Pycnodysostosis with heterozygous beta-thalassemia

Benz, G.; Schmid-Rüter, E

doi:10.1007/bf00973984

Cited by 13 publications

(14 citation statements)

References 11 publications

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“…Comparison of the single-channel conductances published previously for PhoE protein [37] and the data presented here reveal a substantial difference. For instance in 1 M KC1, an average single-channel conductance of 1.8 nS has been reported [37] whereas 0.66 nS was found for the pMRO5-encoded protein in the present manuscript (Table 4).…”

Section: Discussionsupporting

confidence: 45%

“…These steps were specific for the presence of the different porins and were not observed if the detergent SDS alone was present in the experiments. These steps showed that PhoE protein encoded by pMRO5 and the mutant porins form defined pores in lipid bilayer membranes, although the single-channel conductances for all pores, including those encoded by pMRO5, were substantially smaller than the values previously published for wild-type PhoE protein [37].…”

Section: In Vitro Pore Function Of the Mutant Porinsmentioning

confidence: 72%

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Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

1987

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Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed [Korteland et al. (1985) Eur. J . Biochem. 152,. To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12 -74 bp were inserted. The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of p-lactam antibiotics. The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages. The functioning of the mutant pores was characterized both in vivo by studying the uptake of P-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films. The pore characteristics changed depending on the nature of the inserted amino acids. Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.The outer membrane of Enterobacteriaceae functions as a barrier for harmful compounds. The membrane contains a number of proteins, that form transmembrane channels, through which small hydrophilic solutes can pass in a diffusion-like process [I]. Under standard laboratory conditions, Escherichia coli K-12 produces two general pore-forming proteins, OmpC protein and OmpF protein. When cells are grown under phosphate limitation, the synthesis of another pore protein, PhoE protein, is induced [2]. This protein forms pores which are more efficient for phosphate-containing compounds and for negatively charged compounds in general in contrast to the cation-selective OmpC and OmpF pores [3, 41. The primary structures of the OmpC, OmpF and PhoE proteins show an extensive homology of approximately 60% [5].In our laboratory we are studying the structure/function relationship of the porins. In addition, we are aiming to use PhoE protein as a carrier for the transport of foreign antigenic determinants to the cell surface. Korteland et al. [6] have isolated five independent TC45-resistant mutants that produce an altered PhoE protein. In all five mutants the arginine residue in position 158 was substituted by a histidine residue. Thus, arginine-158 is involved in the receptor function of the PhoE protein for phage TC45 and the region around this residue is probably cell-surface-exposed. OmpC protein has an insertion of 14 amino acids in this region when compared to the PhoE protein [5]. Protease-accessibility experiments demonstrated the presence of this insert at the cell surface [7].Correspond...

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Section: Discussionsupporting

confidence: 45%

Section: In Vitro Pore Function Of the Mutant Porinsmentioning

confidence: 72%

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

1987

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“…The synthetic phospholipids DOPC and DOPE were mixed in a 1:1 molar (total concentration, 10 mg/ml) ratio, dissolved in n-decane, and then used to form planar bilayers (39,40). Bilayers were formed by painting lipids using a "brush technique" (41) on a 0.2-mm aperture in a partition separating two 4-ml chambers containing 10 mM HEPES, pH 7.0, 0.1 M KCl, 23°C.…”

