دورية أكاديمية
Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions.
العنوان: | Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions. |
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المؤلفون: | Lunde NN; Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway., Haugen MH; Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway., Bodin Larsen KB; Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway., Damgaard I; Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway; Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway., Pettersen SJ; Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway., Kasem R; Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway., Rut W; Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wroclaw, Poland., Drag M; Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wroclaw, Poland., Poreba M; Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wroclaw, Poland; Program in Tumor Microenvironment and Cancer Immunology, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, USA., Johansen HT; Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway., Solberg R; Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway; Program in Tumor Microenvironment and Cancer Immunology, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, USA. Electronic address: rigmor.solberg@farmasi.uio.no. |
المصدر: | Biochimie [Biochimie] 2017 Aug; Vol. 139, pp. 27-37. Date of Electronic Publication: 2017 May 17. |
نوع المنشور: | Journal Article |
اللغة: | English |
بيانات الدورية: | Publisher: Editions Scientifiques Elsevier Country of Publication: France NLM ID: 1264604 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1638-6183 (Electronic) Linking ISSN: 03009084 NLM ISO Abbreviation: Biochimie Subsets: MEDLINE |
أسماء مطبوعة: | Publication: Paris : Editions Scientifiques Elsevier Original Publication: Paris. |
مواضيع طبية MeSH: | Protein Processing, Post-Translational*, Cystatin M/*pharmacology , Cysteine Endopeptidases/*metabolism , Glycosaminoglycans/*metabolism , Mannose/*chemistry, Cysteine Endopeptidases/chemistry ; Glycosylation ; HCT116 Cells ; HEK293 Cells ; Humans |
مستخلص: | 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 of glycosaminoglycans did not facilitate autoactivation of unglycosylated prolegumain. Glycosylated prolegumain was internalized and processed to the mature active form, but no internalization of unglycosylated prolegumain was observed. A Cy5-labelled legumain specific activity-based probe (MP-L09) was synthesized and shown to be a novel tool to study intracellular legumain. Also, internalization of mature legumain (36 kDa) was visualized both alone and complexed with probe. Contrary to the importance of legumain glycosylation, both glycosylated and unglycosylated cystatin E/M showed similar capacity to inhibit legumain. In conclusion, glycosylation of prolegumain is necessary for correct processing to active forms and internalization, whereas the inhibitory property of cystatin E/M is independent of the glycosylation status. (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.) |
فهرسة مساهمة: | Keywords: Activity-based probe; Asparaginyl endopeptidase (AEP); Cystatin E/M; Glycosylation; Internalization; Legumain |
المشرفين على المادة: | 0 (CST6 protein, human) 0 (Cystatin M) 0 (Glycosaminoglycans) EC 3.4.22.- (Cysteine Endopeptidases) EC 3.4.22.34 (asparaginylendopeptidase) PHA4727WTP (Mannose) |
تواريخ الأحداث: | Date Created: 20170522 Date Completed: 20170912 Latest Revision: 20170912 |
رمز التحديث: | 20231215 |
DOI: | 10.1016/j.biochi.2017.05.009 |
PMID: | 28528272 |
قاعدة البيانات: | MEDLINE |
تدمد: | 1638-6183 |
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DOI: | 10.1016/j.biochi.2017.05.009 |