Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences ...in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3- and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.
The ability to selectively detect and target cancer cells that have undergone an epithelial-mesenchymal transition (EMT) may lead to improved methods to treat cancers such as pancreatic cancer. The ...remodeling of cellular glycosylation previously has been associated with cell differentiation and may represent a valuable class of molecular targets for EMT.
As a first step toward investigating the nature of glycosylation alterations in EMT, we characterized the expression of glycan-related genes in three in-vitro model systems that each represented a complementary aspect of pancreatic cancer EMT. These models included: 1) TGFβ-induced EMT, which provided a look at the active transition between states; 2) a panel of 22 pancreatic cancer cell lines, which represented terminal differentiation states of either epithelial-like or mesenchymal-like; and 3) actively-migrating and stationary cells, which provided a look at the mechanism of migration. We analyzed expression data from a list of 587 genes involved in glycosylation (biosynthesis, sugar transport, glycan-binding, etc.) or EMT. Glycogenes were altered at a higher prevalence than all other genes in the first two models (p<0.05 and <0.005, respectively) but not in the migration model. Several functional themes were shared between the induced-EMT model and the cell line panel, including alterations to matrix components and proteoglycans, the sulfation of glycosaminoglycans; mannose receptor family members; initiation of O-glycosylation; and certain forms of sialylation. Protein-level changes were confirmed by Western blot for the mannose receptor MRC2 and the O-glycosylation enzyme GALNT3, and cell-surface sulfation changes were confirmed using Alcian Blue staining.
Alterations to glycogenes are a major component of cancer EMT and are characterized by changes to matrix components, the sulfation of GAGs, mannose receptors, O-glycosylation, and specific sialylated structures. These results provide leads for targeting aggressive and drug resistant forms of pancreatic cancer cells.
We developed a practical strategy for serum protein profiling using antibody microarrays and applied the method to the identification of potential biomarkers in prostate cancer serum. Protein ...abundances from 33 prostate cancer and 20 control serum samples were compared to abundances from a common reference pool using a two‐color fluorescence assay. Robotically spotted microarrays containing 184 unique antibodies were prepared on two different substrates: polyacrylamide based hydrogels on glass and poly‐1‐lysine coated glass with a photoreactive cross‐linking layer. The hydrogel substrate yielded an average six‐fold higher signal‐to‐noise ratio than the other substrate, and detection of protein binding was possible from a greater number of antibodies using the hydrogels. A statistical filter based on the correlation of data from “reverse‐labeled” experiment sets accurately predicted the agreement between the microarray measurements and enzyme‐linked immunosorbent assay measurements, showing that this parameter can serve to screen for antibodies that are functional on microarrays. Having defined a set of reliable microarray measurements, we identified five proteins (von Willebrand Factor, immunoglobulinM, Alpha1‐antichymotrypsin, Villin and immunoglobulinG) that had significantly different levels between the prostate cancer samples and the controls. These developments enable the immediate use of high‐density antibody and protein microarrays in biomarker discovery studies.
Patients with pemphigus vulgaris (PV) harbor antibodies reactive against self-antigens expressed at the surface of keratinocytes, primarily desmoglein (Dsg) 3 and, to a lesser extent, Dsg1. ...Conventionally, only antibodies targeting these molecules have been thought to contribute to disease pathogenesis. This notion has been challenged by a growing pool of evidence that suggests that antibodies toward additional targets may play a role in disease. The aims of this study were to (i) establish high-throughput protein microarray technology as a method to investigate traditional and putative autoantibodies (autoAbs) in PV and (ii) use multiplexed protein array technology to define the scope and specificity of the autoAb response in PV. Our analysis demonstrated significant IgG reactivity in patients with PV toward the muscarinic acetylcholine receptor subtypes 3, 4, and 5 as well as thyroid peroxidase. Furthermore, we found that healthy first- and second-degree relatives of patients with PV express autoAbs toward desmoglein and non-Dsg targets. Our analysis also identified genetic elements, particularly HLA, as key drivers of autoAb expression. Finally, we show that patients with PV exhibit significantly reduced IgM reactivity toward disease-associated antigens relative to controls. The use of protein microarrays to profile the autoAb response in PV advanced the current understanding of disease and provided insight into the complex relationship between genetics and disease development.
Molecular indicators to specify the risk posed by a pancreatic cyst would benefit patients. Previously we showed that most cancer-precursor cysts, termed mucinous cysts, produce abnormal glycoforms ...of the proteins MUC5AC and endorepellin. Here we sought to validate the glycoforms as a biomarker of mucinous cysts and to specify the oligosaccharide linkages that characterize MUC5AC. We hypothesized that mucinous cysts secrete MUC5AC displaying terminal N-acetylglucosamine (GlcNAc) in either alpha or beta linkage. We used antibody-lectin sandwich assays to detect glycoforms of MUC5AC and endorepellin in cyst fluid samples from three independent cohorts of 49, 32, and 66 patients, and we used monoclonal antibodies to test for terminal, alpha-linked GlcNAc and the enzyme that produces it. A biomarker panel comprising the previously-identified glycoforms of MUC5AC and endorepellin gave 96%, 96%, and 87% accuracy for identifying mucinous cysts in the three cohorts with an average sensitivity of 92% and an average specificity of 94%. Glycan analysis showed that MUC5AC produced by a subset of mucinous cysts displays terminal alpha-GlcNAc, a motif expressed in stomach glands. The alpha-linked glycoform of MUC5AC was unique to intraductal papillary mucinous neoplasms (IPMN), whereas terminal beta-linked GlcNAc was increased in both IPMNs and mucinous cystic neoplasms (MCN). The enzyme that synthesizes alpha-GlcNAc, A4GNT, was expressed in the epithelia of mucinous cysts that expressed alpha-GlcNAc, especially in regions with high-grade dysplasia. Thus IPMNs secrete a gastric glycoform of MUC5AC that displays terminal alpha-GlcNAc, and the combined alpha-GlcNAc and beta-GlcNAc glycoforms form an accurate biomarker of mucinous cysts.
