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Cited by 280Neutrophil, Myeloperoxidase and Oxidative MechanismsRedox biology and oxidative stressNitric Oxide and Endothelin Effects

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Proteins Homologous to p47 and p67 Support Superoxide Production by NAD(P)H Oxidase 1 in Colon Epithelial Cells

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Geiszt, Miklós, Lekstrom, Kristen, Witta, Jassir, Leto, Thomas L.·2003·Journal of Biological Chemistry
DOI 10.1074/jbc.m301289200
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Abstract

Superoxide production by phagocytes involves activation of a multi-component NADPH oxidase. Recently, several homologues of the catalytic component of the phagocyte oxidase, gp91phox, were identified in various tissues. Here we describe two proteins, p41 and p51, with significant homology to two cytosolic components of the phagocytic oxidase, p47phox and p67phox. Like p47phox, p41 contains an amino-terminal Phox homology domain, two SH3 domains, and a conserved carboxyl-terminal, proline-rich motif. Similarly, p51 is homologous to p67phox, containing four amino-terminal tetratrico-peptide repeats, a conserved "activation domain" motif, a PB1 domain, and a carboxyl-terminal SH3 domain. The highest levels of p41 transcript are detected in the colon and in other gastrointestinal tissues that express Nox1, the predominant gp91phox homologue in these tissues. In contrast, the p51 transcript showed a more widespread expression pattern, suggesting that it may support other tissue-specific oxidases. Mouse colon in situ hybridization detected both transcripts in the epithelial cells of colon crypts. Heterologous co-expression of p41 and p51 significantly enhances the superoxide-generating activity of Nox1-expressing cells; thus, p41 and p51 appear to be novel regulators of Nox1. These proteins also support the activity of gp91phox, albeit at much lower levels than the cytosolic phox counterparts. Our results suggest colon epithelial cells contain a multi-component NAD(P)H oxidase with a molecular architecture similar to the phagocytic oxidase.

