Chromosomal sublocalization of the human p97 melanoma antigen

Summary The antigen p97 is a tumor-associated antigen that was first identified in human melanomas using monoclonal antibodies. Recently, p97 mRNA was purified and cloned, and a p97 cDNA clone was synthesized. By using the technique of in situ chromosomal hybridization, we have localized the p97 gene to human chromosome No. 3, at bands q28 to q29. p97 belongs to a superfamily of iron-binding proteins that have amino acid homology; other members of this family include transferrin (TF), lactotransferrin, and ovotransferrin. Based upon the shared amino acid homology and upon the observation that the nucleotide sequence is internally duplicated in these genes, it has been proposed that the TF superfamily arose from a common ancestral duplicated gene. The TF gene has also been mapped to the long arm of chromosome No. 3 at bands q21 to q23.     

Binding of phosphonate chelating agents and pyrophosphate to apotransferrin.

Difference ultraviolet spectroscopy has been used to monitor the binding of a series of phosphonate ligands to human apotransferrin. The ligands consist of pyrophosphate as well as the phosphonic acids (aminomethyl)phosphonic acid (AMPA), (hydroxymethyl)phosphonic acid (HMP), (phosphonomethyl)-iminodiacetic acid (PIDA), N,N-bis(phosphonomethyl)glycine (DPG), and nitrilotris(methylenephosphonic acid) (NTP). Equilibrium constants have been measured for the sequential binding of two ligands per molecule of apotransferrin. In addition, site-specific equilibrium constants have been measured for the binding of AMPA, HMP, and PIDA to the vacant binding site of both forms of monoferric transferrin. Since titrations of diferric transferrin produce no difference UV spectrum, it is proposed that the primary binding site for phosphonic acids includes the protein groups that bind the synergistic bicarbonate anion that is required for formation of a stable ferric transferrin complex. It is further proposed that those ligands with two phosphonate groups can simultaneously bind to cationic amino acid side chains that extend into the cleft between the two domains of each lobe of transferrin. From an inspection of the ferric transferrin crystal structure, the most likely anion binding residues in the cleft are Arg-632 and Lys-534 in the C-terminal lobe and Lys-206 and Lys-296 in the N-terminal lobe.     

The p97 antigen is mapped to the q24-qter region of chromosome 3; the same region as the transferrin receptor.

Since the p97 antigen, a membrane-associated iron-binding protein, has extensive amino acid sequence with homology with transferrin, is functionally related to the transferrin receptor, and has been previously mapped to chromosome 3, we have performed additional studies for regional mapping of the gene expressing p97 antigen. In these experiments, Chinese hamster-human cell lines were chosen that contained a large spectrum of autosomal human chromosomes, but mainly consisted of clones expressing all or a part of chromosome 3. These cell lines included a clone that previously allowed for mapping of human transferrin receptor to q22-qter region. Human p97 expression was assessed by specific binding of [125I]monoclonal antibody 96.5, and human transferrin receptor expression was tested by specific [125I]human transferrin binding and [125I]monoclonal antibody OKT-9 specific for human transferrin receptor. Based on these analyses, both human p97 antigenic expression and human transferrin receptor are mapped concordantly to the q24-qter region. These data and previous reports, therefore, suggest that the related iron-transport proteins are closely linked and may be under coordinate regulation. However, studies of several cell lines that exhibit up-regulation of human transferrin receptor expression with cellular proliferation, and down-regulation of receptor with increased transferrin-iron in the media, showed no change in expression of p97 antigen. p97 antigenic expression increased when melanocyte-stimulating hormone was added to a human melanoma cell line in tissue culture. These latter studies suggest that in mammalian cells the two proteins do not show coordinate regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure, dynamics and interactions of p47, a major adaptor of the AAA ATPase, p97

p47 is a major adaptor molecule of the cytosolic AAA ATPase p97. The principal role of the p97-p47 complex is in regulation of membrane fusion events. Mono-ubiquitin recognition by p47 has also been shown to be crucial in the p97-p47-mediated Golgi membrane fusion events. Here, we describe the high-resolution solution structures of the N-terminal UBA domain and the central domain (SEP) from p47. The p47 UBA domain has the characteristic three-helix bundle fold and forms a highly stable complex with ubiquitin. We report the interaction surfaces of the two proteins and present a structure for the p47 UBA-ubiquitin complex. The p47 SEP domain adopts a novel fold with a betabetabetaalphaalphabeta secondary structure arrangement, where beta4 pairs in a parallel fashion to beta1. Based on biophysical studies, we demonstrate a clear propensity for the self-association of p47. Furthermore, p97 N binding abolishes p47 self-association, revealing the potential interaction surfaces for recognition of other domains within p97 or the substrate.     

