scholarly journals Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

2006 ◽  
Vol 75 (1) ◽  
pp. 74-82 ◽  
Author(s):  
Jere W. McBride ◽  
C. Kuyler Doyle ◽  
Xiaofeng Zhang ◽  
Ana Maria Cardenas ◽  
Vsevolod L. Popov ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Antibody ◽  
Tandem Repeats ◽  
Ehrlichia Canis ◽  
Small Subset ◽  
Amino Acid Homology ◽  
Amino Terminal ◽  
Neutral Sugars ◽  
Carboxyl Terminal ◽  
Species Specific

ABSTRACT Ehrlichia canis has a small subset of major immunoreactive proteins that includes a 19-kDa protein that elicits an early Ehrlichia-specific antibody response in infected dogs. We report herein the identification and molecular characterization of this highly conserved 19-kDa major immunoreactive glycoprotein (gp19) ortholog of the Ehrlichia chaffeensis variable-length PCR target (VLPT) protein. E. canis gp19 has substantial carboxyl-terminal amino acid homology (59%) with E. chaffeensis VLPT and the same chromosomal location; however, the E. chaffeensis VLPT gene (594 bp) has tandem repeats that are not present in the E. canis gp19 gene (414 bp). Consistent with other ehrlichial glycoproteins, the gp19 protein exhibited a larger-than-predicted mass (∼3 kDa), O-linked glycosylation sites were predicted in an amino-terminal serine/threonine/glutamate (STE)-rich patch (26 amino acids), carbohydrate was detected on the recombinant gp19 protein, and the neutral sugars glucose and galactose were detected on the recombinant amino-terminal polypeptide. E. canis gp19 composition consists of five predominant amino acids, cysteine, glutamate, tyrosine, serine, and threonine, concentrated in the STE-rich patch and a carboxyl-terminal domain predominated by cysteine and tyrosine (55%). The amino-terminal STE-rich patch contained a major species-specific antibody epitope strongly recognized by serum from an E. canis-infected dog. The recombinant glycopeptide epitope was substantially more reactive with antibody than the synthetic (nonglycosylated) peptide, and periodate treatment of the recombinant glycopeptide epitope reduced its immunoreactivity, demonstrating the importance of a carbohydrate immunodeterminant(s). The gp19 protein was present on reticulate and dense-cored cells, and it was found extracellularly in the fibrillar matrix and associated with the morula membrane, the host cell cytoplasm, and the nucleus.

scholarly journals Tyrosine-Phosphorylated Ehrlichia chaffeensis and Ehrlichia canis Tandem Repeat Orthologs Contain a Major Continuous Cross-Reactive Antibody Epitope in Lysine-Rich Repeats

2011 ◽  
Vol 79 (8) ◽  
pp. 3178-3187 ◽  
Author(s):  
Jere W. McBride ◽  
Xiaofeng Zhang ◽  
Abdul Wakeel ◽  
Jeeba A. Kuriakose
Keyword(s):  
Amino Acids ◽  
Tandem Repeat ◽  
Tandem Repeats ◽  
Ehrlichia Canis ◽  
Small Subset ◽  
Ehrlichia Chaffeensis ◽  
Whole Cell ◽  
Content Type ◽  
Cell Lysates ◽  
Species Specific

ABSTRACTA small subset of major immunoreactive proteins have been identified inEhrlichia chaffeensisandEhrlichia canis, including three molecularly and immunologically characterized pairs of immunoreactive tandem repeat protein (TRP) orthologs with major continuous species-specific epitopes within acidic tandem repeats (TR) that stimulate strong antibody responses during infection. In this study, we identified a fourth major immunoreactive TR-containing ortholog pair and defined a major cross-reactive epitope in homologous nonidentical 24-amino-acid lysine-rich TRs. Antibodies from patients and dogs with ehrlichiosis reacted strongly with recombinant TR regions, and epitopes were mapped to the N-terminal TR region (18 amino acids) inE. chaffeensisand the complete TR (24 amino acids) inE. canis. Two less-dominant epitopes were mapped to adjacent glutamate/aspartate-rich and aspartate/tyrosine-rich regions in the acidic C terminus ofE. canisTRP95 but not inE. chaffeensisTRP75. Major immunoreactive proteins inE. chaffeensis(75-kDa) andE. canis(95-kD) whole-cell lysates and supernatants were identified with TR-specific antibodies. Consistent with other ehrlichial TRPs, the TRPs identified in ehrlichial whole-cell lysates and the recombinant proteins migrated abnormally slow electrophoretically a characteristic that was demonstrated with the positively charged TR and negatively charged C-terminal domains.E. chaffeensisTRP75 andE. canisTRP95 were immunoprecipitated with anti-pTyr antibody, demonstrating that they are tyrosine phosphorylated during infection of the host cell.

