scholarly journals Molecular Cloning of a Trypanosoma cruzi Cell Surface Casein Kinase II Substrate, Tc-1, Involved in Cellular Infection

2006 ◽  
Vol 74 (7) ◽  
pp. 3922-3929 ◽  
Author(s):  
Swinburne A. J. Augustine ◽  
Yuliya Y. Kleshchenko ◽  
Pius N. Nde ◽  
Siddharth Pratap ◽  
Edward A. Ager ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Trypanosoma Cruzi ◽  
Blot Analysis ◽  
Transmembrane Domain ◽  
Amino Acid Level ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Host Cells ◽  
Direct Immunofluorescence

ABSTRACT In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.

scholarly journals Molecular cloning of a novel Trypanosoma cruzi cell surface casein kinase II substrate that mediates cellular invasion

2007 ◽  
Vol 21 (6) ◽  
Author(s):  
Swinburne A.J. Augustine ◽  
Kaneatra J Simmons ◽  
Pius N Nde ◽  
M Nia Madison ◽  
Yuliya Y Kleshchenko ◽  
...  
Keyword(s):  
Cell Surface ◽  
Trypanosoma Cruzi ◽  
Molecular Cloning ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Cellular Invasion

scholarly journals Molecular Cloning of a Trypanosoma cruzi Cell Surface Casein Kinase II Substrate, Tc-1, Involved in Cellular Infection

2006 ◽  
Vol 74 (10) ◽  
pp. 6022-6022
Author(s):  
Swinburne A. J. Augustine ◽  
Yuliya Y. Kleshchenko ◽  
Pius N. Nde ◽  
Siddharth Pratap ◽  
Edward A. Ager ◽  
...  
Keyword(s):  
Cell Surface ◽  
Trypanosoma Cruzi ◽  
Molecular Cloning ◽  
Casein Kinase Ii ◽  
Casein Kinase

Salamander rods and cones contain distinct transducin alpha subunits

2000 ◽  
Vol 17 (6) ◽  
pp. 847-854 ◽  
Author(s):  
JAMES C. RYAN ◽  
SERGEY ZNOIKO ◽  
LIN XU ◽  
ROSALIE K. CROUCH ◽  
JIAN-XING MA
Keyword(s):  
Amino Acid ◽  
Blot Analysis ◽  
Cellular Localization ◽  
Amino Acid Level ◽  
Single Copy ◽  
Lower Vertebrate ◽  
Rods And Cones ◽  
Sequence Identity ◽  
Outer Segments ◽  
Cone Pigments

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin α subunits (Gαt) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of Gαt. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander Gαt-1 has 91.2–93.7% amino acid sequence identity to mammalian rod Gαt subunits and 79.7–80.9% to mammalian cone Gαts. The salamander Gαt-2 has 86.2–87.9% sequence identity to mammalian cone Gαts and 78.9–80.9% to mammalian rod Gαts at the amino acid level. The Gαt-1 cDNA encodes 350 amino acids while the Gαt-2 cDNA encodes 354 residues, which is typical for rod and cone Gαts, respectively, and we thus identified the Gαt-1 as rod and Gαt-2 as cone Gαt. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone Gαt subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod Gαt and 2.6 kb for cone Gαt. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod Gαt is localized to rods, predominantly in the outer segments; similarly, cone Gαt is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

scholarly journals Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei.

1990 ◽  
Vol 10 (9) ◽  
pp. 4506-4517 ◽  
Author(s):  
M G Lee ◽  
B E Bihain ◽  
D G Russell ◽  
R J Deckelbaum ◽  
L H Van der Ploeg
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Transmembrane Domain ◽  
Density Lipoprotein ◽  
Surface Protein ◽  
Integral Membrane Protein ◽  
Cell Surface Receptor ◽  
Flagellar Pocket ◽  
Cytoplasmic Extension

We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.

Molecular cloning of casein kinase II alpha subunit from Dictyostelium discoideum and its expression in the life cycle

1992 ◽  
Vol 12 (12) ◽  
pp. 5711-5723
Author(s):  
U Kikkawa ◽  
S K Mann ◽  
R A Firtel ◽  
T Hunter
Keyword(s):  
Life Cycle ◽  
Dictyostelium Discoideum ◽  
Cdna Clone ◽  
Blot Analysis ◽  
Vegetative Growth ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Developmental Expression ◽  
Alpha Subunit ◽  
Alpha Gene

A Dictyostelium discoideum cDNA encoding an alpha-type subunit of casein kinase II was isolated, and its cDNA was used to study developmental expression of casein kinase II during the Dictyostelium life cycle. The 1.3-kb cDNA insert contained an open reading frame of 337 amino acids (M(r) 39,900). The deduced amino acid sequence has high homology with those of casein kinase II alpha subunits from other species. Genomic Southern blot analysis suggested that there is a single gene encoding casein kinase II alpha subunit in D. discoideum. Northern (RNA) blot analysis showed that the casein kinase II alpha-subunit gene is expressed constitutively as a 1.9-kb mRNA throughout vegetative growth and multicellular development. Casein kinase purified from normal vegetative cells contained a major protein band of approximately 36 kDa, which was recognized by antisera raised against rat testis casein kinase II. Comparison of the in vitro transcription/translation product of the alpha-subunit cDNA clone and the purified 36-kDa protein by partial proteolysis indicated that the isolated cDNA clone encodes the Dictyostelium casein kinase II alpha subunit. No protein corresponding to a beta subunit was detected in purified casein kinase. Immunoblot analysis using anti-rat casein kinase II sera showed that the alpha subunit of casein kinase II is expressed constitutively like its mRNA during the life cycle of D. discoideum. Casein kinase II activity measured by using a specific peptide substrate paralleled the level of alpha subunit detected by immunoblotting during the life cycle, with a maximum variation of approximately 2-fold. We were unable to obtain disruptants of the casein kinase II alpha gene, suggesting that there is a single casein kinase II alpha gene, which is essential for vegetative growth of D. discoideum.

