Lisa A. Lambert

Molecular evolution and characterization of hepcidin gene products in vertebrates

Antimicrobial peptides (AMPs) include a diverse group of gene-encoded molecules that play a role in innate defense in many organisms. Evolutionary analyses of the AMP genes can be challenging because of gene duplication and diversification. Recently discovered, hepcidins are small, cysteine-rich antimicrobial peptides that also function as hormonal regulators of iron homeostasis. In this paper we investigated the organization, expression and molecular evolution of hepcidin. From searches of the literature and public genomic databases we collected 68 different hepcidin gene products from 51 different species, all among the vertebrates. Although some species have multiple hepcidin homologues, we suggest that each contains only one copy that functions as an iron regulator. Despite the recent report of hepcidin sequences in the pigeon (Fu, Y.M., Li, S.P., Wu, Y.F., Chang, Y.Z., 2007. Identification and expression analysis of hepcidin-like cDNAs from pigeon (Columba livia). Mol. Cell. Biochem. 305, 191-197.), searches of the chicken genomic, EST, and HTGS databases did not reveal any evidence of the presence of this gene in birds. This, along with the absence of reported avian transferrin receptor 2 and hemojuvelin sequences, suggests that iron homeostasis in birds may be regulated by an alternative mechanism.

Molecular evolution of the transferrin family and associated receptors

BACKGROUND In vertebrates, serum transferrins are essential iron transporters that have bind and release Fe(III) in response to receptor binding and changes in pH. Some family members such as lactoferrin and melanotransferrin can also bind iron while others have lost this ability and have gained other functions, e.g., inhibitor of carbonic anhydrase (mammals), saxiphilin (frogs) and otolith matrix protein 1 (fish). SCOPE OF REVIEW This article provides an overview of the known transferrin family members and their associated receptors and interacting partners. MAJOR CONCLUSIONS The number of transferrin genes has proliferated as a result of multiple duplication events, and the resulting paralogs have developed a wide array of new functions. Some homologs in the most primitive metazoan groups resemble both serum and melanotransferrins, but the major yolk proteins show considerable divergence from the rest of the family. Among the transferrin receptors, the lack of TFR2 in birds and reptiles, and the lack of any TFR homologs among the insects draw attention to the differences in iron transport and regulation in those groups. GENERAL SIGNIFICANCE The transferrin family members are important because of their clinical significance, interesting biochemical properties, and evolutionary history. More work is needed to better understand the functions and evolution of the non-vertebrate family members. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders.

Molecular Evolution of Hemojuvelin and the Repulsive Guidance Molecule Family

Repulsive guidance molecules (RGMs) are found in vertebrates and chordates and are involved in embryonic development and iron homeostasis. Members of this family are GPI-linked membrane proteins that contain an N-terminal signal peptide, a C-terminal propeptide, and a conserved RGD motif. Vertebrates are known to possess three paralogues; RGMA and RGMB (sometimes called Dragon) are expressed in the nervous system and are thought to play various roles in neural development. Hemojuvelin (HJV; also called repulsive guidance molecule c, RGMC) is the third member of this family, and mutations in this gene result in a form of juvenile hemochromatosis (type 2A). Phylogenetic analyses of 55 different RGM family sequences from 21 different species support the existence of a novel gene, found only in fish, which we have labeled RGMD. The pattern of conserved residues in each family identifies new candidates for important functional roles, including ligand binding.

Molecular Evolution of the Transferrin Receptor/Glutamate Carboxypeptidase II Family

The transferrin receptor family is represented by at least seven different homologous proteins in primates. Transferrin receptor (TfR1) is a type II membrane glycoprotein that, as a cell surface homodimer, binds iron-loaded transferrin as part of the process of iron transfer and uptake. Other family members include transferrin receptor 2 (TfR2), glutamate carboxypeptidase II (GCP2 or PSMA), N-acetylated α-linked acidic dipeptidase-like protein (NLDL), N-acetylated α-linked acidic dipeptidase 2 (NAALAD2), and prostate-specific membrane antigen-like protein (PMSAL/GCPIII). We compared 86 different sequences from 24 different species, from mammals to fungi. Through this comparison, we have identified several highly conserved residues specific to each family not previously associated with clinical mutations. The evolutionary history of the TfR/GCP2 family shows repeated episodes of duplications consistent with recent theories that nondispensable, slowly evolving genes are more likely to form multiple gene families.

The structure and evolution of the murine inhibitor of carbonic anhydrase: A member of the transferrin superfamily

The original signature of the transferrin (TF) family of proteins was the ability to bind ferric iron with high affinity in the cleft of each of two homologous lobes. However, in recent years, new family members that do not bind iron have been discovered. One new member is the inhibitor of carbonic anhydrase (ICA), which as its name indicates, binds to and strongly inhibits certain isoforms of carbonic anhydrase. Recently, mouse ICA has been expressed as a recombinant protein in a mammalian cell system. Here, we describe the 2.4 Å structure of mouse ICA from a pseudomerohedral twinned crystal. As predicted, the structure is bilobal, comprised of two α‐β domains per lobe typical of the other family members. As with all but insect TFs, the structure includes the unusual reverse γ‐turn in each lobe. The structure is consistent with the fact that introduction of two mutations in the N‐lobe of murine ICA (mICA) (W124R and S188Y) allowed it to bind iron with high affinity. Unexpectedly, both lobes of the mICA were found in the closed conformation usually associated with presence of iron in the cleft, and making the structure most similar to diferric pig TF. Two new ICA family members (guinea pig and horse) were identified from genomic sequences and used in evolutionary comparisons. Additionally, a comparison of selection pressure (dN/dS) on functional residues reveals some interesting insights into the evolution of the TF family including that the N‐lobe of lactoferrin may be in the process of eliminating its iron binding function.

