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Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase.

Nicoll WS, Sacci JB, Rodolfo C, Di Giacomo G, Piacentini M, Holland ZJ, Doerig C, Hollingdale MR, Lanar DE - Malar. J. (2011)

Bottom Line: The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro.Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo.While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

View Article: PubMed Central - HTML - PubMed

Affiliation: USMMVP, Malaria Department, NMRC, Silver Spring, MD 20910, USA. mikedc110@gmail.com

ABSTRACT

Background: Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined.

Methods: Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts.

Results: This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo.

Conclusions: While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

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Analysis of cross-linking site. A. PAGE analysis of LSA-NRC TG2-cross-linking in the absence (i) or presence (ii) of peptide corresponding to the major repeat sequence of LSA-1. B. RP-HPLC analysis of a peptide corresponding to the major repeat sequence of LSA-1 before (i) and after (ii) gpTG2 treatment for 2 h at 100 μg/ml gpTG2. Position of monomers [retention time 23.3 min] (1), dimers [retention time 24.5 min] (2) and trimers [retention time 25.6 min] (3) are indicated. (ii). C. Tertiary structure of a single LSA-1 major repeat as predicted by Robetta modeling. Arrows indicate glutamines and lysines predicted to be involved in TG2 mediated cross-linking. D. PAGE analysis of gpTG2 cross-linking of LSA-NRC-C (i) and LSA-NRC-N (ii). * indicates band formed by the gpTG2 enzyme (MW - 76.6 kDa).
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Figure 3: Analysis of cross-linking site. A. PAGE analysis of LSA-NRC TG2-cross-linking in the absence (i) or presence (ii) of peptide corresponding to the major repeat sequence of LSA-1. B. RP-HPLC analysis of a peptide corresponding to the major repeat sequence of LSA-1 before (i) and after (ii) gpTG2 treatment for 2 h at 100 μg/ml gpTG2. Position of monomers [retention time 23.3 min] (1), dimers [retention time 24.5 min] (2) and trimers [retention time 25.6 min] (3) are indicated. (ii). C. Tertiary structure of a single LSA-1 major repeat as predicted by Robetta modeling. Arrows indicate glutamines and lysines predicted to be involved in TG2 mediated cross-linking. D. PAGE analysis of gpTG2 cross-linking of LSA-NRC-C (i) and LSA-NRC-N (ii). * indicates band formed by the gpTG2 enzyme (MW - 76.6 kDa).

Mentions: To assess whether the predicted TG2 glutamine substrate in the LSA-1 repeat region was in fact a TG2 substrate, LSA-NRC was incubated with TG2 and a LSA-1 repeat peptide containing a single repeat unit. As can be seen in Figure 3A, inclusion of the peptide resulted in blocking the shift in mobility, suggesting a reduction in intra-LSA-NRC cross-linking. Interestingly, the mobility of LSA-NRC-peptide decreased over time suggesting that multiple repeat peptides are being successively crosslinked to the LSA-NRC monomer, gradually increasing its molecular weight.


Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase.

Nicoll WS, Sacci JB, Rodolfo C, Di Giacomo G, Piacentini M, Holland ZJ, Doerig C, Hollingdale MR, Lanar DE - Malar. J. (2011)

Analysis of cross-linking site. A. PAGE analysis of LSA-NRC TG2-cross-linking in the absence (i) or presence (ii) of peptide corresponding to the major repeat sequence of LSA-1. B. RP-HPLC analysis of a peptide corresponding to the major repeat sequence of LSA-1 before (i) and after (ii) gpTG2 treatment for 2 h at 100 μg/ml gpTG2. Position of monomers [retention time 23.3 min] (1), dimers [retention time 24.5 min] (2) and trimers [retention time 25.6 min] (3) are indicated. (ii). C. Tertiary structure of a single LSA-1 major repeat as predicted by Robetta modeling. Arrows indicate glutamines and lysines predicted to be involved in TG2 mediated cross-linking. D. PAGE analysis of gpTG2 cross-linking of LSA-NRC-C (i) and LSA-NRC-N (ii). * indicates band formed by the gpTG2 enzyme (MW - 76.6 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037345&req=5

Figure 3: Analysis of cross-linking site. A. PAGE analysis of LSA-NRC TG2-cross-linking in the absence (i) or presence (ii) of peptide corresponding to the major repeat sequence of LSA-1. B. RP-HPLC analysis of a peptide corresponding to the major repeat sequence of LSA-1 before (i) and after (ii) gpTG2 treatment for 2 h at 100 μg/ml gpTG2. Position of monomers [retention time 23.3 min] (1), dimers [retention time 24.5 min] (2) and trimers [retention time 25.6 min] (3) are indicated. (ii). C. Tertiary structure of a single LSA-1 major repeat as predicted by Robetta modeling. Arrows indicate glutamines and lysines predicted to be involved in TG2 mediated cross-linking. D. PAGE analysis of gpTG2 cross-linking of LSA-NRC-C (i) and LSA-NRC-N (ii). * indicates band formed by the gpTG2 enzyme (MW - 76.6 kDa).
Mentions: To assess whether the predicted TG2 glutamine substrate in the LSA-1 repeat region was in fact a TG2 substrate, LSA-NRC was incubated with TG2 and a LSA-1 repeat peptide containing a single repeat unit. As can be seen in Figure 3A, inclusion of the peptide resulted in blocking the shift in mobility, suggesting a reduction in intra-LSA-NRC cross-linking. Interestingly, the mobility of LSA-NRC-peptide decreased over time suggesting that multiple repeat peptides are being successively crosslinked to the LSA-NRC monomer, gradually increasing its molecular weight.

Bottom Line: The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro.Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo.While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

View Article: PubMed Central - HTML - PubMed

Affiliation: USMMVP, Malaria Department, NMRC, Silver Spring, MD 20910, USA. mikedc110@gmail.com

ABSTRACT

Background: Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined.

Methods: Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts.

Results: This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo.

Conclusions: While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

Show MeSH
Related in: MedlinePlus