| Research Abstract |
Purpose : As recombinant proteins produced by the techniques of genetic engineering often have incomplete carbohydrate chains, there is a need to control artificially the structure of the carbohydrate chains as well as the process of genetic engineering. Therefore, using proteoglycan of extracellular matrix as a model, we have planned a series of studies aimed at the establishment of glycotechnology techniques of manipulate the glycosaminoglycans bearing physiological domain structures.
Experimental Methods and Results :
Enzymes that act on glycosaminoglycan chains, equivalent to the restriction enzymes and ligases used in genetic engineering, have not been available untill now. Therefore, we have focused on the transglycosylation reaction as a reverse reaction of the hydrolysis that is catalyzed by all glycosidases. Using chondroitin sulfate and hyaluronic acid as glycosaminoglycan chains, the optimum conditions for the transglycosylation reaction of testicular hyaluronidase as a repres
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entative endo-type glycosidase were investigated, and some conditions which efficiently accelerated the reaction were founded.
Subsequently, it was found that some oligosaccharides longer than octasaccharide labeled with a fluorescent compound (2-aminopyridine) at the reducing terminals were available as donors, whereas certain oligosaccharides longer than hexasaccharide, which were produced from 4-methylumbelliferyl-beta-D xyloside as a primer in the incubation medium of cultured human skin fibroblasts, were available as for acceptors.
After incubation of these donors and acceptors with hyaluronidase under the optimum conditions, it was observed that the chain length of glycosaminoglycans increased until at least docosaccharide, and that unnatural glycosaminoglycan chains, previously unknown, were synthesized as planned.
Discussion : The success of this random reconstruction method for glycosaminoglycan chains is considered to be a step foward the establishment of a protocol for glycotechnological procedures. Less
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