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Figures

FIG. 1 Comparison of the deduced amino acid sequence of human p41 (GenBank TM accession number AY255768) with its phagocyte oxidase homologue, p47 phox . A , the sequence alignment denotes identical residues by ':' and conservative substitutions by '.'. Structural domains are designated as follows: PX domains ( dotted underline ), P XX P motif ( double underline ), SH3 domains ( dashed underline ), proline-rich motifs ( boxed sequence ), phosphorylated serines in p47 phox ( single underline ). B , schematic representation of structural domains within p41 and p47 phox . The phosphorylated serine/arginine-rich (autoinhibitory) domain is unique to p47 phox .
FIG. 1 Comparison of the deduced amino acid sequence of human p41 (GenBank TM accession number AY255768) with its phagocyte oxidase homologue, p47 phox . A , the sequence alignment denotes identical residues by ':' and conservative substitutions by '.'. Structural domains are designated as follows: PX domains ( dotted underline ), P XX P motif ( double underline ), SH3 domains ( dashed underline ), proline-rich motifs ( boxed sequence ), phosphorylated serines in p47 phox ( single underline ). B , schematic representation of structural domains within p41 and p47 phox . The phosphorylated serine/arginine-rich (autoinhibitory) domain is unique to p47 phox .CC-BY · From the PDF
FIG. 2 Detection of p41 mRNA in colon epithelium. A , Northern blotting of human tissues, showing high expression in the colon. Lanes were loaded with 2 𝛍 g of poly(A) + RNA. B , Northern blot detection of Nox1 and p41 mRNAs ( upper and lower panels , respectively) in human gastro-intestinal tissues. C , in situ hybridization of mouse colon cross-sections to [ 35 S]UTP-labeled p41 antisense RNA probe, showing specific expression in colon crypts. Shown is a bright-field image of Giemsa-stained tissue, superimposed with an epi-illumination dark-field image, in which the light reflected from exposed silver grain signal appears red. D , control p41 sense-strand probe in situ hybridization performed identically to C , showing the absence of specific signal.
FIG. 2 Detection of p41 mRNA in colon epithelium. A , Northern blotting of human tissues, showing high expression in the colon. Lanes were loaded with 2 𝛍 g of poly(A) + RNA. B , Northern blot detection of Nox1 and p41 mRNAs ( upper and lower panels , respectively) in human gastro-intestinal tissues. C , in situ hybridization of mouse colon cross-sections to [ 35 S]UTP-labeled p41 antisense RNA probe, showing specific expression in colon crypts. Shown is a bright-field image of Giemsa-stained tissue, superimposed with an epi-illumination dark-field image, in which the light reflected from exposed silver grain signal appears red. D , control p41 sense-strand probe in situ hybridization performed identically to C , showing the absence of specific signal.CC-BY · From the PDF
FIG. 3 Comparison of deduced amino acid sequences of p51 and p67 phox . A , alignment of murine and human p51 sequences (GenBank TM accession numbers AY255770 and AY255769, respectively) with that of human p67 phox , showing conserved tetratrico-peptide repeats ( TPR ; dashed underline ), activation domain motifs ( boxed ), SH3 domains ( underlined ), and PB1 domains ( italics , underlined ). B , schematic representation of structural domains in murine and human p51 homologues of p67 phox . Human and murine p51 exhibit the greatest structural differences within central regions. Both homologues lack a central SH3 domain.
FIG. 3 Comparison of deduced amino acid sequences of p51 and p67 phox . A , alignment of murine and human p51 sequences (GenBank TM accession numbers AY255770 and AY255769, respectively) with that of human p67 phox , showing conserved tetratrico-peptide repeats ( TPR ; dashed underline ), activation domain motifs ( boxed ), SH3 domains ( underlined ), and PB1 domains ( italics , underlined ). B , schematic representation of structural domains in murine and human p51 homologues of p67 phox . Human and murine p51 exhibit the greatest structural differences within central regions. Both homologues lack a central SH3 domain.CC-BY · From the PDF
Image 4
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FIG. 5 p41 and p51 together support superoxide release by Nox1 and gp91phox-expressing cells. A , chemiluminescence assays of superoxide release by CaCo2 cells transfected 24 h with p41, p51, or both cDNAs. B , superoxide release by Nox1-expressing NIH-3T3 cells transiently transfected (24 h) with p41 and p51 or their phox homologues, p47 phox and p67 phox . C , superoxide release by gp91 phox -expressing K562 cells transiently transfected (66 h) with p41 and p51 or their phox homologues, p47 phox and p67 phox . Data represent the mean of triplicate assays. Similar results were obtained in four separate transfection experiments.
FIG. 5 p41 and p51 together support superoxide release by Nox1 and gp91phox-expressing cells. A , chemiluminescence assays of superoxide release by CaCo2 cells transfected 24 h with p41, p51, or both cDNAs. B , superoxide release by Nox1-expressing NIH-3T3 cells transiently transfected (24 h) with p41 and p51 or their phox homologues, p47 phox and p67 phox . C , superoxide release by gp91 phox -expressing K562 cells transiently transfected (66 h) with p41 and p51 or their phox homologues, p47 phox and p67 phox . Data represent the mean of triplicate assays. Similar results were obtained in four separate transfection experiments.CC-BY · From the PDF
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  1. Teshima, 2000, Am. J. Physiol., 279, G1169

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Metadata

Authors
Geiszt, M., Lekstrom, K., Witta, J., & Leto, T. L.
Affiliations
  • Semmelweis University (HU)
  • National Institutes of Health (US)
  • Uniformed Services University of the Health Sciences (US)
Corresponding author
Leto, Thomas L.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2003-05
Volume
278
Issue
22
Pages
20006-20012
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m301289200
OpenAlex
W2022385815
Cited by
280
References
28
Citations by year
2026: 1 · 2025: 4 · 2024: 3 · 2023: 5 · 2022: 7 · 2021: 2 · 2020: 5 · 2019: 10 · 2018: 5 · 2017: 5 · 2016: 3 · 2015: 4 · 2014: 11 · 2013: 10 · 2012: 15
FWCI
10.24
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
Keywords
NOX1, p41, p47phox, p67phox, superoxide production, colonic epithelial cells, gp91phox, phox homology domain, SH3 domain, PB1 domain, colonic crypts, gastrointestinal tissue
MeSH terms
Amino Acid Sequence, Animals, Colon, DNA-Binding Proteins, Epithelial Cells, Molecular Sequence Data, Phosphoproteins, RNA, Messenger, Superoxides, Transcription Factors, Trans-Activators, Genes, Tumor Suppressor, Sequence Homology, Amino Acid, In Situ Hybridization, NADPH Oxidases, Tumor Suppressor Proteins, Mice

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