scholarly journals Ehrlichia canis gp200 Contains Dominant Species-Specific Antibody Epitopes in Terminal Acidic Domains

2007 ◽  
Vol 75 (10) ◽  
pp. 4900-4908 ◽  
Author(s):  
Kimberly A. Nethery ◽  
C. Kuyler Doyle ◽  
Xiaofeng Zhang ◽  
Jere W. McBride
Keyword(s):  
Amino Acids ◽  
Specific Antibody ◽  
Amino Acid Content ◽  
Ehrlichia Canis ◽  
Biophysical Properties ◽  
B Cell Epitopes ◽  
Immunoreactive Protein ◽  
Recombinant Polypeptides ◽  
Species Specific ◽  
Antibody Epitopes

ABSTRACT Species-specific antibody epitopes within several major immunoreactive protein orthologs of Ehrlichia species have recently been identified and molecularly characterized. In this study, dominant B-cell epitopes within the acidic (pI 5.35) ankyrin repeat-containing 200-kDa major immunoreactive protein (gp200) of Ehrlichia canis were defined. The E. canis gp200 gene (4,263 bp; 1,421 amino acids) was cloned and expressed as four (N-terminal, 1,107 bp; N-internal, 910 bp; C-internal, 1,000 bp; and C-terminal, 1,280 bp) overlapping recombinant proteins. The N-terminal, C-internal, and C-terminal polypeptides (369, 332, and 426 amino acids, respectively) were strongly recognized by antibody, and the major epitope(s) in these polypeptides was mapped to four polypeptide regions (40 to 70 amino acids). Smaller overlapping recombinant polypeptides (14 to 15 amino acids) spanning these regions identified five strongly immunoreactive species-specific epitopes that exhibited conformational dependence. The majority of the epitopes (four) were located in two strongly acidic (pI 4 to 4.9) domains in the distal N- and C-terminal regions of the protein flanking the centralized ankyrin domain-containing region. The amino acid content of the epitope-containing domains included a high proportion of strongly acidic amino acids (glutamate and aspartate), and these domains appear to have important biophysical properties that influence the antibody response to gp200.

Epitope mapping of monoclonal antibodies to bovine prolactin

1992 ◽  
Vol 263 (3) ◽  
pp. E520-E525 ◽  
Author(s):  
J. G. Scammell ◽  
D. N. Luck ◽  
D. L. Valentine ◽  
M. Smith
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Epitope Mapping ◽  
Inhibitory Concentration ◽  
Specific Region ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Species Specific

The epitopes recognized by three monoclonal antibodies generated to sheep prolactin were determined by evaluating their cross-reactivities by immunodot analysis with 14 mutants of bovine prolactin, in which individual amino acids had been deleted or substituted. Mutations were made throughout the molecule and included disruption of the amino-terminal, carboxyl-terminal, and central disulfide loops. Lack of immunoreactivity was taken as an indication that the site of mutation was part of the epitope. Antibody 6F11 reacted with all bovine prolactin mutants tested, except those in which the carboxyl-terminal cysteine (position 199) was substituted by a serine. Antibodies 5G2 and 4C10 reacted with all of the bovine prolactin mutants, except those in which the amino-terminal cysteine (position 4) was substituted by a serine. Western blot analysis of sheep, squirrel monkey, and rat prolactins with the monoclonal antibodies revealed that 5G2 and 4C10 were specific for sheep prolactin, whereas antibody 6F11 cross-reacted with prolactins from all three species. The mitogenic activity of sheep or rat prolactin in the Nb2 bioassay was determined in the presence of the antibodies to determine whether the epitopes were part of the functional domains of these prolactins. The bioactivity of sheep prolactin (0.4 ng/ml) was unaffected by the monoclonal antibodies [0.01-1 microgram immunoglobulin G (IgG)/ml], whereas the bioactivity of rat prolactin (1.25 ng/ml) was inhibited by 6F11 with an apparent 50% inhibitory concentration of 0.25 microgram IgG/ml. These results indicate that monoclonal antibodies 5G2 and 4C10 cross-react with a species-specific region of the amino-terminal disulfide loop of bovine prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional Analysis of a Peptide Sequence (Thr323 - Cys337) In ADAMTS13 Disintegrin-Like Domain Revealed Two Discontinuous Sequences Involved In the Interaction with VWF

