Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase.
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.
USMMVP, Malaria Department, NMRC, Silver Spring, MD 20910, USA. mikedc110@gmail.com
William S
WS
Nicoll
John B
JB
Sacci
Carlo
C
Rodolfo
Giuseppina
G
Di Giacomo
Mauro
M
Piacentini
Zoe Jm
ZJ
Holland
Christian
C
Doerig
Michael R
MR
Hollingdale
David E
DE
Lanar
D000818:Animals; D000953:Antigens, Protozoan / Q000378:metabolism*; D006790:Host-Parasite Interactions; D006801:Humans; D008099:Liver / Q000469:parasitology* / Q000473:pathology; D016778:Malaria, Falciparum / Q000469:parasitology*; D051379:Mice; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D010963:Plasmodium falciparum / Q000472:pathogenicity*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases / Q000378:metabolism*
D000953:Antigens, Protozoan; C000717608:TGM2 protein, human; C067302:liver stage-specific antigen, Plasmodium; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases
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