Recombinant H‐chain ferritins: Effects of changes in the 3‐fold channels

Treffry, Amyra; Harrison, Pauline M.; Luzzago, Alessandra

doi:10.1016/0014-5793(89)81350-x

Cited by 69 publications

(37 citation statements)

References 15 publications

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“…Site-directed mutagenesis and overproduction of the HuHF variant in E. Coli and puri®cation of the ferritins were performed as described by Treffry et al (1989). The substitution of Lys86 by Gln was made to enable crystallisation by creating metal-bridge crystal contacts as described by Lawson et al (1991) since it had proved impossible to obtain X-ray quality crystals of wild-type HuHF.…”

Section: Materials and Methods Proteinsmentioning

confidence: 99%

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Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution 1 1Edited by R. Huber

Hempstead

Yewdall

Fernie

et al. 1997

Journal of Molecular Biology

296

182

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Section: Materials and Methods Proteinsmentioning

confidence: 99%

“…HoLF (homopolymer) was cloned and overproduced in E. coli as described by Takeda et al (1993) and puri®ed according to Treffry et al (1989). Reduction with sodium dithionite was performed as by Bauminger et al (1991).…”

Section: Materials and Methods Proteinsmentioning

confidence: 99%

Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution 1 1Edited by R. Huber

Hempstead

Yewdall

Fernie

et al. 1997

Journal of Molecular Biology

296

182

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“…Site-directed mutagenesis, overexpression of rHuHF and its variants in E. coli and purification of the ferritins were performed as in [34]. Similar procedures to those described previously were used for production of recombinant human L-chain ferritin (rHuLF) [32] and Ec-FTN [4].…”

Section: Methodsmentioning

confidence: 99%

“…19 (3) 19 (3) 46 (7) 58 (5) 31 (5) 38 (3) 20 (3) 18 (3) 10 (3) 8 (3) 12 (2) 11 (1) 8 (4) 11(2) 11 (5) 11 (3) 21 (5) 22 (3) Table 5 Mossbauer spectroscopic analysis at 90 K of the products formed by aerobic oxidation of 57Fe(ll) in the presence of various apoferritins Iron has been removed from all samples by sodium dithionite reduction [13] prior to the addition of 57FeS04. 34 Fe atoms/molecule, protein, 6.7 mg/ml in 0.1 M Mops buffer, pH 7.0. (2) 21 (2) 22 (4) 48 (4) (3) clusters in Ec-FTN compared with those in rHuHF, possibly owing to the different types of clusters present.…”

Section: Time Course Of Fe(iii) Transfer From Rhuhf Donor To Rhuhf Ormentioning

confidence: 99%

Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli

Bauminger

Treffry

Hudson

et al. 1994

Biochemical Journal

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Iron that has been oxidized by H-chain ferritin can be transferred into other ferritin molecules before it is incorporated into mature ferrihydrite iron cores. Iron(III) dimers are formed at the ferroxidase centres of ferritin H chains at an early stage of Fe(II) oxidation. Mössbauer spectroscopic data now show that the iron is transferred as monomeric species arising from dimer dissociation and that it binds to the iron core of the acceptor ferritin. Human H-chain ferritin variants containing altered threefold channels can act as acceptors, as can the ferritin of Escherichia coli (Ec-FTN). A human H-chain ferritin variant with a substituted tyrosine (rHuHF-Y34F) can act as a donor of Fe(III). Since an Fe(III)-tyrosinate (first identified in bullfrog H-chain ferritin) is absent from variant rHuHF-Y34F, the Fe(III) transferred is not derived from this tyrosinate complex. Mössbauer parameters of the small iron cores formed within Ec-FTN are significantly different from those of mammalian ferritins. Analysis of the spectra suggests that they are derived from both ferrihydrite and non-ferrihydrite components. This provides further evidence that the ferritin protein shell can influence the structure of its iron core.

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“…Residues in the D helix contribute to hydrophilic channels of three-fold symmetry found in apoferritin, and a decrease in rate of iron uptake in point mutants in residues 131 or 134 of the D helix have previously been reported (30). However, an alteration in residues that line the hydrophilic channel is unlikely to explain our results, since residues that line these channels, including Asp-131 and Glu-134, are conserved in mouse and human ferritin H. An alternative possibility is that conformational differences underlie the differences in catalytic activity between mouse and human ferritin H. For example, mutations in the loop that connects the D and E helix have been reported to alter the conformation of the ferritin protein (31), an effect that might in turn influence its iron uptake properties.…”

Section: Fig 5 Density Gradient Analysis Of Interspecies Ferritin Hmentioning

confidence: 99%

Role of H and L Subunits in Mouse Ferritin

Rucker

Torti

Torti³

1996

Journal of Biological Chemistry

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Ferritin is an iron-binding protein composed of two subunits, H and L. Twenty-four of these subunits assemble to form apoferritins whose subunit composition varies in a characteristic way in different tissues. Using recombinant proteins, we have assessed the role of H and L subunits in mouse ferritin function and compared these to human ferritin subunits. We report that mouse ferritin subunits exhibit considerable functional similarity to their human counterparts, including a prominent role of the H subunit in the facilitation of rapid iron uptake, and a key role of amino acid residues Glu-62 and His-65 in this process. In addition, amino acid residues important to assembly of the protein are conserved between mouse and human, permitting the formation of fully functional hybrid proteins containing both mouse and human subunits. However, murine and human ferritin H subunits also evidenced substantial functional differences; murine ferritin H showed a consistent reduction in iron uptake activity relative to human ferritin H. Creation of chimeric human/mouse ferritin H subunits by "helix swapping" mapped the domain of the protein critical to this activity difference to the DE helix. These findings suggest a novel functional role for carboxyl-terminal domains of the ferritin H subunit.Ferritin is a protein which has as its principal function the intracellular storage of iron in a nontoxic and bioavailable form (see Refs. 1 and 2 for review). Ferritin is ubiquitously distributed in the animal and plant kingdoms and has recently been described in bacteria (3). Mammalian ferritin is composed of two subunits, termed H and L. Twenty-four of these subunits assemble to form the apoferritin protein.In mammals, ferritin is found in most tissues. However, the composition of ferritin varies in a consistent and tissue-specific way. For example, liver contains ferritin that is predominantly of the L subunit type, whereas heart contains ferritin rich in the H subunit. This biodistribution, as well as the evolutionary conservation of a dual subunit structure, supports the hypothesis that the H and L ferritin subunits may play different and complementary roles within the protein (4). Experiments using H-rich and L-rich ferritin prepared from natural sources (5, 6), as well as recent work with recombinant proteins (7-9), have supported this concept of a functional distinction between ferritin H and L subunits and revealed a prominent role of the H subunit in rapid iron oxidation (7), and the involvement of the L subunit in protein stability and iron mineralization (8, 9).Mouse cells and mouse models have been widely used to study iron homeostasis in health and disease (e.g. Refs. 10 -14). For the most part, however, biological inferences concerning ferritin function and iron metabolism in these models have been based on analogy to human ferritin. Indeed, substantial sequence similarity exists between mouse and human ferritin subunits; mouse L and human L exhibit 82% similarity (15), whereas human and mouse ferritin H are 93% identic...

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Recombinant H‐chain ferritins: Effects of changes in the 3‐fold channels

Cited by 69 publications

References 15 publications

Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution 1 1Edited by R. Huber

Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution 1 1Edited by R. Huber

Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli

Role of H and L Subunits in Mouse Ferritin

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