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2200-2200
Author(s):  
Atsuko Igari ◽  
Takanori Moriki ◽  
Terumichi Nakagawa ◽  
Yusuke Yamaguchi ◽  
Mitsuru Murata
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Synthetic Peptides ◽  
Inhibitory Effect ◽  
Peptide Sequence ◽  
Recent Analysis ◽  
Inhibitory Effects ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Carboxyl Terminal

Abstract Abstract 2200 ADAMTS13 specifically cleaves multimeric von Willebrand factor (VWF) into smaller molecules to reduce its high reactivity with platelets. The disintegrin-like (D) domain, adjacent to the catalytic domain of ADAMTS13, plays an important role in the process of VWF cleavage. In this study, we aimed to elucidate critical peptide sequences in D-domain involved in the interaction with VWF. A series of partially overlapping peptide sequences, approximately 20 amino acids in length, covering the D-domain, were synthesized and the inhibitory effects on the catalytic activity of plasma ADAMTS13 was examined using FRETS-VWF73 assay. Consequently, some synthetic peptides were selected and the minimal length necessary for the inhibitory effect was determined as TFAREHLDMCQALSC (peptide323-337). Removal of the amino-terminal threonine diminished the inhibitory effect moderately, although deletion of the carboxyl-terminal cysteine abolished it completely. According to the amino acids alignment of ADAMTS family, this peptide sequence is not conserved, highlighting the specific role in the interaction with its substrate. From the recent analysis of crystal structure, amino-terminal half of the peptide323-337, TFAREHL (323-329), was disordered and designated as the variable (V) loop, which creates one of VWF-binding exosites (Akiyama, et al. Proc Natl Acad Sci USA. 2009; 106:19274-9). We hypothesized that the amino-terminal amino acids of the peptide323-337 contribute to VWF binding, whereas the carboxyl-terminal amino acids allow the structural stability of the peptide conformation. To evaluate the effect of carboxyl-terminal cysteine at 337, other synthetic peptides with alanine, serine, glycine or phenylalanine instead of the cysteine (C337A, C337S, C337G, or C337F) were tested about their inhibitory effects on the catalytic activity. Interestingly, C337A, C337S, C337G peptides exhibited slightly weaker inhibitory effects on VWF73 catalysis, although C337F peptide showed stronger inhibition than wild-type sequence, suggesting that the residue 337 regulates the characteristics of the peptide323-337. From the results of peptide screening, the amino- and carboxyl-terminal amino acids of the peptide323-337, TFAREHLDMCQALSC, likely play key roles in the inhibitory effects; therefore, the middle part of the sequence, HLDMC, was replaced by 5 alanines (AAAAA) or reversed sequence CMDLH. Surprisingly, the converted peptides still retained the equivalent level of inhibitory effects, indicating both sides of the amino- and carboxyl-terminal amino acids were especially significant in the interaction with VWF. In conclusion, we characterized the peptide sequence, TFAREHLDMCQALSC (323-337), in D-domain. The peptide clearly inhibited the cleavage of VWF73 and the both sides of amino- and carboxyl-terminal amino acids seemed especially important. The peptide sequence is supposed to bind to VWF for the precise cleavage in the process of proteolysis. By modifying this peptide sequence, such variant ADAMTS13 as gain-of-function recombinants might be developed, leading to an alternative anti-thrombotic drug. Disclosures: No relevant conflicts of interest to declare.

