Background: Both the cysteine protease legumain and its inhibitor cystatin E/M are endogenously glycosylated. However, little is known about the role of the carbohydrate groups for the functions of these proteins. In this study we have compared glycosylated and unglycosylated forms and studied their functions.
Methods: HEK293 cells stably over-expressing either legumain (M38L) or cystatin E/M (M4C) were used, secreting high amounts of the respective proteins into the conditioned media. In addition, the colorectal cancer cell line HCT116 was used. Cells treated with tunicamycin, an N-linked glycosylation inhibitor, secreted unglycosylated prolegumain or cystatin E/M.
Results: Glycosylated legumain or cystatin E/M were incubated with the deglycosylating enzymes PNGase F or Endo H and both proteins were shown to be N-glycosylated. The carbohydrates on legumain were hybrid or high mannose, whereas cystatin E/M was characterized as complex mannose. Lysates of M38L cells after treatment with tunicamycin showed reduced legumain activity and intracellular accumulation of the unglycosylated proform (47 kDa). HCT116 was used to perform a more detailed study of unglycosylated forms of legumain and low tunicamycin concentrations showed a gradual decrease in cellular legumain activity as well as increased secretion.
While glycosylated prolegumain was able to autoactivate at pH 4.0, the unglycosylated form did not undergo autoactivation. Addition of glycosaminoglycans did not accelerate autoactivation of unglycosylated prolegumain, as previously shown for the glycosylated form. Interestingly, both glycosylated and unglycosylated forms of cystatin E/M showed similar inhibition of purified legumain.
Conclusion: Glycosylation of prolegumain is necessary for correct processing and activation, whereas the inhibitory property of cystatin E/M is independent of the glycosylation status.