An amino acid region at the N-terminus of rat hepatoma alpha1-->2 fucosyltransferase modulates enzyme activity and interaction with lipids: strong preference for glycosphingolipids containing terminal Galbeta1-->3GalNAc-structures.
An amino acid region at the N-terminus of rat hepatoma alpha1-->2 fucosyltransferase modulates enzyme activity and interaction with lipids: strong preference for glycosphingolipids containing terminal Galbeta1-->3GalNAc-structures.
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大鼠肝癌 α1-->2 岩藻糖基转移酶 N 末端的氨基酸区域调节酶活性以及与脂质的相互作用：强烈偏好含有末端 Galbeta1-->3GalNAc 结构的鞘糖脂。
DOI：
10.1021/bi0102104
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Holmes,EH
中科院分区：
文献类型：
--
作者：
Sherwood,AL;Stroud,MR;Levery,SB;Holmes,EH
在Fischer 344大鼠化学性肝癌发生的早期阶段，在饲料中添加0.03%N-2-乙酰氨基芴（AAF）诱导了肝实质细胞中的GDP-岩藻糖：GM 1 α1→2岩藻糖基转移酶（FucT）。这种酶在正常大鼠肝组织中检测不到，但在许多大鼠肝癌细胞系中高度表达，包括大鼠肝癌H35细胞。研究了天然大鼠肝癌H35细胞α1→2FucT、表达的重组全长H35细胞α1→2FucT和N端缺失前27个氨基酸残基的截短形式（包括酶的胞质和跨膜结构域）的酶性质和受体特异性。结果表明，重组全长酶的比活性比截短的酶高80倍以上。天然和重组全长酶在不存在去污剂或磷脂的情况下显示出显著的活性，并且在Triton CF-54去污剂存在下显示出最佳活性。截短的酶被CHAPSO最佳活化，在其不存在时几乎没有活性。这些研究结果与先前的研究一致，证明了该酶的最佳活性需要一个无菌环境，并表明N-末端跨膜结构域在维持活性构象或允许与受体糖脂有效相互作用中是重要的。全长和截短的酶不仅将岩藻糖转移到GM 1和去唾液酸GM 1（Gg 4），而且还转移到半乳糖基球糖苷（Gb 5）。观察到向乳糖和新乳糖系列受体的微弱或不可检测的转移，表明对末端Galβ1→3GalNAc-结构的强烈偏好。表达的重组全长α1→2FucT产生的两种反应产物（岩藻糖基-GM 1和岩藻糖基-Gb 5）的结构通过1H-NMR光谱分析明确证实。
A GDP-fucose:GM1α1→2 fucosyltransferase (FucT) is induced during early stages of chemical hepatocarcinogenesis in parenchymal cells of Fischer 344 rats fed a diet supplemented with 0.03%N-2-acetylaminofluorene (AAF). This enzyme is undetectable in normal rat liver tissues but is highly expressed in many rat hepatoma cell lines, including rat hepatoma H35 cells. Enzymatic properties and acceptor specificity of native rat hepatoma H35 cell α1→2FucT, expressed recombinant full-length H35 cell α1→2FucT, and a truncated form missing the first 27 amino acid residues from the N-terminus, comprising the cytoplasmic and transmembrane domains of the enzyme, were studied. The results indicate that the recombinant full-length enzyme has a specific activity over 80-fold higher than the truncated enzyme. Both the native and recombinant full-length enzymes display significant activity in the absence of detergent or phospholipid and optimal activity in the presence of Triton CF-54 detergent. The truncated enzyme is optimally activated by CHAPSO, showing little activity in its absence. These findings are in agreement with previous studies demonstrating a requirement of a lipidic environment for optimal activity with this enzyme and suggest that the N-terminal transmembrane domain is important either in the maintenance of an active conformation or in allowing efficient interaction with acceptor glycolipids. Both the full-length and truncated enzymes transfer fucose not only to GM1and asialo-GM1(Gg4) but also to galactosyl globoside (Gb5) as well. Weak or undetectable transfer to lacto- and neolacto-series acceptors was observed, demonstrating a strong preference for terminal Galβ1→3GalNAc- structures. The structures of two reaction products generated by expressed recombinant full-length α1→2FucT, which are known to be important tumor-associated antigens (fucosyl-GM1and fucosyl-Gb5), were unambiguously confirmed by1H-NMR spectral analysis.