PubMed Health⌕ Search

Biomedical subjects

D M Engman

Publications and source records attributed to D M Engman.

33 records · Page 2Linked to original sources
PubMed1994

Expression and localization of Trypanosoma cruzi hsp60.

A 60-kDa heat shock protein (hsp60) is involved in mitochondrial protein folding and assembly of oligomeric protein complexes in the mitochondrial matrix. Here we report the isolation of Trypanosoma cruzi hsp60 cDNAs, the determination of the organization and chromosomal location of the genes, and the assessment of the heat-regulated expression and subcellular location of the protein. T. cruzi hsp60 is encoded by a multigene family organized in two allelic direct tandem arrays on a chromosome of 1.6 Mb. The regulation of hsp60 expression by heat is complex. While the hsp60 mRNA level is 6-fold higher at 37 degrees C than at either 26 degrees C, the hsp60 protein level remains essentially constant across all temperatures examined. Further analysis of the protein by two-dimensional immunoblotting revealed the existence of multiple isoforms that, with increasing temperature, shift in relative abundance from the more basic to the more acidic. A combination of immunofluorescence microscopy and cell fractionation was used to show that hsp60 is distributed throughout the matrix of the mitochondrion--a location distinct from that of the 70-kDa mitochondrial hsp, mtp70, which is associated with the kinetoplast.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and characterization of the 78-kilodalton glucose-regulated protein of Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the etiologic agent of Chagas' disease, an illness responsible for morbidity and death among millions of Latin Americans. Mice also develop this disease when infected with T. cruzi and are a useful model organism for the study of parasite-specific immune responses. To identify immunogenic T. cruzi antigens, serum from an infected mouse was used to isolate clones from a T. cruzi epimastigote cDNA expression library. One of these clones was found to encode the 78-kDa glucose-regulated protein (grp78), the endoplasmic reticular member of the 70-kDa heat shock protein (hsp70) family. Like the mammalian and yeast grp78s, the T. cruzi protein contains an endoplasmic reticular leader peptide and a carboxyl-terminal endoplasmic reticular retention sequence. T. cruzi grp78 is encoded by a tandemly arranged family of three genes located on a chromosome of 1.6 Mb. The effects on grp78 expression of heat shock and tunicamycin treatment, the latter of which specifically stimulates mammalian grp78, were investigated. While the level of the grp78 protein remained constant under all circumstances, grp78 mRNA was unaffected by heat shock but induced fivefold by tunicamycin. Finally, we found that grp78 is the most immunogenic of the T. cruzi heat shock proteins we have characterized, reacting strongly in immunoblots with sera from infected mice.

Amino Acid Sequence↗
PubMed1993

A mitochondrial heat shock protein from Crithidia fasciculata.

MCP72 is a mitochondrial hsp70 protein from the trypanosomatid Crithidia fasciculata. An MCP72 cDNA clone was isolated from a C. fasciculata cDNA library by screening with antiserum specific for the homologous protein of Trypanosoma cruzi [9]. The MCP72 cDNA encodes a polypeptide of 663 amino acids which is 84% identical to the Trypanosoma cruzi protein and 56% identical to the Escherichia coli hsp70 protein DnaK. MCP72 is less similar to other hsp70 proteins. Native MCP72 was purified to homogeneity by ATP-agarose affinity chromatography. Comparison of its N-terminal amino acid sequence with that deduced from the cDNA sequence shows that 20 amino acid residues had been cleaved from the N-terminus; this sequence probably represents a mitochondrial import signal which is cleaved during translocation into the mitochondrion. Fluorescence microscopy, using antibodies specific for MCP72, indicates that the protein is concentrated in a region of the mitochondrial matrix which surrounds the kinetoplast.

Adenosine Triphosphatases↗
PubMed1992

83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases.

A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids.

Adenosine Triphosphatases↗
PubMed1990

Human humoral immunity to hsp70 during Trypanosoma cruzi infection.

