The role of the membrane-bound tumour antigen, melanotransferrin (p97), in iron uptake by the human malignant melanoma cell.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue with several characteristics in common with serum Tf. MTf is found at high levels in melanoma cells and previous studies have shown that MTf can bind Fe. In addition, Chinese hamster ovary cells transfected with MTf transport Fe from 59Fe-citrate at greater rates than control cells. However, the role of MTf in the Fe uptake process of human melanoma cells remains unknown. In the present study we have characterized the role of MTf in Fe uptake by SK-Mel-28 melanoma cells in order to understand its function. Initial studies examined whether modulation of intracellular Fe levels using the Fe chelator desferrioxamine (DFO) or the Fe donor ferric ammonium citrate (FAC) could change MTf mRNA levels. In contrast to transferrin receptor (TfR) mRNA that increased after exposure to DFO and decreased after incubation with FAC, there was no change in MTf mRNA levels. In addition, compared to control cells, there was no alteration of 125I-labelled anti-MTf mAb-binding in cells exposed to DFO or FAC, suggesting no change in the number of MTf sites. Further studies examined the ability of DFO and FAC to modulate Fe uptake from 59Fe-citrate which is bound by MTf. In contrast to the effect of DFO or FAC at increasing and decreasing Fe uptake from 59Fe-Tf, respectively, DFO had no influence on 59Fe-citrate uptake, whereas FAC markedly increased it. Collectively, these studies suggest that MTf is not regulated in a manner similar to the TfR in response to cellular Fe levels. MTf can be removed from the membrane by phosphatidylinositol-specific phospholipase C (PtdIns-PLC). Preincubation of melanoma cells with PtdIns-PLC reduced anti-MTf mAb binding to 3% of the control, while PtdIns-PLC only slightly reduced 59Fe uptake from 59Fe-citrate. These results suggest that MTf played only a minor role in Fe uptake from 59Fe-citrate by these cells. The expression of MTf mRNA (poly A+) was also examined in 50 human tissues and found to be markedly different to Tf mRNA or TfR mRNA. Surprisingly, MTf mRNA expression was widespread in normal tissues, and was observed at its highest levels in the salivary gland. In contrast to expectations, MTf mRNA expression was generally greater in adult than fetal tissues.     

The transferrin homologue, melanotransferrin (p97), is rapidly catabolized by the liver of the rat and does not effectively donate iron to the brain

Melanotransferrin (MTf) or melanoma tumor antigen p97 is a membrane-bound transferrin (Tf) homologue that binds iron (Fe). This protein is also found as a soluble form in the plasma (sMTf) and was suggested to be an Alzheimer's disease marker. In addition, sMTf has been recently suggested to cross the blood-brain barrier (BBB) and accumulate in the brain of the mouse following intravenous infusion. Considering the importance of this observation to the physiology and pathophysiology of the BBB and the function of sMTf in vivo, we investigated the uptake and distribution of 59Fe-125I-sMTf and compared it to 59Fe-125I-Tf that were injected intravenously in rats. Studies were also performed to measure 59Fe and 125I-protein uptake by reticulocytes using these radiolabelled proteins. The results showed that sMTf was rapidly catabolized, mainly in the liver and to a lesser extent by the kidneys. The 59Fe was largely retained by these organs but the 125I was released into the plasma. Only a small amount of 125I-sMTf or its bound 59Fe was taken up by the brain, less than that from 59Fe-125I-Tf. There was much less 59Fe uptake by erythropoietic organs (spleen and femurs) from 59Fe-sMTf than from 59Fe-Tf, and no evidence of receptor-mediated uptake of sMTf was obtained using reticulocytes. It is concluded that compared to Tf, sMTf plays little or no role in Fe supply to the brain and erythropoietic tissue. However, a small amount of sMTf was taken up from the plasma by the brain and a far greater amount by the liver.     

