Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase
Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase
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DOI：
10.2337/diabetes.49.10.1627
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发表时间：
2000-10-01
期刊：
影响因子：
7.7
通讯作者：
Van Schaftingen, E
中科院分区：
文献类型：
--
作者：
Delpierre, G;Rider, MH;Van Schaftingen, E
果糖胺被认为在糖尿病并发症的发展中起着重要作用。除了最近有迹象表明这些化合物可以转化为3-磷酸果糖胺外，人们对这些化合物在哺乳动物组织中代谢的反应知之甚少。本工作的目的是确定和表征负责这种转化的酶。红细胞提取物可催化人工合成的果糖胺1-脱氧-1-吗啉果糖的磷酸化，该酶经Blue Sepharose、Q Sepharose和Sephacryl S-200柱层析纯化约2,500倍，并与35,000-M-r蛋白交配。用纳米电喷雾电离质谱仪从该蛋白质中提取部分胰蛋白酶多肽序列，从而能够识别相应的人和小鼠的cDNA。这两个基因编码309个氨基酸的蛋白质，与几个细菌基因组序列预测的未知功能蛋白质有89%的同源性，并在大肠杆菌中表达和纯化。它们按亲和力递减的顺序催化DMF、果糖赖氨酸、果糖甘氨酸和果糖的磷酸化。对磷酸化的DMF和磷酸化的果糖甘氨酸的核磁共振分析表明，磷酸结合在1-脱氧果糖部分的第三个碳上，果糖胺-3-激酶的生理功能可能是启动导致果糖赖氨酸和糖化蛋白脱糖的过程。
Fructosamines are thought to play an important role in the development of diabetic complications. Little is known about reactions that could metabolize these compounds in mammalian tissues, except for recent indications that they can be converted to fructosamine 3-phosphates. The purpose of the present work was to identify and characterize the enzyme responsible for this conversion. Erythrocyte extracts were found to catalyze the ATP-dependent phosphorylation of 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine, The enzyme responsible for this conversion was purified similar to 2,500-fold by chromatography on Blue Sepharose, Q Sepharose, and Sephacryl S-200 and shown to copurify with a 35,000-M-r protein. Partial sequences of tryptic peptides were derived from the protein by nanoelectrospray-ionization mass spectrometry, which allowed for the identification of the corresponding human and mouse cDNAs. Both cDNAs encode proteins of 309 amino acids, showing 89% identity with each other and homologous to proteins of unknown function predicted from the sequences of several bacterial genomes, Both proteins were expressed in Escherichia coli and purified. They were shown to catalyze the phosphorylation of DMF, fructoselysine, fructoseglycine, and fructose in order of decreasing affinity. They also phosphorylated glycated lysozyme, though not unmodified lysozyme, Nuclear magnetic resonance analysis of phosphorylated DMF and phosphorylated fructoseglycine showed that the phosphate was bound to the third carbon of the 1-deoxyfructose moiety, The physiological function of fructosamine-3-kinase may be to initiate a process leading to the deglycation of fructoselysine and of glycated proteins.