Section: Purification Of E2r135-thementioning

confidence: 99%

Structure of the Complex of the Colicin E2 R-domain and Its BtuB Receptor

Sharma¹,

Yamashita²,

Zhalnina

et al. 2007

Journal of Biological Chemistry

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The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-Å crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg 452 at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.Protein transport across membranes in organelles and bacteria is known to involve multiprotein complexes (1, 2). Colicin import across the outer membrane of Escherichia coli has also been inferred to involve such a translocon (3-8). An experimentally useful attribute of colicin uptake for studies on protein transport across membranes is that the end result is cytotoxicity. Colicins are plasmid-encoded bactericidal proteins that are released in response to stress, enter the bacterial cell by appropriating its outer membrane nutrient-uptake machinery, and provide an advantage to colicin-resistant cells in the competition for nutrition (9). Colicins are produced in complex with a small immunity (ϳ10 kDa) protein that binds to, and prevents, the colicin from killing the producing cell (10, 11). Nuclease colicins consist of three domains: an N-terminal T (translocation)-domain that functions in the import of the colicin across the outer membrane, a C-terminal C (catalysis or channel)-domain that contains the cytotoxic activity, and a central R (receptor-binding)-domain that functions in irreversible attachment to an outer membrane receptor.Colicins have been divided into two groups, A and B, based on the intracellular protein translocation network that is utilized. "Group A" colicins utilize the Tol proteins TolA, TolB, TolQ, TolR, and Pal (4, 12, 13), whereas "Group B" colicins utilize the Ton proteins, TonB, ExbB, and ExbD (5, 14), to enter the bacterial cytoplasmic compartment and/or insert into the cytoplasmic membra...

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“…Concerning ion selectivity, the OmpP2 pores showed a small preference for cations over anions for KCl and potassium acetate (P c /P a ϭ 1.5 and 2.5, respectively) and were slightly anion-selective for LiCl (P c /P a ϭ 0.8). Compared with the partially highly cation-selective general diffusion pores of enteric bacteria such as E. coli and Salmonella typhimurium with P c /P a ratios between ϳ4 and ϳ20 for KCl, the selectivity of OmpP2 porin is not very pronounced (40,50). Although, in enteric bacteria, the OM acts as a filter against negatively charged harmful compounds such as bile salts, OmpP2 porin might not select passing compounds according to their charge.…”

Section: Discussionmentioning

confidence: 99%

“…This result suggests that OmpP2 could be a channel specific for nicotinamide compounds containing a binding site for NAD and NMN in a similar way as LamB and ScrY are carbohydrate-specific channels with a binding site for malto-oligosaccharides (25,40,41). To test this hypothesis, we performed similar titration experiments as described previously for specific porins of Escherichia coli (24,33).…”

Section: Nucleotide Specificity Of Ompp2 Porin Of H Influenzaementioning

confidence: 90%

Porin OmpP2 of Haemophilus influenzae Shows Specificity for Nicotinamide-derived Nucleotide Substrates

Andersen

Maier

Kemmer

et al. 2003

Journal of Biological Chemistry

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Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with K S values of ϳ8 and 4 mM, respectively, in 0.1 M KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.The only known natural habitat of Haemophilus influenzae is the human nasopharynx. H. influenzae is Gram-negative and is able to grow under facultative anaerobic conditions. It is also a human pathogen and is responsible for significant morbidity and mortality in young children (1, 2). To cultivate H. influenzae, a complex medium is required; and if not blood-based, it must contain two growth factors: NAD and hemin (3). In the nasopharynx, H. influenzae finds a relatively constant environment, and the organism has retained a certain degree of flexibility in its ability to utilize nutrients (4) by possessing some limiting metabolic pathway redundancy (5).Early biochemical investigations have established that NMN 1 and nicotinamide riboside (NR) can substitute for NAD, whereas nicotinamide, niacin, or other nicotine-based intermediates of the Preiss-Handler pathway cannot (6 -8). The NAD dependence of H. influenzae was confirmed by the absence of genes encoding the enzymes necessary for the de novo biosynthesis of NAD (9). Accumulation of nicotinamide nucleotides derived from NAD or NR has been demonstrated in H. influenzae and Haemophilus parainfluenzae (10, 11). For H. parainfluenzae, the K m for transport is ϳ0.55 M for NAD and 0.14 M for NR, and the V max for NR is about four times that of NAD (11). This implies that NR is the substrate for an as yet unidentified inner membrane transporter. Recently, we presented data showing that two gene products appear to be involved in the NAD utilization pathway of H. influenzae (12)(13)(14). The gene products were identified as the hel-encoded outer membrane lipoprotein e(P4) and a periplasmic protein termed NadN. Knockout mutations of both genes resulted in growth-deficient phenotypes depending on the...

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Pycnodysostosis with heterozygous beta-thalassemia

Cited by 13 publications

References 11 publications

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

Structure of the Complex of the Colicin E2 R-domain and Its BtuB Receptor

Porin OmpP2 of Haemophilus influenzae Shows Specificity for Nicotinamide-derived Nucleotide Substrates

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