RESUMO
A Trypanosoma cruzi gene, PKB, coding for a putative protein kinase was cloned and sequenced. Analysis of the sequence showed that the encoded protein (called PKB) corresponds to a relatively novel subgroup of Ser/Thr protein kinases denominated protein kinases B (PKB), related to A and C protein kinases (RAC), or protein kinases of the transforming retrovirus AKT8 (Akt) in which the catalytic domains show similarity to corresponding domains of protein kinases A and protein kinases C. Unlike mammalian enzymes belonging to the same subgroup, PKB did not have a pleckstrin (PH)-homologous domain. PKB was expressed in Escherichia coli and the recombinant protein was found to be a Thr-specific protein kinase that required Mn2+ for activity and used ATP as phosphate donor (Km = 1.8 microM). Classical protein kinase A and protein kinase C modulators and inhibitors were found to have only marginal or no effect on PKB activity. Antisera raised against the recombinant protein recognized PKB in Western blotting analysis of cell extracts as a membrane bound protein. Evidence was obtained suggesting the presence of a Cys-linked acyl anchor. Northern and Western blotting analysis showed that PKB was constitutively expressed in the lag, exponential and stationary phases of T. cruzi epimastigote growth, as well as in the amastigote and metacyclic trypomastigote stages of differentiation. This is the first description of the existence of a protein kinase B in trypanosomatid protozoa.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , DNA de Protozoário , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Temperatura , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Lectinas/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma cruzi/química , 1-Desoxinojirimicina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Calreticulina , Sequência de Carboidratos , Clonagem Molecular , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Inibidores de Glicosídeo Hidrolases , Glicosilação , Hexosaminidases/farmacologia , Soros Imunes , Lectinas/efeitos dos fármacos , Lectinas/genética , Dados de Sequência Molecular , Testes de Precipitina , Dobramento de Proteína , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Frações Subcelulares , alfa-GlucosidasesRESUMO
Rat brain glycogen branching enzyme was partially purified in order to elucidate its mechanism of action. The alpha1,4-alpha1,6-glucan polysaccharide was synthesized using rat brain branching enzyme under two different elongation conditions: Glc-1-P and phosphorylase or UDP-Glc and glycogen synthase. The products obtained demonstrated that the cpolysaccharides synthesized (pattern of the spectra obtained in the presence of Krisman's reagent, lambda max, parameter A and R, % beta-amylolysis and degree of branching) under different incubation times are nearly constant. These results imply that the degree of branching of a polysaccharide depends only on the enzyme specificity.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Encéfalo/enzimologia , Absorção , Animais , Glucofosfatos/metabolismo , Fosforilases/metabolismo , Polissacarídeos/biossíntese , Ratos , Ratos Wistar , beta-Amilase/metabolismoRESUMO
It has been proposed that the UDP-Glc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked Glc1Man7-9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin-like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP-Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides.
Assuntos
Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Glucose/metabolismo , Glucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Trypanosoma cruzi/metabolismo , 1-Desoxinojirimicina/farmacologia , Animais , Asparagina/metabolismo , Transporte Biológico , Sequência de Carboidratos , Compartimento Celular , Glicosilação , Lisossomos/metabolismo , Dados de Sequência Molecular , Oligossacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários , Fatores de Tempo , Trypanosoma cruzi/enzimologiaRESUMO
Epimastigotes of the American Trypanosome Trypanosoma rangeli contain a very low cysteine proteinase (CP) activity. The enzyme was purified to homogeneity by affinity chromatography on ConA-Sepharose and Cystatin-Sepharose. This CP had a similar apparent molecular mass and an identical N-terminal sequence (15 amino acids) as compared with cruzipain from Trypanosoma cruzi; cross-reacted immunologically with the latter enzyme, it was inhibited by E-64 and TLCK, but not by PMSF, o-phenanthroline or Pepstatin, and was able to use the same substrates, although with different order of effectiveness and optimum pH.
Assuntos
Cisteína Endopeptidases/isolamento & purificação , Trypanosoma/enzimologia , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/química , Ativação Enzimática , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
Cruzipain is a lysosomal enzyme of the flagellate Trypanosoma cruzi. It has three potential asparagine-glycosylation sites, two in the catalytic domain and one in the C-terminal domain. The latter appeared to have both high mannose- and complex-type oligosaccharides, whereas the catalytic domain only had compounds of the former type. The partial susceptibility of the complex-type compounds to endo-beta-N-acetylglucosaminidase H and their relative mannose and galactose content indicate that they had hybrid/monoantennary and biantennary structures. The same pattern of high mannose-type compounds was found at both domains, thus indicating that in cruzipain molecules having only high mannose-type compounds, all oligosaccharides were equally exposed to processing glycosidases and glycosyltransferases. As heterogenity of the protein C-terminal domain has already been detected, it is suggested that this feature might elicit an increased accessibility to processing enzymes responsible for complex-type oligosaccharide formation in certain cruzipain molecules or, alternatively, that a second glycosylation site with increased accessibility might be present in certain cruzipain molecules. Furthermore, the presence of complex-type oligosaccharides strongly suggests that, as in mammalian cells, T. cruzi lysosomal enzymes traverse the entire Golgi apparatus up to the trans-Golgi cisternae and the trans-Golgi network before reaching lysosomes.
Assuntos
Cisteína Endopeptidases/química , Oligossacarídeos/química , Trypanosoma cruzi/enzimologia , Animais , Asparagina/química , Sítios de Ligação , Sequência de Carboidratos , Catálise , Glicosilação , Complexo de Golgi/enzimologia , Manose/química , Dados de Sequência Molecular , Estrutura Molecular , Proteínas de ProtozoáriosRESUMO
The major cysteine proteinase from Trypanosoma cruzi, cruzipain, can be obtained essentially homogeneous, starting from crude epimastigote extracts, in one step, by affinity chromatography on Cystatin-Sepharose (specific for cysteine proteinases) or ConA-Sepharose (specific for high mannose N-linked glycoproteins). The methods offer considerable potential for enzyme purification from scarce sources, such as other parasite stages or radioactively labelled material with high specific radioactivity.