Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox

Akira Yamauchi, Lixin Yu, Andy J.G. Pötgens, Futoshi Kuribayashi, Hiroyuki Nunoi, Shiro Kanegasaki, Dirk Roos, Harry L. Malech, Mary C. Dinauer, Michio Nakamura

Research output: Contribution to journalArticle

55 Scopus citations

Abstract

Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X-linked chronic 23 disease by flow-cytometry. Therefore, were-evaluated the location of the 7D5 epitope using gene-engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the ∼58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycintreated PLB-985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.

Original languageEnglish
Pages (from-to)249-257
Number of pages9
JournalMicrobiology and Immunology
Volume45
Issue number3
DOIs
StatePublished - Jan 1 2001
Externally publishedYes

Keywords

  • 7D5
  • NADPH oxidase
  • Phagocytes
  • gp91

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