Characterization Of HmFucT:an α1,2-fucosyltransferase From Helicobacter Mustelae And Synthesis Of H-type Ⅴ Blood Group Antigen

Fucosylation of the oligosaccharides and proteins is through glycosyltransferases, which transfer fucoses to the oligosaccharides or proteins. The fucosylation structure plays important roles in a variety of physiological and pathological processes of eukaryotes, including neural development, fertilization, cell adhesion, inflammation reactions and tumor metastasis and so on. For example, fucosylation occurs during the processes of the formation of ABO blood antigens, and they are important for the human physiology. Athough fucosylation occurs not so widespread in prokaryotes, compared with eukaryotes. Fucosylation occurs in gram negative bacteria’s LPS and associated with the formation of the o-antigen. O-antigen is the important compontent of the LPS, and is related with the effective colonization of the host and the resistance to the immune responses of the host.FucTs transfer L-Fucose from GDP-Fucose to the corresponding receptors to form α1,2, a1,3, a1,4, a1,6connection. According to the similarity of the amino acid sequence, a1,2FucTs belongs to glycosytransferase family11, and a1,2-FucTs from Human, other manmals, viruses, plants, and bacteria also belong to the family. Several of the genes responsible for eukaryotic a1,2-FucTs have been cloned and characterized. Compared with their mamamalian counterparts, bacterial glycosytransferase are more easily overexpressed as soluble and active forms without complicated gene manipulation techniques. Furthermore, they appear to have broader substrate specificity. Therefore, explore and find more bacterial glycosytransferase has the very good industrial prospect.We study a kind of α1,2-FucTs from Helicobacter mustelae, Helicobacter mustelae belongs to Helicobacter, and is from the stomach of the ferret. And it is the second member. The first one is Helicobacter Pylori, which is separated from the stomach of human. Helicobacter mustelae is asscoiated with chronic gastritis, and can be the mode of pathogenic strains. Some study showed that Helicobacter mustelae expresses human blood group A epitope. On the analysis of sequence of information of Helicobacter mustelae, Nozomu Kamada found a DNA encoding the protein having al,2-FucT activity.By constructing different expression plasmids, at last, the soluble expression level was enhanced by fusion with GST. Ninety percent purity of GST-HmFucT fusion protein was obtained via GST affinity chromatography. Inaddition, results showed that HmucT’s activity was independent of divalent metal ions such as Mn2+, this result showed that HmFucT belongs to GTB family. And we use GST-HmFucT to synthsis H-type V antigen in mg-scale. And we find a method that sperate Fucα1,2lactose-S-ph and lactose-S-ph by HPLC, this method laid the foundation for the research of the Characteriaztion of HmFucT.