Three-dimensional structure of mare diferric lactoferrin at 2.6 A resolution.

Lactoferrin is a monomeric glycoprotein with a molecular mass of approximately 80 kDa. The three-dimensional structure of mare diferric lactoferrin (mlf) has been determined at 2.6 A resolution. The protein crystallizes in the space group P 212121with a=85.2 A, b=99.5 A, c=103.1 A with a solvent content of 55 % (v/v). The structure was solved by the molecular replacement method using human diferric lactoferrin as the model. The structure has been refined using XPLOR to a final R -factor of 0.194 for all data in the 15-2.6 A resolution range. The amino acid sequence of mlf was determined using a cDNA method. The final refined model comprises 5281 protein atoms, 2 Fe3+, 2 CO32-and 112 water molecules. The overall folding of mlf is similar to that of other proteins of the transferrin family. The protein folds into two globular lobes, N and C. The lobes are further divided into two domains, N1 and N2, and C1 and C2. The iron-binding cleft is situated between the domains in each lobe. The N lobe appears to be well ordered and is more stable than the C lobe in mlf unlike in other lactoferrins, where the C lobe is the more stable. The opening of the binding cleft in the N lobe of mlf is narrower than those in other proteins of the transferrin family. This is very unusual and is found only in mare lactoferrin. Apart from certain hydrophobic interactions at the mouth of the cleft, one salt-bridge (Lys301 . . . . . . . . Glu216) crosses between the two walls of the cleft. The two lobes are connected covalently by a three-turn alpha-helix involving residues 334-344. The N lobe displays a highly ordered structure with appreciably low temperature factors. The iron coordination is more symmetrical in the N lobe than in the C lobe. There are only 16 intermolecular hydrogen bonds in the structure of mlf.     

Iron release from transferrin, its C-lobe, and their complexes with transferrin receptor: presence of N-lobe accelerates release from C-lobe at endosomal pH

Human transferrin, like other members of the transferrin class of iron-binding proteins, is a bilobal structure, the product of duplication and fusion of an ancestral gene during the course of biochemical evolution. Although the two lobes exhibit 45% sequence identity and identical ligand structures of their iron-binding sites (one in each lobe), they differ in their iron-binding properties and their responsiveness to complex formation with the transferrin receptor. A variety of interlobe interactions modulating these iron-binding functions has been described. We have now studied the kinetics of iron release to pyrophosphate from the isolated recombinant C-lobe and from that lobe in the intact protein, each free and bound to receptor. The striking finding is that the rates of iron release at the pH of the endosome to which transferrin is internalized by the iron-dependent cell are similar in the free proteins but 18 times faster from full-length monoferric transferrin selectively loaded with iron in the C-lobe than from isolated C-lobe when each is complexed to the receptor. The possibility that the faster release in the receptor complex of the full-length protein at endosomal pH contributes to the evolutionary advantage of the bilobal structure is considered.     

Calorimetric studies of melanotransferrin (p97) and its interaction with iron

The mammalian molecule melanotransferrin (mTf), also called p97, is a member of the transferrin family of molecules. It exists in both secreted and glycosylphosphatidylinositol-anchored forms and is thought to play a role in angiogenesis and in transporting iron across the blood brain barrier. The binding affinity of iron to this molecule has not been formally established. Here, the binding of ferric ion (chelated with a 2-fold molar ratio of nitrilotriacetate) to mTf has been studied using isothermal titration calorimetry and differential scanning calorimetry. One iron-binding site was determined for mTf with similar binding characteristics to other transferrins. In the absence of bicarbonate, binding occurs quickly with an apparent association constant of 2.6 x 10(7) M(-1) at 25 degrees C. The presence of bicarbonate introduces kinetic effects that prevent direct determination of the apparent binding constant by isothermal titration calorimetry. Differential scanning calorimetry thermograms of mTf unfolding in the presence and absence of iron were therefore used to determine the apparent binding constant in the bicarbonate-containing system; at pH 7.5 and 25 degrees C, iron binding occurs in a 1:1 ratio with a K(app) of 4.4 x 10(17) M(-1). This affinity is intermediate between the high and low affinity lobes of transferrin and suggests that mTf is likely to play a significant role in iron transport where the high affinity lobe of transferrin is occupied or where transferrin is in proportionally low concentrations.     

