The human CD10 lacking an N-glycan at Asn628 is deficient in surface expression and neutral endopeptidase activity |
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Authors: | Ban Sato Yohko U. Katagiri Kazutoshi Iijima Hiroyuki Yamada Satsuki Ito Nana Kawasaki Hajime Okita Junichiro Fujimoto Nobutaka Kiyokawa |
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Affiliation: | 1. Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157–8535, Japan;2. Division of Biological Chemistry and Biologicals, National Institute of Health Science, 1-18-1 Kami-yoga, Setagaya-ku, Tokyo 158–8501, Japan;3. Clinical Research Center, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157–8535, Japan |
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Abstract: | BackgroundCD10, 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 pre-B-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography.MethodsIn 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.ResultsCD10 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 Asn628-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities.ConclusionsN-glycosylation at Asn628 is essential not only for NEP activities, but also for surface expression.General significanceQuality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane. |
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Keywords: | CD10 N-glycan Neutral endopeptidase Glycopeptidase F Site-directed mutagenesis Common acute lymphoblastic leukemia antigen (CALLA) |
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