LSA-NRC is susceptible to TG2 cross-linking by both gpTG2 and hTG2 in vitro. As a monomer LSA-NRC is highly soluble, but upon cross-linking, LSA-NRC rapidly comes out of solution and is seen as a flocculent mass under in vitro cross-linking conditions. This is consistent with ultrastructural observations [19, 23] that described LSA-1 in 6 day post-infection primate liver sections as a 'fluffy flocculent mass'.
The presence of a potential CK2 phosphorylation site within the LSA-1 repeat region that overlaps the TG2 cross-linking site suggested that TG2-mediated cross-linking of LSA-1 may be regulated through CK2 phosphorylation. However, although this study demonstrated that LSA-NRC is phosphorylated in vitro by CK2 of parasite origin, this phosphorylation does not affect TG2 mediated cross-linking under our experimental conditions. However, it cannot be ruled out that phosphorylation has an effect on cross-linking, but that the proportion of phosphorylated substrate is too small in our conditions to allow detection in the cross-linking assay.
Tertiary structural Robetta modelling  predicted that each LSA-1 repeat sequence exists as a single α-helix resulting in an extended α-helical arrangement. This is consistent with previous analysis of the LSA-1 repeat peptides by circular dichroism suggesting that the repeat region of LSA-1 is an uninterrupted stretch of α-helices reaching a length of 220 nm . The α-helix model produced by Robetta modelling in this study showed that a Gln-Lys pair protrudes on either side of the repeat helix. By orientating successive LSA-1 molecules in opposite directions these pairs could bind to each other forming TG2-cross-linked bonds between molecules resulting in a flexible matrix type arrangement as seen with the transglutaminase-mediated cross-linking of fibrin during blood clotting . Incubation of the LSA-1 repeat peptide with gpTG2 resulted primarily in the formation of peptide dimers with very few trimers or tetramers, indicating that the majority of cross-linking was occurring at only one site on the peptide and that once this is bound no further cross-linking occurs. Further evidence indicating that the primary cross-linking site is the repeat region was provided by attempts to crosslink LSA-NRC-N and LSA-NRC-C proteins that lack any repeats: neither of these proteins was able to form multimers after incubation with TG2. However, LSA-NRC-C did show an increase in mobility during SDS-PAGE analysis suggesting that intramolecular cross-linking was occurring and leading to speculation that intramolecular cross-linking of the C-terminal of LSA-NRC may be responsible for the increased mobility seen in the full length LSA-NRC.
Obtaining human or primate livers infected with early stages of P. falciparum is either impossible or prohibitively expensive. Therefore, analysis of infected human liver sections derived from chimeric mice infected with P. falciparum [40, 41] has proved invaluable. That TG2-specific cross-linking does occur in vivo and that the location of this cross-linking is closely associated with that of LSA-1 was shown by incubating tissue sections derived from these livers with two different monoclonal antibodies that are specific to the very unique bond formed by the TG2 cross-linking, the ε-(γ-glutamyl)lysine cross-bridge. While this model system does provide tissue sections for analysis, the infection rate is not sufficient to allow purification of native LSA-1, and thus biochemical or biophysical analysis that would show that native LSA-1 is cross-linked by TG2. However, the in vitro data coupled with the in vivo co-localization of the unique ε-(γ-glutamyl)lysine cross-bridge with the LSA-1 tissue localization pattern observed strongly suggests the two are associated in vivo.
This then leads to speculation as to why LSA-1 needs to be cross-linked during infection. The internal repeat unit of LSA-1, about 85 copies of a 17 amino acid unit containing the TG2 substrate motif would suggest that its function is important. A typical P. falciparum infection involves the migration of the P. falciparum sporozoites through a number of liver cells prior to actually infecting a hepatocyte and forming a parasitophorous vacuole . Cellular damage to the liver has been shown to result in up regulation of TG2 expression in the damaged tissue . Additionally, TG2 activity has been shown to be present in P. falciparum and Plasmodium gallinaceum infected red blood cells . Thus it is likely that TG2 activity would be found at the site of P. falciparum infection. A P. falciparum infected hepatocyte experiences major internal reorganization as the parasite schizonts undergo massive expansion, with tens of thousands of merozoites being made in each infected cell. It is reasonable to speculate that in order to maximize the survival rate of the merozoites it would be advantageous for the parasite to maintain structural integrity of the host cell for as long as is feasibly possible. Construction of a dense cytoskeletal matrix formed with crosslinked LSA-1 would be possible to create a strong flexible cell that would allow rapid expansion but minimize the chance of rupture. However, if this were the case, why is LSA-1 protein not found in most other Plasmodium species? It is possible that a similar flocculent material seen in other Plasmodium species is functionally analogous to LSA-1, but differs in sequence, and a possible functional ortholog, identified by synteny mapping  in Plasmodium berghei, that may play a similar role.
It has recently been shown in P. berghei that merozoites are released in 'merosomes' - clusters of merozoites that bud off from the main hepatocyte, taking a protective layer of the host membrane with them . Prior to merosome formation, Plasmodium liver stages seem to protect the host cell from apoptosis  through hepatocyte growth factor (HGF) signaling of its receptor MET, but may undergo autophagy induced by the huge growth of the liver stage parasite . HGF/MET signaling may also occur during sporozoite invasion of hepatocytes, again blocking apoptosis. LSA-1, or analogous flocculent material, may therefore play a vital role in maintaining cell integrity during autophagy and merosome formation, TG2 has been shown to play an essential role in conferring resistance to damage in the liver . Plasmodium falciparum may be using this response to maintain the structural integrity of the infected hepatocyte. Reinforcement of the cell by a LSA-1 matrix could play a role in reducing the chance of hepatocyte death by apoptosis.
These studies suggest that recombinant LSA-1 is a TG2 substrate in vitro and that the unique modification made by TG2 to the protein can be detected in vivo in a pattern consistent with LSA-1 protein localization; this is the first study suggesting a functional role for LSA-1.