STUDIES ON THE MECHANISM OF SOMATOSTATIN ACTION ON INSULIN RELEASE

1977 ◽  
Vol 85 (3) ◽  
pp. 579-586 ◽  
Author(s):  
S. Efendić ◽  
P. E. Lins ◽  
R. Luft ◽  
H. Sievertsson ◽  
G. Westin-Sjödal
Keyword(s):  
Amino Acids ◽  
Insulin Release ◽  
Cyclic Peptide ◽  
The Other ◽  
Perfused Rat Pancreas ◽  
Rat Pancreas ◽  
Amino Terminal ◽  
Structure Activity ◽  
Carboxyl Terminal ◽  
Relationship Of

ABSTRACT Eighteen analogues of somatostatin have been used in order to elucidate the structure-activity relationship of the peptide on the release of insulin and glucagon from the isolated perfused rat pancreas. Neither the amino terminal nor a free carboxyl terminal seemed to be essential for the activity of the cyclic peptide. Addition of amino acids to the amino terminal did not decrease the activity. On the other hand, minor changes in the structure of linear somatostatin, which lead to the loss of ability to form a cyclic peptide, impaired the activity. Deletion of Asn5 was accompanied by decreased action on glucagon but not on insulin release. It seems that the major actions of somatostatin on the pancreas are bound to the amino acid sequence 4–13 in the molecule and to the ability of the molecule to cyclize.

scholarly journals Evolution of PTH assays

2006 ◽  
Vol 50 (4) ◽  
pp. 621-627 ◽  
Author(s):  
José Gilberto H. Vieira ◽  
Ilda Kunii ◽  
Sônia Nishida
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
First Generation ◽  
Recent Observation ◽  
Cost Benefit ◽  
Molecular Forms ◽  
Specific Antibodies ◽  
Amino Terminal ◽  
2Nd Generation ◽  
Carboxyl Terminal

PTH metabolism is complex and the circulating forms include the intact 1-84 molecule as well as several carboxyl-terminal fragments. The first generation of PTH assays included several types of competitive assays, with specificities that spanned carboxyl, mid-region and amino-terminal portions of the molecule. The limitations of these assays and the methodological evolution led to the description of 2nd generation non-competitive immunometric assays for PTH in the late 80's, based on the recognition of the PTH molecule by two different antibodies, one directed against de amino-terminal and other against the carboxyl-terminal segments. The observation that in some circumstances "long" carboxyl-terminal segments were also measured by 2nd generation assays led to the development of 3rd generation assays based on amino-terminal specific antibodies that are specific for the first amino acids, measuring only the molecular forms that activate PTH1R. The practical and cost-benefit advantages of these assays are still debatable. The recent observation that carboxyl-terminal fragments of PTH have biological activity via a distinct receptor than PTH1R, points to the future need of more than one assay in order to evaluate parathyroid hormone function.

gamma-tubulin in trypanosomes: molecular characterisation and localisation to multiple and diverse microtubule organising centres

1997 ◽  
Vol 110 (2) ◽  
pp. 157-168 ◽  
Author(s):  
V. Scott ◽  
T. Sherwin ◽  
K. Gull
Keyword(s):  
Amino Acids ◽  
Cell Body ◽  
Mammalian Cells ◽  
Amino Acid Identity ◽  
Full Length ◽  
Small Subset ◽  
Tubulin Gene ◽  
Amino Terminal ◽  
Degenerate Oligonucleotide ◽  
Gamma Tubulin

A genomic clone from Trypanosoma brucei, which contains a full length gamma-tubulin gene, was isolated using degenerate oligonucleotide primers. The sequence of this clone predicts a protein of 447 amino acids having a high degree of homology with gamma-tubulins from human and Xenopus laevis (67.2% amino acid identity) and only 57.7% identity with the Plasmodium falciparum gamma-tubulin. Northern blot analysis of poly(A)+ selected RNA from a procyclic culture detects a major transcript of approximately 2.2 kb plus a minor transcript of approximately 3.6 kb. A fusion protein comprising almost the full length gamma-tubulin gene product (amino acids 8–447) plus an amino-terminal histidine tag has been expressed and purified from Escherichia coli and used to raise a polyclonal antibody. Immunofluorescence, using this antibody, shows classical centrosomal localisation in mammalian cells. In T. brucei gamma-tubulin is present in the basal bodies which subtend the flagellum and also at the anterior tip of the cell body where many minus ends of microtubules are located. Furthermore the antibody reveals a small subset of the sub-pellicular microtubules and a discrete dot within the nucleus which alters form with progression through the mitotic cycle. Evidence is also presented for discrete punctate staining within the microtubules of the cell body which may represent the presence of gamma-tubulin on the ends of individual microtubules. Our results indicate that gamma-tubulin is associated with diverse microtubule organising centres and structures in trypanosomes.