Immunologic screening of cDNA expression libraries has been widely used for the identification of DNA sequences encoding the immunologically relevant proteins of many pathogenic microorganisms. For reasons that are not entirely clear, sequences encoding 70-kDa heat shock and related proteins (hsp70), which are among the most highly conserved proteins known, have routinely been identified by this approach. Consequently, hsp70 proteins have been proposed to be involved in the autoimmune processes thought responsible for the pathogenesis of the diseases caused by some of these organisms, e.g., chronic Trypanosoma cruzi infection (Chagas' disease). Therefore, we investigated whether hsp70 might be a specific target of the human humoral immune response to T. cruzi infection, and, if so, whether humoral autoimmunity to hsp70 might play a role in pathogenesis. We found that hsp70 is indeed a major polypeptide Ag in Chagas' disease, but that the antibodies to T. cruzi hsp70 do not react with human hsp70--even though the proteins display 73% amino acid sequence identify. These results indicate that self-tolerance to hsp70 is maintained during chronic T. cruzi infection and strongly argue against a role for humoral autoimmunity to hsp70 in the pathogenesis of Chagas' disease.

Amino Acid Sequence↗
PubMed1990

The gene family encoding eggshell proteins of Schistosoma japonicum.

The four closely related genes encoding eggshell proteins in the human parasite Schistosoma japonicum are described. A cDNA and a genomic DNA library were constructed and members of the eggshell protein gene family isolated. The four genes in this family do not contain introns, and differ in organization and nucleotide sequence from the related set of genes in Schistosoma mansoni and Schistosoma haematobium. The coding sequences of two of the S. japonicum genes and their flanking regions were determined. Transcription start sites for these genes were shown by primer extension analysis to occur 47 and 50 nucleotides in front of the start codon. A female-specific component in nuclear extracts binds to a DNA fragment containing conserved sequences upstream of the transcription start sites. The deduced protein sequences of 207 and 212 amino acids are composed of 50% glycine with continuous glycine regions as long as 11 residues. In vitro translations of male and female RNAs revealed female-specific translation products, the sizes of which were consistent with the eggshell proteins.

Amino Acid Sequence↗
PubMed1989

A novel flagellar Ca2+-binding protein in trypanosomes.

A 24-kDa protein of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is recognized by antisera from both humans and experimental animals infected with this organism. Near its C terminus are two regions that have sequence similarity with several Ca2+-binding proteins and that conform to the "E-F hand" Ca2+-binding structure. We expressed a cDNA encoding this protein in Escherichia coli and showed that both the recombinant protein and the 24-kDa native trypanosome protein do indeed bind Ca2+. The protein's low Ca2+-binding capacity (less than 2 mol of Ca2+/mol of protein) and high Ca2+-binding affinity (apparent Kd less than 50 microM Ca2+) are consistent with binding of Ca2+ via the E-F hand structures. Immunofluorescence assays using a mouse antiserum directed against the fusion protein localized the native protein to the trypanosome's flagellum. The protein's abundance, Ca2+-binding property, and flagellar localization suggest that it participates in molecular processes associated with the high motility of the parasite.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of mtp70, a mitochondrial member of the hsp70 family.

We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family. Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication. This relationship to DnaK is especially relevant in view of the intracellular location of the protein. Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa. Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs. In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E. coli.

Amino Acid Sequence↗
PubMed1988

Ubiquitin genes in trypanosomatidae.

