Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant.
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.
Penha
Luciana L
LL
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.
Mendonça-Previato
Lucia
L
Previato
Jose O
JO
Scharfstein
Julio
J
Heise
Norton
N
Lima
Ana Paula C de A
AP
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D002244:Carbon / Q000737:chemistry; D014355:Chagas Disease / Q000378:metabolism; D003001:Cloning, Molecular; D018387:Codon, Initiator; D005663:Furans / Q000737:chemistry; D005690:Galactose / Q000737:chemistry; D005816:Genetic Complementation Test; D018320:Leishmania major; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010733:Phosphoglucomutase / Q000737:chemistry* / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014349:Trypanosoma cruzi / Q000378:metabolism*
D018387:Codon, Initiator; D005663:Furans; D012333:RNA, Messenger; D002244:Carbon; D010733:Phosphoglucomutase; D005690:Galactose