Journal
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
Volume 1820, Issue 11, Pages 1715-1723Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbagen.2012.06.017
Keywords
CD10; N-glycan; Neutral endopeptidase; Glycopeptidase F; Site-directed mutagenesis; Common acute lymphoblastic leukemia antigen (CALLA)
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Funding
- Health and Labour Sciences Research Grants [H22-011]
- National Center for Child Health and Development [22A-5]
- National Institute of Biomedical Innovation (NIBIO) [10-41, 10-42, 10-43, 10-44, 10-45]
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Background: CD10, also known as neprilysin or enkephalinase exhibiting neutral endopeptidase (NEP) activity, is expressed by B-lineage hematopoietic cells as well as a variety of cells from normal tissues. It cleaves peptides such as cytokines to act for terminating inflammatory responses. Although CD10 molecules of the human preB-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography. Methods: In order to investigate the role of N-glycans in the full expression of NEP activity, we modified N-glycans by treatment of NALM6 cells with various glycosidases or alter each of the consensus N-glycosylation sites by generating site-directed mutagenesis and compared the NEP activities of the sugar-altered CD10 with those of intact CD10. Results: CD10 of the human B-cell line NALM-6 was dominantly localized in raft microdomains and heterogeneously N-glycosylated. Although neither desialylation nor further degalactosylation caused defective NEP activity, removal of only a small part of N-glycans by treatment with glycopeptidase F under non-denaturing conditions decreased NEP activity completely. All of the three consensus sites of CD10 in HEK293 cells introduced with wild type-CD10 were confirmed to be N-glycosylated. Surface expression of N-glycan at Asn(628)-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities. Conclusions: N-glycosylation at Asn(628) is essential not only for NEP activities, but also for surface expression. General significance: Quality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane. (C) 2012 Elsevier B.V. All rights reserved.
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