Earticle

Carbohydrate microarray might have powerful potential for assaying substrate specificity or activity of carbohydrate-processing enzymes such as glycosyltransferase and glycosidase. However, carbohydrate microarray system has been rarely utilized because complex chemical reactions are demanded for microarray preparation. Recently, we have been developed a simple and cost-effective method for fabrication of carbohydrate microarray which uses the reaction of amine-conjugated carbohydrates with glass surface. In the present work, we employed the developed carbohydrate microarray system to rapidly determine substrate specificity or activity of glycosyltransferase. Pasteurella multocidaoriginated sialyltransferase was used as a target glycosyltransferase because it is a wonderful target to prove the potential of carbohydrate microarray. Collectively, the functional carbohydrate microarray would be a powerful analytical tool to screen substrate specificity or other properties of carbohydrate-processing enzymes.

Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by the ectopic expression of the four reprogramming transcription factors (OCT4, SOX2, c-MYC and KLF4). Although several minor differences between iPSCs and embryonic stem cells (ESCs) have been reported, they share lots of properties including the surface markers for their identifications. Several glycomics studies have been already carried out to elucidate the glycan profiles of ESCs. However, no investigation about the glycosylation patterns of iPSCs has been reported yet. In the present study, we investigated the changes of N-glycosylation patterns induced by the introduction of four reprogramming transcription factors. After each reprogramming factor was introduced into human GP2-293 cells using a retroviral vector, its profile of N-glycans obtained from whole cell lysates was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the effects caused by three different combinatorial introductions of reprogramming factors (OCT4/SOX2, OCT4/SOX2/KLF4, and OCT4/SOX2/KLF4/c-MYC) were also investigated. Also, quantitative real-time polymerase chain reactions were performed to analyze the expression levels of glycosyltransferase genes related with the changed glycan structures. Our results suggested that the portions of sialylated complex-type glycans attached with fucose were increased in the cells transfected with reprogramming factors including OCT4 or SOX2. Through this study, we sought to predict glycosylation patterns changed by the introduction of reprogramming factors, which would contribute to discover novel glycan markers specific for pluripotent stem cells.