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Structural properties of 2/2 hemoglobins: the group III protein from Helicobacter hepaticus.

ABSTRACT: The ?-proteobacterium Helicobacter hepaticus (Hh) contains a gene coding for a hemoglobin (Hb). The protein belongs to the 2/2 Hb lineage and is representative of group III, a set of Hbs about which little is known. An expression and purification procedure was developed for Hh Hb. Electronic absorption and nuclear magnetic resonance (NMR) spectra were used to characterize ligation states of the ferric and ferrous protein. The pK(a) of the acid/alkaline transition of ferric Hh Hb was 7.3, an unusually low value. NMR analysis of the cyanomet complex showed the orientation of the heme group to be reversed when compared with most group I and group II 2/2 Hbs. Ferrous Hh Hb formed a stable cyanide complex that yielded NMR spectra similar to those of the carbonmonoxy complex. All forms of Hh Hb were self-associated at NMR concentrations. Comparison was made to the related Campylobacter jejuni 2/2 Hb (Ctb), and the amino acid conservation pattern of group III was reinspected to help in the generalization of structure-function relationships.

SUBMITTER: Nothnagel HJ

PROVIDER: S-EPMC3077074 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Structural properties of 2/2 hemoglobins: the group III protein from Helicobacter hepaticus.

Nothnagel Henry J HJ Winer Benjamin Y BY Vuletich David A DA Pond Matthew P MP Lecomte Juliette T J JT

IUBMB life 20110301 3

The ε-proteobacterium Helicobacter hepaticus (Hh) contains a gene coding for a hemoglobin (Hb). The protein belongs to the 2/2 Hb lineage and is representative of group III, a set of Hbs about which little is known. An expression and purification procedure was developed for Hh Hb. Electronic absorption and nuclear magnetic resonance (NMR) spectra were used to characterize ligation states of the ferric and ferrous protein. The pK(a) of the acid/alkaline transition of ferric Hh Hb was 7.3, an unus ...[more]

PMID: 21445851

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Project description:Helicobacter hepaticus can lead to chronic hepatitis and hepatocellular carcinoma in certain strains of mice. Until now the pathogenic role of Helicobacter species on human liver tissue is still not clarified though Helicobacter species identification in human liver cancer was successful in case controlled studies. Therefore we established an in vitro model to investigate the interaction of primary human hepatocytes (PHH) with Helicobacter hepaticus. Successful co-culturing of PHH with Helicobacter hepaticus was confirmed by visualization of motile bacteria by two-photon-microscopy. Isolated human monocytes were stimulated with PHH conditioned media. Changes in mRNA expression of acute phase cytokines and proteins in PHH and stimulated monocytes were determined by Real-time PCR. Furthermore, cytokines and proteins were analyzed in PHH culture supernatants by ELISA. Co-cultivation with Helicobacter hepaticus induced mRNA expression of Interleukin-1 beta (IL-1?), Tumor necrosis factor-alpha, Interleukin-8 (IL-8) and Monocyte chemotactic protein-1 (MCP-1) in PHH (p<0.05) resulting in a corresponding increase of IL-8 and MCP-1 concentrations in PHH supernatants (p<0.05). IL-8 and IL-1? mRNA expression was induced in monocytes stimulated with Helicobacter hepaticus infected PHH conditioned media (p<0.05). An increase of Cyclooxygenase-2 mRNA expression was observed, with a concomitant increase of prostaglandin E2 concentration in PHH supernatants at 24 and 48 h (p<0.05). In contrast, at day 7 of co-culture, no persistent elevation of cytokine mRNA could be detected. High expression of intercellular adhesion molecule-1 on PHH cell membranes after co-culture was shown by two-photon-microscopy and confirmed by flow-cytometry. Finally, expression of Cytochrome P450 3A4 and albumin mRNA were downregulated, indicating an impairment of hepatocyte synthesis function by Helicobacter hepaticus presence. This is the first in vitro model demonstrating a pathogenic effect of a Helicobacter spp. on human liver cells, resulting in an inflammatory response with increased synthesis of inflammatory mediators and consecutive monocyte activation.

Dataset Information

Structural properties of 2/2 hemoglobins: the group III protein from Helicobacter hepaticus.

Publications

Structural properties of 2/2 hemoglobins: the group III protein from Helicobacter hepaticus.

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