Evidence of Fe3+ interaction with the plug domain of the outer membrane transferrin receptor protein of Neisseria gonorrhoeae: implications for Fe transport.

Neisseria gonorrhoeae is an obligate pathogen that hijacks iron from the human iron transport protein, holo-transferrin (Fe(2)-Tf), by expressing TonB-dependent outer membrane receptor proteins, TbpA and TbpB. Homologous to other TonB-dependent outer membrane transporters, TbpA is thought to consist of a β-barrel with an N-terminal plug domain. Previous reports by our laboratories show that the sequence EIEYE in the plug domain is highly conserved among various bacterial species that express TbpA and plays a crucial role in iron utilization for gonococci. We hypothesize that this highly conserved EIEYE sequence in the TbpA plug, rich in hard oxygen donor groups, binds with Fe(3+) through the transport process across the outer membrane through the β-barrel. Sequestration of Fe(3+) by the TbpA-plug supports the paradigm that the ferric iron must always remain chelated and controlled throughout the transport process. In order to test this hypothesis here we describe the ability of both the recombinant wild-type plug, and three small peptides that encompass the sequence EIEYE of the plug, to bind Fe(3+). This is the first report of the expression/isolation of the recombinant wild-type TbpA plug. Although CD and SUPREX spectroscopies suggest that a non-native structure is observed for the recombinant plug, fluorescence quenching titrations indicate that the wild-type recombinant TbpA plug binds Fe (3+) with a conditional log K(d) = 7 at pH 7.5, with no evidence of binding at pH 6.3. A recombinant TbpA plug with mutated sequence (NEIEYEN → NEIAAAN) shows no evidence of Fe(3+) binding under our experimental set up. Interestingly, in silico modeling with the wild-type plug also predicts a flexible loop structure for the EIEYE sequence under native conditions which once again supports the Fe(3+) binding hypothesis. These in vitro observations are consistent with the hypothesis that the EIEYE sequence in the wild-type TbpA plug binds Fe(3+) during the outer membrane transport process in vivo.

A novel murine protein with no effect on iron homoeostasis is homologous with transferrin and is the putative inhibitor of carbonic anhydrase

In a search for genes that modify iron homoeostasis, a gene (1300017J02Rik) was located immediately upstream of the murine TF (transferrin) gene. However, expression of the 1300017J02Rik gene product was not responsive to a number of modulators of iron metabolism. Specifically, expression was not altered in mouse models of iron disorders including mice with deficiencies in the haemochromatosis protein Hfe, the recombination-activating protein, Rag, beta2-microglobulin, TF, ceruloplasmin or Hb, or in mice with microcytic anaemia. Additionally, neither lipopolysaccharide nor hypoxia treatment resulted in any significant changes in the 1300017J02Rik expression level. The genomic DNA sequence suggested that the 1300017J02Rik gene product might be a protein equivalent to the pICA {porcine ICA [inhibitor of CA (carbonic anhydrase)]}. The coding region for the murine 1300017J02Rik gene was placed into the pNUT expression vector. Transformed BHK cells (baby-hamster kidney cells) were transfected with this plasmid, resulting in secretion of recombinant mICA (murine ICA) into the tissue culture medium. Following purification to homogeneity, the yield of mICA from the BHK cells was found to be considerably greater (at least 4-fold) than the yield of pICA from a previously reported Pichia pastoris (yeast) expression system. MS showed that the recombinant mICA was a glycoprotein that associated with CA in a 1:1 stoichiometry. Despite its high sequence similarity to TF, titration experiments showed that mICA was unable to bind iron specifically. Although enzymatic assays revealed that mICA was able to inhibit CA, it is unclear if this is its sole or even its major function since, to date, humans and other primates appear to lack functional ICA. Lastly, we note that this member of the TF superfamily is a relatively recent addition resulting from a tandem duplication event.

Bordetella pertussis FbpA binds both unchelated iron and iron siderophore complexes

Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with μM affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe3+ and indirectly as a ferric siderophore complex.

Molecular evolution and selection pressure in alpha-class carbonic anhydrase family members

Carbonic anhydrases (CA) are ubiquitous, and their involvement in diseases such as hypertension, diabetes, and glaucoma is well known. Most members of this family of metalloenzymes convert carbon dioxide to bicarbonate with the help of a Zn(2+) cofactor. While the expression patterns and kinetic activities of many of these isozymes have been studied, little is known about the differences in the conservation patterns of individual residues. To better understand the molecular evolution of the CA gene family, we created multiple sequence alignments and analyzed the selection pressure (dN/dS ratios) on surface and active site residues in 248 mammalian sequences of the 14 known family members. Using the values found for amino acids of known functional importance (i.e. the three histidines that bind the zinc cofactor) as our baseline, we were able to identify other regions of possible structural and functional importance.