Molecular structure of serum transferrin at 3.3-A resolution

Serum transferrin is a metal-binding glycoprotein, molecular weight ca. 80,000, whose primary function is the transport of iron in the plasma of vertebrates. The X-ray crystallographic structure of diferric rabbit serum transferrin has been determined to a resolution of 3.3 A. The molecule has a beta alpha structure of similar topology to human lactoferrin and is composed of two homologous lobes that each bind a single ferric ion. Each lobe is further divided into two dissimilar domains, and the iron-binding site is located within the interdomain cleft. The iron is bound by two tyrosines, a histidine, and an aspartic acid residue. The location of the 19 disulfide bridges is described, and their possible structural roles are discussed in relation to the transferrin family of proteins. Mapping of the intron/exon splice junctions onto the molecule provides some topological evidence in support of the putative secondary role for transferrin in stimulating cell proliferation.     

A novel iron uptake mechanism mediated by GPI-anchored human p97.

The established process for iron uptake into mammalian cells involves transferrin and its receptor. Here, the role of the glycosyl-phosphatidylinositol (GPI)-linked transferrin homologue, melanotransferrin or p97, was studied using CHO cell lines defective in the transferrin receptor (TR) and transfected with human TR and/or human p97. The presence of p97 doubled the iron uptake, which could be explained by the binding of one atom of iron to one molecule of p97. The internalization of iron was shown to be temperature sensitive and saturated at a media iron concentration of 2.5 micrograms/ml with a Vmax of 0.1 pmol Fe/10(6) cell/min and a Km of 2.58 microM for p97. Treatment of the cells with either phosphatidylinositol-phospholipase C or monoclonal antibodies against p97 resulted in over a 50% reduction and a 47% increase in the iron uptake respectively. These data identify p97 as a unique cell surface GPI-anchored, iron binding protein involved in the transferrin-independent uptake of iron in mammals.     

Molecular Cloning and Characterization of Transferrin from Wax Moth, Galleria mellonella

Complete mRNA sequence of transferrin from Galleria mellonella was obtained, and compared with those of other species. Until now, two types of insect transferrin were reported. Transferrins in cockroach and termite have two iron binding sites while those in most other insect groups, studied for the protein, have only one. It was suggested that the presence of two types of transferrin was related with transferrin evolution, because vertebrate transferrins have two iron binding sites, called N and C terminal lobe. It was shown that G. mellonella transferrin also has only one iron binding site (N terminal lobe), and the deduced amino acid sequence was most similar to those of Manduca sexta and Bombyx mori.     

Nicastrin, a presenilin-interacting protein, contains an aminopeptidase/transferrin receptor superfamily domain

Nicastrin, a protein implicated in Alzheimer's disease, has a domain that is found in the aminopeptidase/transferrin receptor superfamily. In nicastrin, this domain might possess catalytic activity (as observed with aminopeptidases) or it could serve merely as a binding domain (with analogy to the transferrin receptors) for the beta-amyloid precursor protein.     

Drosophila melanogaster transferrin. Cloning, deduced protein sequence, expression during the life cycle, gene localization and up-regulation on bacterial infection.

Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron-binding site only in the N-terminal lobe. The C-terminal lobe lacks iron-binding residues found in other transferrins, and has large deletions which make it much smaller than functional C-terminal lobes in other transferrins. In-situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1-2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.     

SVIP is a novel VCP/p97-interacting protein whose expression causes cell vacuolation

VCP/p97 is involved in a variety of cellular processes, including membrane fusion and ubiquitin-dependent protein degradation. It has been suggested that adaptor proteins such as p47 and Ufd1p confer functional versatility to VCP/p97. To identify novel adaptors, we searched for proteins that interact specifically with VCP/p97 by using the yeast two-hybrid system, and discovered a novel VCP/p97-interacting protein named small VCP/p97-interacting protein (SVIP). Rat SVIP is a 76-amino acid protein that contains two putative coiled-coil regions, and potential myristoylation and palmitoylation sites at the N terminus. Binding experiments revealed that the N-terminal coiled-coil region of SVIP, and the N-terminal and subsequent ATP-binding regions (ND1 domain) of VCP/p97, interact with each other. SVIP and previously identified adaptors p47 and ufd1p interact with VCP/p97 in a mutually exclusive manner. Overexpression of full-length SVIP or a truncated mutant did not markedly affect the structure of the Golgi apparatus, but caused extensive cell vacuolation reminiscent of that seen upon the expression of VCP/p97 mutants or polyglutamine proteins in neuronal cells. The vacuoles seemed to be derived from endoplasmic reticulum membranes. These results together suggest that SVIP is a novel VCP/p97 adaptor whose function is related to the integrity of the endoplasmic reticulum.     

Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.     

Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization

Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.     

