Penha Luciana L, Mendonça-Previato Lucia, Previato Jose O, Scharfstein Julio, Heise Norton, Lima Ana Paula C de A
Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde-Bloco G, Universidade Federal do Rio de Janeiro, 21944-970, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, Brazil.
Glycobiology. 2005 Dec;15(12):1359-67. doi: 10.1093/glycob/cwj023. Epub 2005 Jul 21.
Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.
克氏锥虫是恰加斯病的病原体，恰加斯病是一种以进行性心肌病和/或消化道去神经化为特征的慢性疾病。该寄生虫表面覆盖有糖缀合物，如粘蛋白型糖蛋白和糖基磷脂酰肌醇（GIPL），其聚糖富含吡喃半乳糖（Galp）和/或呋喃半乳糖（Galf）残基。这些分子与寄生虫对哺乳动物细胞的附着和侵袭以及感染期间宿主免疫反应的调节有关。在克氏锥虫中，半乳糖（Gal）的生物合成依赖于尿苷二磷酸（UDP）-葡萄糖（UDP-Glc）通过UDP-Gal 4-表异构酶催化的NAD依赖性还原反应转化为UDP-Gal。磷酸葡萄糖变位酶（PGM）是该代谢途径中的关键酶，催化6-磷酸葡萄糖（Glc-6-P）和1-磷酸葡萄糖（Glc-1-P）的相互转化，然后Glc-1-P再转化为UDP-Glc。我们在此报告了克氏锥虫PGM的克隆，即编码克氏锥虫PGM的基因，以及该功能性酶在酿酒酵母中的异源表达。克氏锥虫PGM是一个单拷贝基因，编码的预测蛋白与硕大利什曼原虫PGM的氨基酸同一性为61%，与酵母酶的氨基酸同一性为43%。PGM RNA的59反式剪接位点定位于起始密码子上游18个碱基对的区域。克氏锥虫PGM在缺乏编码PGM两种同工型的基因（pgm1Delta/pgm2Delta）的酿酒酵母缺失突变体中的表达挽救了以Gal作为唯一碳源进行细胞生长时诱导的致死表型。