A ubiquitin encoding cDNA from Trypanosoma cruzi, the protozoan cause of Chagas' disease, was isolated by immunoscreening a lambda gt11 expression library with serum from a mouse chronically infected with this parasite. The cDNA encodes a precursor protein consisting of four tandem repeats of ubiquitin differing from that of Saccharomyces cerevisiae in four positions, followed by an unrelated 52-amino acid tail containing a putative metal and nucleic acid binding domain. Southern and Northern blots of DNA and RNA from various strains of T. cruzi and from several African trypanosome and Leishmania isolates revealed dramatic differences in the numbers and sizes of ubiquitin genes and transcripts. One of the T. cruzi isolates examined has as many as 10 ubiquitin genes, while a strain of Leishmania donovani appears to have only 1. Forty or more tandemly arranged ubiquitin coding repeats are present in some genes, while others have only two or three. Evidence for stage-specific expression of a ubiquitin transcript was found in one strain, but no stress-induced changes in the pattern of transcripts were detected in the one isolate examined. Thus the cellular requirements for ubiquitin in trypanosomatids can be supplied by diversely organized genes containing highly variable numbers of ubiquitin coding repeats.

Amino Acid Sequence↗
PubMed1987

Trypanosoma cruzi exhibits inter- and intra-strain heterogeneity in molecular karyotype and chromosomal gene location.

Molecular karyotypes of 6 strains and 6 clones of Trypanosoma cruzi were determined using orthogonal-field-alternation gel electrophoresis. At least 15 different chromosome-sized DNA molecules, ranging in size from less than 200 kilobase pairs to greater than 2000 kilobase pairs, were resolved for each of the isolates examined. Many of the bands were present in different relative intensities suggesting that the number of individual chromosomes per organism may be considerably higher. Significant inter- and intra-strain differences in molecular karyotype and in the chromosomal locations of the genes for the spliced leader, tubulins, 5S ribosomal RNA and a heat shock protein were found. These marked chromosomal differences among T. cruzi strains and clones may be related to the high degree of phenotypic heterogeneity previously found in this parasite.

Animals↗
PubMed1986

The 35-nucleotide spliced leader sequence is common to all trypanosome messenger RNA's.

In Trypanosomatidae the messenger RNA's (mRNA's) that code for the variant surface glycoproteins (VSG's), tubulins, calmodulin, and at least a subset of other proteins contain a common 35-nucleotide leader sequence at their 5' ends. Hybrid-arrested in vitro translation has been used to show that all mRNA's in both African and South American trypanosomes contain this 35-nucleotide sequence. Oligonucleotides complementary to this sequence blocked translation of all trypanosome mRNA's in a rabbit reticulocyte lysate system, but did not inhibit translation of mRNA's from other organisms lacking this sequence. An oligonucleotide complementary to the VSG mRNA downstream from the spliced leader sequence arrested only VSG synthesis. Thus, the 35-nucleotide leader sequence is a general feature of all trypanosome mRNA's. The high specificity of oligonucleotides complementary to the spliced leader for their target sequence suggests that analogues permeable to the cell membrane may be useful in the treatment of trypanosomal infections.

Base Sequence↗
PubMedDate not supplied

Comparison of the 24 kDa flagellar calcium-binding protein cDNA of two strains of Trypanosoma cruzi.

DNA sequences encoding the 24 kDa flagellar calcium binding protein (FCaBP) of two strains of Trypanosoma cruzi were found to differ at fourteen positions, six of which result in amino acid differences. Four of the amino acid differences are located within the calcium-binding domains of FCaBP; however, none is predicted to affect the calcium-binding ability of the protein. Chromosomes harboring the FCaBP gene clusters differ in size among T. cruzi strains.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular comparison of the mitochondrial and cytoplasmic hsp70 of Trypanosoma cruzi, Trypanosoma brucei and Leishmania major.

We compared the expression and localization of the mitochondrial and cytoplasmic hsp70 of the protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. The mitochondrial protein is encoded by multiple mRNA in all species, while the cytoplasmic protein is encoded by a single mRNA. In all three species, the mitochondrial hsp70 is concentrated in the kinetoplast, a submitochondrial structure that houses the unusual DNA (kDNA) that characterizes this group of organisms, while the cytoplasmic protein is distributed throughout the cell. These results suggest that, in all kinetoplastid species, mt-hsp70 has a specific function in kDNA biology, possibly in the processes of kDNA replication, RNA editing or kinetoplast structure.

Animals↗