The crystal structure of iron-free human serum transferrin provides insight into inter-lobe communication and receptor binding

Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-A resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7A by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4 degrees and 49.5 degrees rotations are required to open the N- and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.     

Evolution reversed: the ability to bind iron restored to the N-lobe of the murine inhibitor of carbonic anhydrase by strategic mutagenesis

The murine inhibitor of carbonic anhydrase (mICA) is a member of the superfamily related to the bilobal iron transport protein transferrin (TF), which binds a ferric ion within a cleft in each lobe. Although the gene encoding ICA in humans is classified as a pseudogene, an apparently functional ICA gene has been annotated in mice, rats, cows, pigs, and dogs. All ICAs lack one (or more) of the amino acid ligands in each lobe essential for high-affinity coordination of iron and the requisite synergistic anion, carbonate. The reason why ICA family members have lost the ability to bind iron is potentially related to acquiring a new function(s), one of which is inhibition of certain carbonic anhydrase (CA) isoforms. A recombinant mutant of the mICA (W124R/S188Y) was created with the goal of restoring the ligands required for both anion (Arg124) and iron (Tyr188) binding in the N-lobe. Absorption and fluorescence spectra definitively show that the mutant binds ferric iron in the N-lobe. Electrospray ionization mass spectrometry confirms the presence of both ferric iron and carbonate. At the putative endosomal pH of 5.6, iron is released by two slow processes indicative of high-affinity coordination. Induction of specific iron binding implies that (1) the structure of mICA resembles those of other TF family members and (2) the N-lobe can adopt a conformation in which the cleft closes when iron binds. Because the conformational change in the N-lobe indicated by metal binding does not impact the inhibitory activity of mICA, inhibition of CA was tentatively assigned to the C-lobe. Proof of this assignment is provided by limited trypsin proteolysis of porcine ICA.     

Crystal Structure of HypA, a Nickel-Binding Metallochaperone for [NiFe] Hydrogenase Maturation

HypA is one of the auxiliary proteins involved in the maturation of [NiFe] hydrogenases. By an unknown mechanism, HypA functions as a metallochaperone in the insertion of the Ni atom into hydrogenases. We have determined the crystal structures of HypA from Thermococcus kodakaraensis KOD1 in both monomeric and dimeric states. The structure of the HypA monomer consists of Ni- and Zn-binding domains. The relative arrangement of the two metal-binding domains has been shown to be associated with local conformations of the conserved Ni-binding motif, suggesting a communication between the Ni- and Zn-binding sites. The HypA dimer has been shown to be stabilized by unexpected domain swapping through archaea-specific linker helices. In addition, the hexameric structure of HypA is formed in the crystal packing. Several hydrogen bonds and hydrophobic interactions stabilize the hexamer interface. These findings suggest the functional diversity of HypA proteins.     

The SHS2 module is a common structural theme in functionally diverse protein groups, like Rpb7p, FtsA, GyrI, and MTH1598/TM1083 superfamilies

Using structural comparisons, we identified a novel domain with a simple fold in the bacterial cell division ATPase FtsA, the archaeo-eukaryotic RNA polymerase subunit Rpb7p, the GyrI superfamily, and the uncharacterized MTH1598/Tm1083-like proteins. The fold contains a core of 3 strands, forming a curved sheet, and a single helix in a strand-helix-strand-strand (SHS2) configuration. The SHS2 domain may exist either in single or duplicate copies within the same polypeptide. The single-copy versions of the domain in FtsA and Rbp7p are most closely related, and appear to mediate protein-protein interactions by means of strand 1, and the loop between strand 2 and strand 3 of the domain. We predict that the interactions between FtsA and its functional partners in bacterial cell division are likely to be similar to the interactions of Rbp7p in the archaeo-eukaryotic RNA polymerase complex. The dimeric versions typified by the GyrI superfamily appear to have been adapted for small-molecule binding. Sequence profiles searches helped us to identify several new versions of the GyrI superfamily, including a family of secreted forms that is found only in animals and the bacterial pathogen Leptospira. Through sequence-structure comparisons, we predict the positions that are likely to be important for ligand specificity in the GyrI superfamily. In the MTH1598/Tm1083-like proteins, a SHS2 domain is inserted into the loop between strand 1 and helix 1 of another SHS2 domain. This has resulted in a structure that has convergent similarities with the Hsp33 and green fluorescent protein folds. The sequence conservation pattern and its phyletic profile suggest that it might function as an enzyme in some conserved aspect of nucleic acid metabolism. Thus, the SHS2 domain is an example of a simple module that has been adapted to perform an entire spectrum of functions ranging from protein-protein interactions to small-molecule recognition and catalysis.