| Fucosyltransferase family is composed of a group of molecules which synthesize carbohydrate chains within various glycoproteins and glycolipids on cell surface. These enzymes are involved in various biological processes, including cell adhesion, lymphocyte homing, leukocyte trafficking, blood canalization, embryo development, etc. Fucosyltransferase is classified into four different subfamilies, the subfamily α1,2, subfamily al,3/4, subfamily a1,6, and subfamily POFUT according to their specific activities. Among these four subfamilies, the biggest subfamily a1,3/4consists of8FUT members, and has been identified as the key enzyme during the synthesis of Lewis antigens (SLex). It has been demonstrated that the expressional alteration of α1,3/4fucosyltransferase correlated with its roles in tumorigenesis, tumor invasion and metastasis.In the first section of this research, we restrict our focus on one α1,3/4fucosyltransferase, FUT6, involved in Hepatocellular Carcinoma (HCC). We investigated the expression pattern, biological function and molecular mechanism of FUT6through performing molecular and cellular experiments.By using quantitative real-time PCR, we firstly found that the-expression of FUT6gene is significantly increased in HCC tissues than that in corresponding nontumorous tissues. Western blot analysis further found that SLex, the product of FUT6, is also specifically up-regulated in HCC tissues. These findings first indicated the potential relationship between FUT6and HCC. To further study how FUT6is involved in hepatocarcinogenesis and tumor development, two representative HCC cell lines SMMC-7721and SK-Hepl were selected in the functional study of FUT6.After cells were transiently transfected with FUT6, the expression of exogenous FUT6correlated with downstream Slex antigens. And expression of FUT6in HCC cells was able to elevate the percentage of cells resting in S phase and inhibited the expression of cell cycle regulator protein p21. In the cell lines stably expressing FUT6, both cell growth ratio in dish and colony-forming ability in soft agar were elevated than those of control cells transfected with empty vector only. Expression of FUT6was also able to enhance the adhesiveness of HCC cells to extracellular matrix protein fibronectin. Moreover, cells stably expressing FUT6formed larger tumors than control cells in nude mice, exhibiting a faster growth ratio in vivo. In order to explore the molecular mechanism accounting for FUT6-mediated cell growth, we investigated a few signaling pathways in stable cell lines. Stable expression of FUT6led to a significant suppression of the expression of a few integrin subunits on cell surface (including integrin α5, β1, αv, β3), an promoted cytoplasmic Akt phosphorylation. A transcriptional suppression of cyclin-dependent kinase inhibitor p21, but not p27, was also observed in FUT6-expressing cells.Taken together, FUT6could promote HCC cell growth both in vitro and in vivo, correlated with its up-regulated expression pattern in HCC tissues. FUT6is likely to activate PI3K/Akt signaling pathway, suppress p21expression level to realize its biological functioin. These findings bring us new contents into current knowledgement of HCC and provide important experimental evidence in developing effective HCC markers and clinical therapeutic targets.In the second section of this work, we carried out a functional analysis of α1,3/4fucosyltransferase using bioinformatics’ methods. Due to the great differences in sequences, expression levels and biological functions among different family members, the findings from functional study of fucosyltransferase through molecular techniques remains rather limited. Analysis based on the conserved motifs of protein sequences may provide us an alternative and effective manner.Here we analyzed the sequences from each of the8FUTs belonging to α1,3/4fucosyltransferase subfamily from five selected species (Homo sapiens, Pan troglodytes, Mus musculus, Xenopus tropicalis, Caenorhabditis elegans). Aided with MEME, an analysis tool different from previous researchers, we found a new conserved motif in this enzyme group. On the basis of sequence alignment, functional site analysis and3D modeling, we hypothesized that this motif has a PKC phosphorylation site. Moreover, this new motif is prominently conserved, reflecting the diverse relationship among the family members.The conformation of constructed3D model showed that this motif is located at the center of donor binding region. It could access to the Cys142active site of donor-receptor binding by folding itself, and forms a ’pocket-like’ substrate binding structure. Based on the protein sequences, we constructed an evolutionary tree of all8members in this subfamily. The phylogeny relationship among each of these members is well consistent to the results from functional diversification presumed by Diverge software.Our finding of the new motif increased the number of the conserved motifs of this subfamily from5to6. We hypothesize that this new motif might play a crucial role in the functional diversification of α1,3/4fucosyltransferase subfamily as the active site of donor-acceptor binding region. Additionally, this motif is likely to be involved in the regulation of PKC signaling pathway. Our work provides a new way for the studies of the function and evolution of α1,3/4fucosyltransferase.
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