Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions

Biochimie
Ngoc Nguyen LundeRigmor Solberg

Abstract

The asparaginyl endopeptidase legumain and its inhibitor cystatin E/M are endogenously glycosylated. However, little is known about the nature of the carbohydrate groups and whether they affect the functions of these proteins. In this study both glycosylated and unglycosylated forms of legumain and cystatin E/M were studied. HEK293 cell lines stably over-expressing legumain or cystatin E/M, and HCT116 cells were used as cell models, and mature legumain was purified from bovine kidneys. To obtain unglycosylated proteins, cells were treated with tunicamycin, an inhibitor of N-linked glycosylation, whereas PNGase F and Endo H were used to characterize the glycosylation types. Cells were incubated with glycosylated, unglycosylated proteins and/or legumain selective activity-based probe, and legumain and/or cystatin E/M was studied by activity measurement, ELISA or immunoblotting in cell lysates or conditioned media. Legumain and probe in whole cells were studied by immunofluorescence. The carbohydrates on legumain were shown to be of the hybrid or high mannose type, whereas cystatin E/M was characterized as complex mannose-linked. While glycosylated prolegumain was able to autoactivate, the unglycosylated form was not, and addition...Continue Reading

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