Ixodes ricinus asparaginyl endopeptidase (IrAE1)
was the first legumain identified from arthropods. IrAE1 has been
characterized as a pivotal cysteine endopeptidase of the multienzyme
hemoglobinolytic machinery in ticks. Genomic and EST data from closely related
American tick Ixodes scapularis
complement IrAE1 with a full–set of nine legumain-like peptidases, IsAE1-9. IsAE1-3
possess the active site triad and are thus capable of protein bond hydrolysis. Following
recent discussion, we propose that isoforms with the active site cysteine
replaced by serine (IsAE4, 5, 8), and those with modified active site histidine
(IsAE4, 7, 9.), may function as protein ligases or carboxylases rather than
peptidases. Expressional qRT-PCR profiling over I. scapularis lifestages and tissues demonstrates that predominant abundance
of IsAE1 in female guts is accompanied by three other legumains tagged as IsAE2,
IsAE4, and IsAE9. Interestingly, IsAE2 replaces IsAE1 as the dominant gut tissue
legumain in tick males not feeding on hosts and instead living from blood reserves
stored from nymphal feeding. Additionally, IsAE9 with modified active site
histidine is also upregulated by bloodfeeding and could possess regulatory roles.
IrAE1 and IrAE2 have nearly identical in-silico
3D model structures, yet have
distinct properties. IrAE1 recombinantly expressed in yeast is self-catalytically
activatable in acidic conditions, and shows a strong preference for asparagine
in P1 position as seen from combinational peptidyl substrate library screenings.
It is also capable of endopeptidolytic activation (in-trans) of Schistosoma
mansoni cathepsin B (SmCB1). In contrast, IrAE2 expressed in yeast does not
exhibit self-catalytic activation as seen from fluorescent peptidyl assays and
is instead activated by IrAE1 in-trans.
In order to ascertain if IrAE1 and IrAE2 have fuctional differences, qRT-PCR, in-vivo LE-28 activity probe labeling
(Laura Edgington, Matthew Bogyo) and RNAi experiments are performed. E. coli expressed IrAE1 and IrAE2 are
also used for rabbit immunization and tested as anti-tick vaccines.