Shared Ly6/uPAR structural features and epitope mapping of CD59 and the Crotalus neutralizing factor (CNF).

Abstract

Structural motifs can be conserved among proteins with low primary-sequence identity, potentially supporting distinct yet related molecular functions. Endogenous phospholipase A2 inhibitors from snake plasma (sbPLIs) include the γ-type inhibitor from Crotalus durissus terrificus, known as Crotalus neutralizing factor (CNF), which protects the snake by selectively inhibiting the lethal and PLA2 activities of crotoxin (CTx). CD59, a complement regulator and single-LU-domain member of the Ly6/uPAR superfamily, shares the three-finger fold with CNF, raising the possibility of structural and functional convergence. Here, CD59 linear peptide arrays produced by SPOT synthesis were probed with CB, the PLA2 subunit of CTx, to identify CD59 regions capable of binding CB. Two reactive clusters were mapped, and derived peptides were synthesized and tested for their ability to inhibit the PLA2 activity of CTx and CB in vitro. Four CD59-derived peptides interacted with CB on the arrays; however, none inhibited PLA2 activity, in contrast to native CNF used as a positive control. These findings indicate that, although CD59 contains linear segments that recognize CB, this interaction is not sufficient to reproduce the PLA2-inhibitory function of CNF, suggesting that any functional interplay between these Ly6/uPAR proteins is likely to depend on more complex structural determinants.