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Identification of functional domains of Bordetella dermonecrotizing toxin.

ABSTRACT: Bordetella dermonecrotizing toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rho. To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutants. Our analyses of the nucleotide and amino acid sequences revealed that the start codon of the DNT gene is a GTG triplet located 39 bp upstream of the reported putative initiation ATG codon; consequently, DNT contains an additional 13 amino acids at its N-terminal end. All of the N-terminally truncated mutants were found to modify Rho. The shortest fragment of DNT possessing the Rho modification activity consists of amino acids from Ile1176 to the C-terminal end. This fragment overlaps the region homologous to Escherichia coli cytotoxic necrotizing factors (CNFs), which show activity similar to that of DNT. The introduction of a mutation at Cys1305 located in the highly conserved region between CNFs and DNT eliminated the activity, indicating that this domain is the catalytic center of DNT. The N-terminal fragment (1 to 531) of DNT failed to modify Rho but reduced the DNT-induced polynucleation in MC3T3-E1 cells when simultaneously added with the holotoxin, suggesting competitive inhibition in the receptor-binding or internalizing step. Our finding that DNT consists of an N-terminal receptor-binding and/or internalizing domain and a C-terminal catalytically active domain may facilitate analysis of the overall action of the toxin on the mammalian target cells.

SUBMITTER: Kashimoto T

PROVIDER: S-EPMC96646 | biostudies-literature | 1999 Aug

REPOSITORIES: biostudies-literature

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Identification of functional domains of Bordetella dermonecrotizing toxin.

Kashimoto T T Katahira J J Cornejo W R WR Masuda M M Fukuoh A A Matsuzawa T T Ohnishi T T Horiguchi Y Y

Infection and immunity 19990801 8

Bordetella dermonecrotizing toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rho. To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutants. Our analyses of the nucleotide and amino acid sequences revealed that the start codon of the DNT gene is a GTG triplet located 39 bp upstream of the reported putative initiation ATG codon; conseque ...[more]

PMID: 10417130

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Project description:Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein-Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152-160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1beta-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-alpha production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-gamma-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Dataset Information

Identification of functional domains of Bordetella dermonecrotizing toxin.

Publications

Identification of functional domains of Bordetella dermonecrotizing toxin.

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