Trypanosoma cruzi: cell surface dynamics in trypomastigotes of different strains

Parasitology ◽  
2019 ◽  
Vol 147 (3) ◽  
pp. 310-321
Author(s):  
Roberta Ferreira Cura das Neves ◽  
Camila Marques Adade ◽  
Anne Cristine Silva Fernandes ◽  
Angela Hampshire Lopes ◽  
Thaïs Souto-Padrón
Keyword(s):  
Cell Surface ◽  
Trypanosoma Cruzi ◽  
Binding Sites ◽  
Host Cells ◽  
Cationized Ferritin ◽  
Surface Dynamics ◽  
Con A ◽  
Low Intensity ◽  
Intense Labelling ◽  
Different Strains

AbstractCapping and shedding of ectodomains in Trypanosoma cruzi may be triggered by different ligands. Here, we analysed the mobility and shedding of cell surface components of living trypomastigotes of the Y strain and the CL Brener clone in the presence of poly-L-lysine, cationized ferritin (CF) and Concanavalin A (Con A). Poly-L-lysine and CF caused intense shedding in Y strain parasites. Shedding was less intense in CL Brener trypomastigotes, and approximately 10% of these parasites did not show any decrease in poly L-lysine or CF labelling. Binding of Con A induced low-intensity shedding in Y strain and redistribution of Con A-binding sites in CL Brener parasites. Trypomastigotes of the Y strain showed intense labelling with anti-〈-galactosyl antibodies, resulting in the lysis of approximately 30% of their population, in contrast with what was observed in CL Brener parasites. Incubation with Con A and CF protected trypomastigotes of the Y strain from lysis by anti-αGal. The last treatment did not interfere with the survival of the CL Brener parasites. This study corroborates with the idea that a ligand can differentially modulate the cell surface of T. cruzi, depending on the strain used, resulting in variable immune system responses and recognition by host cells.

scholarly journals Determinants of Tetherin Antagonism in the Transmembrane Domain of the Human Immunodeficiency Virus Type 1 Vpu Protein

2010 ◽  
Vol 84 (24) ◽  
pp. 12958-12970 ◽  
Author(s):  
Raphaël Vigan ◽  
Stuart J. D. Neil
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Cell Surface ◽  
Human Immunodeficiency Virus Type ◽  
Transmembrane Domain ◽  
Human Tetherin ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Tetherin Antagonism

ABSTRACT Tetherin (BST2/CD317) potently restricts the particle release of human immunodeficiency virus type 1 (HIV-1) mutants defective in the accessory gene vpu. Vpu antagonizes tetherin activity and induces its cell surface downregulation and degradation in a manner dependent on the transmembrane (TM) domains of both proteins. We have carried out extensive mutagenesis of the HIV-1 NL4.3 Vpu TM domain to identify three amino acid positions, A14, W22, and, to a lesser extent, A18, that are required for tetherin antagonism. Despite the mutants localizing indistinguishably from the wild-type (wt) protein and maintaining the ability to multimerize, mutation of these positions rendered Vpu incapable of coimmunoprecipitating tetherin or mediating its cell surface downregulation. Interestingly, these amino acid positions are predicted to form one face of the Vpu transmembrane alpha helix and therefore potentially contribute to an interacting surface with the transmembrane domain of tetherin either directly or by modulating the conformation of Vpu oligomers. While the equivalent of W22 is invariant in HIV-1/SIVcpz Vpu proteins, the positions of A14 and A18 are highly conserved among Vpu alleles from HIV-1 groups M and N, but not those from group O or SIVcpz that lack human tetherin (huTetherin)-antagonizing activity, suggesting that they may have contributed to the adaption of HIV-1 to human tetherin.

Isolation, sequencing, and disruption of the CKA1 gene encoding the alpha subunit of yeast casein kinase II

1988 ◽  
Vol 8 (11) ◽  
pp. 4981-4990
Author(s):  
J L Chen-Wu ◽  
R Padmanabha ◽  
C V Glover
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Product ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Protein Sequencing ◽  
Alpha Subunit ◽  
Yeast Genome ◽  
Gene Encoding ◽  
Alpha Gene

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which must be encoded by separate genes (R. Padmanabha and C. V. C. Glover, J. Biol. Chem. 262:1829-1835, 1987). The gene encoding the 42-kilodalton alpha subunit has been isolated by screening a yeast genomic library with oligonucleotide probes synthesized on the basis of the N-terminal amino acid sequence of the polypeptide. This gene (designated CKA1) contains an intron-free open reading frame of 372 amino acid residues. The deduced amino acid sequence is 67% identical to the alpha subunit of Drosophila melanogaster casein kinase II. The CKA1 gene product appears to be distantly related to other known protein kinases but exhibits highest similarity to the CDC28 gene product and its homolog in other species. Gene replacement techniques have been used to generate a null cka1 mutant allele. Haploid and diploid strains lacking a functional CKA1 gene appear to be phenotypically wild type, presumably because of the presence of the alpha' gene. Interestingly, the CKA1 gene appears to be single copy in the yeast genome; i.e., the alpha' gene, whose existence is known from biochemical studies and protein sequencing, cannot be detected by low-stringency hybridization.

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