for understanding the functions of glycans in these cellular processes. Recently, a technique called lectin microarray was developed for rapid profiling of glycosylation, although its use was mainly restricted to glycoproteins of cell lysates, and thus unable to profile the intact cell surface glycans. Here we report a simple and sensitive procedure based on this technology for direct analysis of the live mammalian cell-surface glycome. Fluorescent-labeled live cells were applied in situ to the established lectin microarray consisting of 43 immobilized lectins with distinctive binding specificities. After washing, bound cells were directly detected by an evanescent-field fluorescence scanner in a liquid phase without fixing and permeabilization. The results obtained by differential profiling of CHO and its glycosylation-defective mutant cells, and splenocytes of wild-type and beta1-3-N-acetylglucosaminyltransferase II knockout mice performed as model experiments agreed well with their glycosylation phenotypes. We also compared cell surface glycans of K562 cells before and after differentiation and found a significant increase in the expression of O-glycans on differentiated cells. These results demonstrate that the technique provides a novel strategy for profiling global changes of the mammalian cell surface glycome.     

Differing lectin binding profiles among human embryonic stem cells and derivatives aid in the isolation of neural progenitor cells

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells.     

Lectin microarray profiling of metastatic breast cancers

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 μm section of a primary breast tumor or <1 μL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.     

Natural glycan microarrays

Glycan microarrays are emerging as increasingly used screening tools with a high potential for unraveling protein-carbohydrate interactions: probing hundreds or even thousands of glycans in parallel, they provide the researcher with a vast amount of data in a short time-frame, while using relatively small amounts of analytes. Natural glycan microarrays focus on the glycans' repertoire of natural sources, including both well-defined structures as well as still-unknown ones. This article compares different natural glycan microarray strategies. Glycan probes may comprise oligosaccharides from glycoproteins as well as glycolipids and polysaccharides. Oligosaccharides may be purified from scarce biological samples that are of particular relevance for the carbohydrate-binding protein to be studied. We give an overview of strategies for glycan isolation, derivatization, fractionation, immobilization and structural characterization. Detection methods such as fluorescence analysis and surface plasmon resonance are summarized. The importance of glycan density and multivalency is discussed. Furthermore, some applications of natural glycan microarrays for studying lectin and antibody binding are presented.     

Glycoprofiling with micro-arrays of glycoconjugates and lectins

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.     

Exquisite specificity of mitogenic lectin from <Emphasis Type="Italic">Cephalosporium curvulum</Emphasis> to core fucosylated N-glycans

Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.     

Ligand Binding and Signaling of Dendritic Cell Immunoreceptor (DCIR) Is Modulated by the Glycosylation of the Carbohydrate Recognition Domain

C-type lectins are innate receptors expressed on antigen-presenting cells that are involved in the recognition of glycosylated pathogens and self-glycoproteins. Upon ligand binding, internalization and/or signaling often occur. Little is known on the glycan specificity and ligands of the Dendritic Cell Immunoreceptor (DCIR), the only classical C-type lectin that contains an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). Here we show that purified DCIR binds the glycan structures Lewis^b and Man₃. Interestingly, binding could not be detected when DCIR was expressed on cells. Since DCIR has an N-glycosylation site inside its carbohydrate recognition domain (CRD), we investigated the effect of this glycan in ligand recognition. Removing or truncating the glycans present on purified DCIR increased the affinity for DCIR-binding glycans. Nevertheless, altering the glycosylation status of the DCIR expressing cell or mutating the N-glycosylation site of DCIR itself did not increase glycan binding. In contrast, cis and trans interactions with glycans induced DCIR mediated signaling, resulting in a decreased phosphorylation of the ITIM sequence. These results show that glycan binding to DCIR is influenced by the glycosylation of the CRD region in DCIR and that interaction with its ligands result in signaling via its ITIM motif.     

Tuning specificity and topology of lectins through synthetic biology

Lectins are non-immunoglobulin and non-catalytic glycan binding proteins that are able to decipher the structure and function of complex glycans. They are widely used as biomarkers for following alteration of glycosylation state in many diseases and have application in therapeutics. Controlling and extending lectin specificity and topology is the key for obtaining better tools. Furthermore, lectins and other glycan binding proteins can be combined with additional domains, providing novel functionalities. We provide a view on the current strategy with a focus on synthetic biology approaches yielding to novel specificity, but other novel architectures with novel application in biotechnology or therapy.     

Differential recognition of natural and remodeled glycotopes by three Diocleae lectins

The carbohydrate specificities of Dioclea grandiflora lectins DGL-I¹ and DGL-II, and Galactia lindenii lectin II (GLL-II) were explored by use of remodeled glycoproteins as well as by the lectin hemagglutinating activity against erythrocytes from various species with different glycomic profiles. The three lectins exhibited differences in glycan binding specificity but also showed overlapping recognition of some glycotopes (i.e. Tα glycotope for the three lectins; IIβ glycotope for DGL-II and GLL-II lectins); in many cases the interaction with distinct glycotopes was influenced by the structural context, i.e., by the neighbouring sugar residues. Our data complement and expand the existing knowledge about the binding specificity of these three Diocleae lectins, and taken together with results of previous studies, allow us to suggest a functional map of the carbohydrate recognition which illustrate the impact of modification of basic glycotopes enhancing, permiting, or inhibiting their recognition by each lectin.