Allen T E, Ullman B
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098.
Nucleic Acids Res. 1993 Nov 25;21(23):5431-8. doi: 10.1093/nar/21.23.5431.
The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a M(r) = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21-23% amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into S phi 606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin.
布氏锥虫及相关寄生虫的次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（HGPRT）是治疗非洲昏睡病和其他几种寄生虫病的合理靶点。为了详细表征布氏锥虫HGPRT酶，在一个4.2 kb的SalI - KpnI基因组插入片段中分离出布氏锥虫hgprt并进行测序。核苷酸序列分析揭示了一个630 bp的开放阅读框，其编码一个210个氨基酸的蛋白质，分子量为23.4 kd。经缺口比对后，布氏锥虫HGPRT与来自人类、曼氏血吸虫和恶性疟原虫的HGPRT在氨基酸序列上有21 - 23%的同一性，主要集中在三个聚集区域，这表明锥虫酶是该组中差异最大的。令人惊讶的是，布氏锥虫HGPRT与原核生物哈氏弧菌的次黄嘌呤磷酸核糖转移酶（HPRT）的同源性高于与真核生物HGPRT的同源性。Northern印迹分析显示有1.4 kb和1.9 kb的两种锥虫转录本，它们在寄生虫的昆虫载体和哺乳动物形式中表达程度相当。将布氏锥虫hgprt插入表达质粒并转化到缺乏HPRT和黄嘌呤 - 鸟嘌呤磷酸核糖转移酶活性的S phi 606大肠杆菌中。可溶性、具有酶活性的重组布氏锥虫HGPRT被高水平表达，并通过GTP - 琼脂糖亲和层析纯化至同质。纯化的重组酶识别次黄嘌呤、鸟嘌呤和别嘌呤醇作为底物，但不识别黄嘌呤或腺嘌呤，并且受到多种核苷酸效应物的抑制。编码布氏锥虫hgprt的分子克隆以及大量同质重组HGPRT酶的可得性，为合理设计治疗非洲昏睡病和其他寄生虫源性疾病的新型治疗药物提供了一个可实验操作的分子和生化系统。
