Gasa1 Gasa2

Veterinary Parasitology

Volume 287, November 2020, 109275

Characterization of GASA-1, a new vaccine candidate antigen of Babesia bovis

Author links open overlay panelDaniela A.FloresabMonicaFlorin-Christensenab

Outline

Cite

https://doi.org/10.1016/j.vetpar.2020.109275Get rights and content

Highlights

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Babesia bovis predicted GPI anchored antigen GASA-1 is highly conserved.

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B. bovis gasa-1 gene has syntenic orthologues in B. bigemina and B. ovata.

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B. bovis merozoites expose GASA-1 B-cell epitopes on their surface.

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B. bovis-infected bovines produce anti-GASA-1 antibodies.

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GASA-1 bears neutralization sensitive B-cell and T-cell epitopes.

Abstract

Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.

Plant Science

Volume 260, July 2017, Pages 71-79

The Arabidopsis GASA10 gene encodes a cell wall protein strongly expressed in developing anthers and seeds

Author links open overlay panelMenelaosTrapalisRoger W.Parish

Outline

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https://doi.org/10.1016/j.plantsci.2017.04.003Get rights and content

Highlights

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GASA10 gene is primarily expressed in developing anthers and seeds.

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GASA10 protein is located in the cell wall.

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Over-expressing GASA10 results in reduced silique elongation.

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GASA10 protein expressed in vitro does not possess antimicrobial activity.

Abstract

The Arabidopsis GASA10 gene encodes a GAST1-like (Gibberellic Acid-Stimulated) protein. Reporter gene analysis identified consistent expression in anthers and seeds. In anthers expression was developmentally regulated, first appearing at stage 7 of anther development and reaching a maximum at stage 11. Strongest expression was in the tapetum and developing microspores. GASA10 expression also occurred throughout the seed and in root vasculature. GASA10 was shown to be transported to the cell wall. Using GASA1 and GASA6 as positive controls, gibberellic acid was found not to induce GASA10 expression in Arabidopsis suspension cells. Overexpression of GASA10 (35S promoter-driven) resulted in a reduction in silique elongation. GASA10 shares structural similarities to the antimicrobial peptide snakin1, however, purified GASA10 failed to influence the growth of a variety of bacterial and fungal species tested. We propose cell wall associated GASA proteins are involved in regulating the hydroxyl radical levels at specific sites in the cell wall to facilitate wall growth (regulating cell wall elongation).

International Journal of Biochemistry

Volume 24, Issue 9, September 1992, Pages 1487-1491

Proteolytic processing sites producing the mature form of human cathepsin D

Author links open overlay panelTakahikoKobayashi1AkiraMakita1

Outline

1. The proteolytic processing sites of human lysosomal aspartic protease cathepsin D at which the intermediate single-chain form was converted into the mature two-chain form were determined.

2. The two chains were isolated by reversed-phase HPLC in order to investigate the cleavage sites of the enzyme.

3. Protein sequencing of the heavy chain, which was presumed to be derived from the C-terminal side in the single-chain enzyme, gave an N-terminal Leu 105. In addition, it revealed that there were also minor sequences, which commenced with Gly 106 and Gly 107.

4. A small C-terminal peptide was isolated from the light chain, which had been digested with two kinds of exogenous proteases. Sequence determination of this peptide, which was characterized as a nonapeptide by mass spectrometry, suggested that the C-terminus of the light chain was Ser 98.

5. These results indicate that a Ser 98-Ala 99 bond and an Ala 104-Leu 105 bond are cleaved to release 6 amino acid residues between the two chains.

The NOD Idd9 Genetic Interval Influences the Pathogenicity of Insulitis and Contains Molecular Variants of Cd30, Tnfr2, and Cd137

Previous analyses of NOD mice have shown that some genes control the development of both insulitis and diabetes, while other loci influence diabetes without reducing insulitis. Evidence for the existence of a gene only influencing diabetes, Idd9 on mouse chromosome 4, is provided here by the development of a novel congenic mouse strain, NOD.B10 Idd9. NOD.B10 Idd9 mice display profound resistance to diabetes even though nearly all develop insulitis. Subcongenic analysis has demonstrated that alleles of at least three B10 genes, Idd9.1, Idd9.2, and Idd9.3 are required to produce Idd9-mediated diabetes resistance. Candidate genes with amino acid differences between the NOD and B10 strains have been localized to the 5.6 cM Idd9.2 interval (Tnfr2, Cd30) and to the 2.0 cM Idd9.3 interval (Cd137).

Female (A) and male (B) NOD, NOD.B10 Idd9, and (NOD × NOD.B10 Idd9)F1 mice were monitored for the development of diabetes for 210 days. For females, comparisons of the 7 month cumulative diabetes frequencies of the two parental strains as well as the F1 strain versus both parental strains had p values of <10−4 (Fisher's exact test). For males, comparison of the two parental strains had a p value of <10−4. The F1 differed from NOD with a p value of 0.0005 and from the NOD.B10 Idd9 strain with a p value of 0.0031.

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