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Biomedical subjects

Yoichiro Koshi

Publications and source records attributed to Yoichiro Koshi.

3 recordsLinked to original sources

A fluorescent lectin array using supramolecular hydrogel for simple detection and pattern profiling for various glycoconjugates.

Because sugar and its derivatives play important roles in various biological phenomena, the rapid and high-throughput analysis of various glycoconjugates is keenly desirable. We describe herein the construction of a novel fluorescent lectin array for saccharide detection using a supramolecular hydrogel matrix. In this array, the fluorescent lectins were noncovalently fixed under semi-wet conditions to suppress the protein denaturation. It is demonstrated by fluorescence titration and fluorescence lifetime experiments that the immobilized lectins act as a molecular recognition scaffold in the hydrogel matrix, similar to that in aqueous solution. That is, a bimolecular fluorescence quenching and recovery (BFQR) method can successfully operate under both conditions. This enables one to fluorescently read-out a series of saccharides on the basis of the recognition selectivity and affinity of the immobilized lectins without tedious washing processes and without labeling the target saccharides. Simple and high-throughput sensing and profiling were carried out using the present lectin array for diverse glycoconjugates, which not only included a simple glucose, but also oligosaccharides, and glycoproteins, and, furthermore, the pattern recognition and profiling of several types of cell lysates were also accomplished.

Fluorescent Dyes↗

Double-modification of lectin using two distinct chemistries for fluorescent ratiometric sensing and imaging saccharides in test tube or in cell.

The site-selective incorporation of two different fluorophores into a naturally occurring protein (lectin, a sugar-binding protein) has been successfully carried out using two distinct orthogonal chemical methods. By post-photoaffinity labeling modification, Con A, a glucose- and mannose-selective lectin, was modified with fluorescein in the proximity of the sugar binding site (Tyr100 site), and the controlled acylation reaction provided the site-selective attachment of coumarin at Lys114. In this doubly modified Con A, the fluorescein emission changed upon the binding to the corresponding sugars, such as the glucose or mannose derivatives, whereas the coumarin emission was constant. Thus, the doubly modified Con A fluorescently sensed the glucose- and mannose-rich saccharides in a ratiometric manner while retaining the natural binding selectivity and affinity, regardless of the double modification. On the benefit of the ratiometric fluorescent analysis using two distinct probes, the sugar trimming process of a glycoprotein can be precisely monitored by the engineered Con A. Furthermore, the doubly modified Con A can be used not only for the convenient fluorescent imaging of saccharides localized on a cell surface, such as the MCF-7, a breast cancer cell having rich high-mannose branch, but also for the ratiometric fluorescent sensing of the glucose concentration inside HepG2 cells. These results demonstrated that the semisynthetic lectin modified doubly by two distinct chemistries is superior to the singly modified one in function, and thus, it may be potentially useful in cell, as well as in test tube.

Biosensing Techniques↗