Figure 5.

Ameba lectins are substrates for EhROM1. (A) Transmembrane sequences (overlined) of ameba lectins EHI_012270 (Hgl) and EHI_044650 contain small residues in their upper regions (boxed) that are hallmarks of rhomboid substrates. The sequence of a glycine-to-valine mutant of EHI_044650 is also shown for comparison (mut). Only TM and surrounding residues are shown; the N-terminal lectin ectodomains are to the left, and cytoplasmic tails are to the right. (B) GFP-tagged E. histolytica heavy-chain lectin (EHI_044650) construct was cotransfected into COS cells with either high or low amounts of EhROM1 (hi or lo, respectively), the EhROM1 serine-to-alanine mutant (SA), or TgROM5 and PfROM4. Media and cell fractions were analyzed by anti-GFP Western, with prestained molecular mass standards denoted to the left of each image. EhROM1, TgROM5, and PfROM4 were able to process the GFP-lectin, as assessed by accumulation of the cleaved form in cell culture media. (C) Cleavage of wild type (WT) versus a glycine-to-valine mutant (mut) EHI_044650 lectin by cellular metalloproteases (MP), EhROM1 (EhR1, wild-type product denoted by arrow), and TgROM5 (TgR5) were compared. Note that the glycine-to-valine mutant abolished cleavage by EhROM1 but not by endogenous cellular metalloproteases (MP). Cells transfected with only the GFP-lectin construct were incubated without metalloprotease inhibitors (MP) to allow processing by cellular metalloproteases (inhibitors were included in the remaining transfections to facilitate detection of rhomboid-cleaved products).