Lectin-Based Protein Microarray for the Glycan Analysis of Colorectal Cancer Biomarkers: The Insulin-Like Growth Factor System.

Lectin-based protein microarrays are used for glycoprofiling of various kinds of biological samples. Here we describe lectin-based microarray assay in the reverse-phase format where glycoprotein samples are spotted onto microarray slide and then are incubated with set of lectins. This configuration allows high-throughput screening of a large cohort of samples by a set of lectins without need of separation of glycans from glycoproteins. We applied the described method for glycan analysis of glycoprotein biomarkers of colorectal cancer associated with the insulin-like growth factor system.

Sensitive glycoprofiling of insulin-like growth factor receptors isolated from colon tissue of patients with colorectal carcinoma using lectin-based protein microarray.

Glycosylation of cell receptors influences their function and development of tumour induces changes in glycosylation. Cell growth depends on the activation of receptors which bind growth factors and the insulin-like growth factor (IGF) receptors are among the most important ones. Usually, only small quantities of isolated receptors are available thus there is a need of suitable assay to study receptors glycosylation. Therefore, we developed a lectin-based reverse-phase protein microarray method for screening the glycosylation pattern of receptors in picomolar (pM) concentrations. The method was applied to glycoprofile IGF1 and IGF2 receptors and the solubilised membrane proteins isolated from tumour and non-tumour colon tissue of patients with colorectal cancer. We found that common to both receptors was partial overlapping of the major glycan structures with those present in the entire glycome of membrane proteins. In contrast, receptors possess higher level of α2,3 sialic acid residues and lower level of tri-/tetra-antennary complex type N-glycans and terminal mannose in high-mannose structures. Increased levels of fucosylation and branched mannose structures were observed in both receptors derived from tumour tissue compared to non-tumour tissue. The described method enabling glycan analysis of receptors has a big application potential in e.g. biomarker research, biology and diagnostics.

Glycoanalysis of the placental membrane glycoproteins throughout placental development.

Structural changes of glycans are observed in different (patho)physiological conditions. Human placental membrane (glyco)proteins were isolated from the first and third trimester placentas of mothers at different ages. By using lectin microarray, we demonstrated that the placental membrane N-glycome contains several N-glycan groups: high mannose, asialylated and sialylated biantennary moieties, bisected, core fucosylated, fucosylated at other positions (bearing terminal and/or antennary Fuc), α2-6 and α2-3 sialylated structures. Employing MALDI-TOF MS enabled identification of over sixty different N-glycan structures in all samples, with 17 moieties exceeding the relative abundance of 2%. The major MS peaks originated from: 1) biantennary complex type N-glycan with a bisecting GlcNAc residue and 2) a core Fuc paucimannosidic and high mannose type structures M3-M9. Age of mothers and the stage of placental development affected N-glycome. The work presented in this article is the first comprehensive mass spectrometric study of the N-glycome of human placental membrane proteins. Our results may be seen as the baseline which can serve for future MALDI MS profiling of the placental membrane N-glycome in different pathophysiological conditions.

Diagnostic Potential of Transferrin Glycoforms-A Lectin-Based Protein Microarray Approach.

PURPOSE: Disease or a specific condition may cause alteration of human transferrin (hTf) glycosylation pattern. A specific analytical platform, lectin-based protein microarray, is designed and optimized for the investigation of hTf glycans, attached to the protein core in their native form. EXPERIMENTAL DESIGN: hTf molecules isolated from healthy persons of different age, diabetes mellitus type 2 (T2DM) or colorectal carcinoma (CRC) patients are used for method validation. Reliability of the results is ensured by three criteria for the evaluation of hTf-lectin interactions: i) signal-to-noise ratio above 3, ii) signal intensity above 250 arbitrary units, and iii) hTf concentration ensuring high sensitivity of the assay. RESULTS: Six lectins, out of fourteen tested, satisfy the criteria. hTf is spotted at concentration of 50 µg mL-L . When physiological samples (isolated hTf) are analyzed, the highest potential to differentiate between population groups expresses Aleuria aurantia (AAL), Triticum vulgaris (WGA) and Phaseolus vulgaris (PHA-E) lectins. The initial amount of hTf which can be analyzed is very low (75 pg). CONCLUSION AND CLINICAL RELEVANCE: Results confirm that a very sensitive, high-throughput lectin-based protein microarray platform can be formulated to detect changes in hTf glycan structures which can be considered as biomarkers of ageing or a disease.