scholarly journals Differentially Expressed and Secreted Major Immunoreactive Protein Orthologs of Ehrlichia canis and E. chaffeensis Elicit Early Antibody Responses to Epitopes on Glycosylated Tandem Repeats

2006 ◽  
Vol 74 (1) ◽  
pp. 711-720 ◽  
Author(s):  
C. Kuyler Doyle ◽  
Kimberly A. Nethery ◽  
Vsevolod L. Popov ◽  
Jere W. McBride
Keyword(s):  
Acute Phase ◽  
Recombinant Proteins ◽  
Tandem Repeats ◽  
Antibody Responses ◽  
Ehrlichia Canis ◽  
Differentially Expressed ◽  
Native Proteins ◽  
Repeat Units ◽  
Canine Monocytic Ehrlichiosis ◽  
Species Specific

ABSTRACT Ehrlichia canis major immunoreactive proteins of 36 and 19 kDa elicit the earliest detectable antibody responses during the acute phase of canine monocytic ehrlichiosis. Genes encoding the major immunoreactive 36-kDa protein of E. canis and the corresponding ortholog of E. chaffeensis (47 kDa) were identified and the proteins characterized. The molecular masses of the strongly immunoreactive recombinant proteins were larger than predicted (26.7 and 32.9 kDa, respectively) but were consistent with those of the corresponding native proteins (36 and 47 kDa). Similar to other reported ehrlichial immunoreactive glycoproteins, carbohydrate was detected on the recombinant expressed proteins, indicating that they were glycoproteins. Both glycoproteins (gp36 and gp47) have carboxy-terminal serine/threonine-rich tandem repeat regions containing repeats that vary in number (4 to 16 repeats) and amino acid sequence among different isolates of each species. E. canis gp36 was recognized by early acute-phase antibodies (day 14), and species-specific antibody epitopes were mapped to C-terminal nonhomologous repeat units of gp36 and gp47. Periodate treatment of recombinant gp36 reduced the antibody reactivity, and nonglycosylated synthetic peptide repeat units from E. canis gp36 and E. chaffeensis gp47 were substantially less immunoreactive than corresponding recombinant peptides, demonstrating that glycans are important epitope determinants that are structurally conserved on the recombinant proteins expressed in Escherichia coli. E. canis gp36 and E. chaffeensis gp47 were differentially expressed only on the surface of dense-cored ehrlichiae and detected in the Ehrlichia-free supernatants, indicating that these proteins are released extracellularly during infection.

scholarly journals Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding.

1996 ◽  
Vol 135 (4) ◽  
pp. 1109-1123 ◽  
Author(s):  
M C Brown ◽  
J A Perrotta ◽  
C E Turner
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Amino Terminal ◽  
Deletion Mutagenesis ◽  
Lim Domains ◽  
Carboxyl Terminal

Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.

Sequence of an acidic ribosomal protein from the jellyfish Polyorchis penicillatus

1991 ◽  
Vol 69 (2-3) ◽  
pp. 211-215 ◽  
Author(s):  
Warren Gallin
Keyword(s):  
Amino Acids ◽  
Ribosomal Protein ◽  
Fruit Fly ◽  
Amino Terminal ◽  
Polyorchis Penicillatus ◽  
Eukaryotic Species ◽  
The Common ◽  
Carboxyl Terminal ◽  
Time Of Divergence ◽  
Acidic Ribosomal Protein

We have isolated a cDNA clone from the jellyfish Polyorchis penicillatus that encodes the homologue of the A1 acidic ribosomal protein previously characterized in human, brine shrimp, fruit fly, and yeast. The sequence of this protein is strongly conserved among the five eukaryotic species for which it has been determined. Conservation is greatest in the amino-terminal 51 amino acids and the carboxyl-terminal 25 amino acids. This suggests that these regions are necessary for interactions with other components of the protein synthetic machinery, while the central part of the protein has a less specific role to play. Comparison of the sequences obtained from the different species indicate that the metazoan lineages all appear to have arisen at approximately the same time and significantly later than the time of divergence of yeast from the common ancestor of the Metazoa.Key words: ribosome, protein synthesis, molecular evolution, nucleic acid sequence.

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