The human CD10 lacking an N-glycan at Asn628 is deficient in surface expression and neutral endopeptidase activity

Ban Sato, Yohko U. Katagiri, Kazutoshi Iijima, Hiroyuki Yamada, Satsuki Ito, Nana Kawasaki, Hajime Okita, Junichiro Fujimoto, Nobutaka Kiyokawa

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 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. 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 Asn628-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities. Conclusions: N-glycosylation at Asn628 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.

Original languageEnglish
Pages (from-to)1715-1723
Number of pages9
JournalBiochimica et Biophysica Acta - General Subjects
Volume1820
Issue number11
DOIs
Publication statusPublished - 2012 Nov
Externally publishedYes

Fingerprint

Neprilysin
Polysaccharides
Glycosylation
HEK293 Cells
Cells
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Cell Line
Mutagenesis
B-Lymphoid Precursor Cells
Glycoside Hydrolases
X ray crystallography
X Ray Crystallography
Cell membranes
Site-Directed Mutagenesis
Sugars
Quality Control
Quality control
Peptide Hydrolases
B-Lymphocytes
Cell Membrane

Keywords

  • CD10
  • Common acute lymphoblastic leukemia antigen (CALLA)
  • Glycopeptidase F
  • N-glycan
  • Neutral endopeptidase
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

The human CD10 lacking an N-glycan at Asn628 is deficient in surface expression and neutral endopeptidase activity. / Sato, Ban; Katagiri, Yohko U.; Iijima, Kazutoshi; Yamada, Hiroyuki; Ito, Satsuki; Kawasaki, Nana; Okita, Hajime; Fujimoto, Junichiro; Kiyokawa, Nobutaka.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1820, No. 11, 11.2012, p. 1715-1723.

Research output: Contribution to journalArticle

Sato, Ban ; Katagiri, Yohko U. ; Iijima, Kazutoshi ; Yamada, Hiroyuki ; Ito, Satsuki ; Kawasaki, Nana ; Okita, Hajime ; Fujimoto, Junichiro ; Kiyokawa, Nobutaka. / The human CD10 lacking an N-glycan at Asn628 is deficient in surface expression and neutral endopeptidase activity. In: Biochimica et Biophysica Acta - General Subjects. 2012 ; Vol. 1820, No. 11. pp. 1715-1723.
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T1 - The human CD10 lacking an N-glycan at Asn628 is deficient in surface expression and neutral endopeptidase activity

AU - Sato, Ban

AU - Katagiri, Yohko U.

AU - Iijima, Kazutoshi

AU - Yamada, Hiroyuki

AU - Ito, Satsuki

AU - Kawasaki, Nana

AU - Okita, Hajime

AU - Fujimoto, Junichiro

AU - Kiyokawa, Nobutaka

PY - 2012/11

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AB - 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 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. 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 Asn628-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities. Conclusions: N-glycosylation at Asn628 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.

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KW - Common acute lymphoblastic leukemia antigen (CALLA)

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KW - Site-directed mutagenesis

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