Changes Due to Ageing in the Glycan Structure of Alpha-2-Macroglobulin and Its Reactivity with Ligands.

Alpha-2-macroglobulin (α2M) is a molecule generally associated with inflammation, and chronic inflammation is associated with ageing and cancer. The degree of inflammation was recently proposed to be considered as a biomarker of biological ageing. In this study, glycans attached to α2M were analysed in a human population of different ages by lectin-based protein microarray. Higher reactivity of α2M with several lectins was detected in older individuals indicating an increased content of specific monosaccharides: α2,6 sialic acid, mannose and N-acetylglucosamine, and multiantennary complex type N-glycans. The increased glycosylation of α2M was accompanied by reduced binding of Zn ions and insulin-like growth factor-binding protein 2 (IGFBP-2). Glycosylation of α2M and its reactivity with IGFBP-2 is similarly affected by ageing and incidence of colon cancer, but the reactivity of α2M with Zn ions is differently affected, as the binding of Zn ions remains unaltered in patients with colon cancer compared to healthy middle-aged individuals. Thus, the binding of IGFBP-2 to α2M seems to be related to structural changes in the glycan moieties of α2M, whereas binding of Zn ions, most likely, is not.

Structural changes of fibrinogen as a consequence of cirrhosis.

Cirrhosis is a disease which may develop as a consequence of various conditions. In advanced liver disease, blood coagulation can be seriously affected. Portal hypertension, vascular abnormalities and/or a dysbalance in coagulation factors may result in bleeding disorders or in the development of thrombosis. Fibrinogen is the main protein involved in clot formation and wound healing. The aim of this work was to analyse the glycosylation pattern of the isolated fibrinogen molecules by lectin-based protein microarray, together with the carbonylation pattern of the individual fibrinogen chains, possible changes in the molecular secondary and tertiary structure and reactivity with the insulin-like growth factor-binding protein 1 (IGFBP-1) in patients with cirrhosis. The results pointed to an increase in several carbohydrate moieties: tri/tetra-antennary structures, Gal ß-1,4 GlcNAc, terminal α-2,3 Sia and α-1,3 Man, and a decrease in core α-1,6 Fuc and bi-antennary galactosylated N-glycans with bisecting GlcNAc. Fibrinogen Aα chain was the most susceptible to carbonylation, followed by the Bß chain. Cirrhosis induced additional protein carbonylation, mostly on the α chain. Spectrofluorimetry and CD spectrometry detected reduction in the α-helix content, protein unfolding and/or appearance of modified amino acid residues in cirrhosis. The amount of complexes which fibrinogen forms with IGFBP-1, another factor involved in wound healing was significantly greater in patients with cirrhosis than in healthy individuals. A more detailed knowledge of individual molecules in coagulation process may contribute to deeper understanding of coagulopathies and the results of this study offer additional information on the possible mechanisms involved in impaired coagulation due to cirrhosis.

Structural and functional changes of fibrinogen due to aging.

Different factors affect coagulation process. Since fibrinogen is the main coagulation factor, the influence of aging on fibrinogen structure and function was investigated in this study. Fibrinogen was isolated from plasma obtained from healthy persons in the age range 21-83 and examined. Lectin microarray analysis demonstrated increased glycosylation of fibrinogen due to aging, with predominant increase in high-mannose or hybrid type N-glycans, as well as tri-/tetraantennary complex N-glycans with greater content of galactose and N-acetylglucosamine residues. Spectrofluorimetric analysis indicated that fibrinogen molecules have more densely packed structure, but there are no additional advanced glycation end products with increasing age. According to the results of functional analysis, fibrinogen molecules isolated from older persons exhibited reduced clotting time, with significant positive correlation with age, but there were no differences in clotting speed, maximal optical density of fibrin clot, diameter of fibrin fibres, fibrin porosity or reactivity with the insulin-like growth factor binding protein 1. Glycosylation changes of fibrinogen in healthy aging most likely affect its structure and function, namely clotting time. Structural and functional studies of proteins in relation to healthy aging contribute to deeper understanding of mechanisms responsible for longevity.