Glycan-binding proteins are commonly used as analytical reagents to detect the levels of specific glycan structures in biological samples. A detailed knowledge of the specificities of glycan-binding ...proteins is required for properly interpreting their binding data. A powerful technology for characterizing glycan-binding specificity is the glycan array. However, the interpretation of glycan-array data can be difficult due to the complex fine specificities of certain glycan-binding proteins. We developed a systematic approach, called outlier-motif analysis, for extracting fine-specificity information from glycan-array data, and we applied the method to the study of four commonly used lectins: two mannose binders (concanavalin A and Lens culinaris) and two galactose binders (Bauhinia purpurea and peanut agglutinin). The study confirmed the known, primary specificity of each lectin and also revealed new insights into their binding preferences. Lens culinaris's main specificity may be non-terminal, α-linked mannose with a single linkage at its 2' carbon, which is more restricted than previous definitions. We found broader specificity for bauhinea purpurea (BPL) than previously reported, showing that BPL can bind terminal N-acetylgalactosamine (GalNAc) and penultimate β-linked galactose under certain limitations. Peanut agglutinin may bind terminal Galβ1,3Gal, a glycolipid motif, in addition to terminal Galβ1,3GalNAc, a common O-linked glycoprotein motif. These results could be used to more accurately interpret data obtained using these well-studied lectins. Furthermore, this study demonstrates a systematic and general approach for extracting fine-specificity information from glycan-array data.
A subset of pancreatic ductal adenocarcinomas (PDACs) is highly resistant to systemic chemotherapy, but no markers are available in clinical settings to identify this subset. We hypothesized that a ...glycan biomarker for PDACs called sialylated tumor-related antigen (sTRA) could be used for this purpose.
We tested for differences between PDACs classified by glycan expression in multiple systems: sets of cell lines, organoids, and isogenic cell lines; primary tumors; and blood plasma from human subjects.
The sTRA-expressing models tended to have stem-like gene expression and the capacity for mesenchymal differentiation, in contrast to the nonexpressing models. The sTRA cell lines also had significantly increased resistance to seven different chemotherapeutics commonly used against pancreatic cancer. Patients with primary tumors that were positive for a gene expression classifier for sTRA received no statistically significant benefit from adjuvant chemotherapy, in contrast to those negative for the signature. In another cohort, based on direct measurements of sTRA in tissue microarrays, the patients who were high in sTRA again had no statistically significant benefit from adjuvant chemotherapy. Furthermore, a blood plasma test for the sTRA glycan identified the PDACs that showed rapid relapse following neoadjuvant chemotherapy.
This research demonstrates that a glycan biomarker could have value to detect chemotherapy-resistant PDAC in clinical settings. This capability could aid in the development of stratified treatment plans and facilitate biomarker-guided trials targeting resistant PDAC.
Lectin–glycan interactions have critical functions in multiple normal and pathological processes, but the binding partners and functions for many glycans and lectins are not known. An important step ...in better understanding glycan–lectin biology is enabling systematic quantification and analysis of the interactions. Glycan arrays can provide the experimental information for such analyses, and the thousands of glycan array datasets available through the Consortium for Functional Glycomics provide the opportunity to extend the analyses to a broad scale. We developed software, based on our previously described Motif Segregation algorithm, for the automated analysis of glycan array data, and we analyzed the entire storehouse of 2883 datasets from the Consortium for Functional Glycomics. We mined the resulting database to make comparisons of specificities across multiple lectins and comparisons between glycans in their lectin receptors. Of the lectins in the database, viral lectins were the most different from other organism types, with specificities nearly always restricted to sialic acids, and mammalian lectins had the most diverse range of specificities. Certain mammalian lectins were unique in their specificities for sulfated glycans. Simple modifications to a lactosamine core structure radically altered the types of lectins that were highly specific for the glycan. Unmodified lactosamine was specifically recognized by plant, fungal, viral, and mammalian lectins; sialylation shifted the binding mainly to viral lectins; and sulfation resulted in mainly mammalian lectins with the highest specificities. We anticipate that this analysis program and database will be valuable in fundamental glycobiology studies, detailed analyses of lectin specificities, and practical applications in translational research.
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.
Carbohydrates have fundamental roles throughout biology, yet they have not been as well studied as proteins and nucleic acids, in part due to limitations in the experimental tools. Improved methods ...for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents, such as lectins and glycan-binding antibodies, is a valuable complement to methods involving mass spectrometry and chromatography. This article addresses two limitations that have prevented the broader experimental use of glycan-binding proteins: sensitivity and availability. The sensitivity limitation stems from the poor affinity that many glycan-binding proteins have as isolated analytical reagents. To address this problem, I propose making use of multivalent interactions between lectins and glycans, mimicking those frequently found in the biological setting. Recent experiments show that a practical technique for producing lectin multimers can significantly improve detection sensitivity. The second limitation, availability, is the difficulty of finding and obtaining glycan-binding proteins that recognize less common or arbitrarily defined glycan structures. To address this problem, I propose translating the wealth of existing glycan array data into a quantitative, searchable database of the specificities of glycan-binding proteins. Such a resource would allow us to more easily identify proteins with defined specificities and perform detailed comparisons between reagents. Solutions to these two limitations could lead to the more effective use of, and a broader range of, glycan-binding reagents.
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