Iron uptake by melanoma cells from the soluble form of the transferrin homologue,melanotransferrin

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue that can also exist in a soluble form (sMTf). Considering the high homology of MTf to Tf, it is possible to suggest that sMTf could bind to the high affinity transferrin receptor 1 (TfR1) or lower affinity TfR2. We have used sMTf labelled with 59Fe to examine its ability to donate Fe to cells. Our experiments demonstrate that sMTf is far less effective than Tf at donating Fe to cells and this does not occur via specific receptors. Indeed, the uptake of sMTf by cells occurred via a non-specific process (e.g. adsorptive pinocytosis).     

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.     

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.     

The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane-bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034-3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK-Mel-28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf-binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe-labelled Tf or sMTf had no effect on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein-free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.     

High transcytosis of melanotransferrin (P97) across the blood-brain barrier

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration- and conformation-dependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.     

Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.     

Mapping the human CAS2 gene, the homologue of the mouse brown (b) locus, to human chromosome 9p22-pter

Melanin biosynthesis is a multistep process with the first step being the conversion of L-tyrosine to L-Dopa catalyzed by the enzyme tyrosinase. The enzymes which catalyze the other steps of melanogenesis are not known. One murine pigmentation gene, the brown (b) locus, when mutated, leads to a brown or hypopigmented coat. The b-locus protein has been shown to display catalase activity. The human b-locus, therefore, is designated as CAS2. We used the mouse b-locus cDNA to isolate the human homologue, which in turn, was used to map the CAS2 locus to a human chromosome. The potential CAS2 protein codes for 527 amino acids containing a putative signal sequence and transmembrane domain. The CAS2 protein has primary and probably secondary structures similar to human tyrosinase. The CAS2 was mapped to human Chromosome 9 by somatic cell hybridization and, more specifically, to 9p22-pter by in situ hybridization. The assignment of CAS2 on the human Chromosome 9 extends this region of known homology on mouse Chromosome 4.     

CD4+ T cell clones specific for the human p97 melanoma-associated antigen can eradicate pulmonary metastases from a murine tumor expressing the p97 antigen.

p97 is a human tumor-associated Ag present on most melanoma cells that represents a possible target for immunologic attack. To evaluate the capacity of T cells reactive with this protein to promote elimination of melanoma cells expressing p97, a murine model was developed by transfecting a C3H/HeN melanoma with the p97 cDNA, generating p97-specific CD4+ T cells by in vivo immunization of C3H/HeN mice with a vaccinia/p97 recombinant virus followed by in vitro cloning with soluble p97 protein, and determining whether these CD4+ T cells could mediate rejection of pulmonary metastases. Characterization of the T cell clones demonstrated the presence of both I-Ak and I-Ek-restricted clones, although the majority of clones recognized p97 in the context of I-Ek. Analysis of clonal specificity using truncated p97 proteins revealed that at least three epitopes were immunogenic, and further studies with overlapping 15-amino acid peptides from a region of the p97 molecule defined by these truncated proteins identified an immunodominant epitope responsible for the majority of the I-Ek response. The T cell clones were not capable of directly recognizing the p97-expressing melanoma cells but responded to the tumor if syngeneic APC were present to process the tumor-derived p97 Ag. The therapeutic efficacy of these CD4+ T cell clones was evaluated in an adoptive therapy model in which mice bearing metastatic pulmonary lesions were treated by i.v. administration of the p97-specific cells. Despite the inability of the CD4+ clones to directly respond to or lyse the tumor cells, the clones were effective in promoting tumor eradication. In vitro studies demonstrated that this may have reflected secretion of lymphokines that activated macrophages to lyse the tumor. The results suggest that noncytolytic p97-specific CD4+ T cell clones can be effective in therapy of pulmonary melanoma metastases. Moreover, if human T cells reactive with the p97 protein could be generated, the expression of this tumor-associated Ag in melanoma cells might be adequate for such T cells to mediate a therapeutic antitumor response.     

Characterization of a recombinant vaccinia virus expressing human melanoma-associated antigen p97.

p97 is a cell surface glycoprotein expressed at high levels in most human melanomas but present only in trace amounts in normal adult tissues. We are interested in exploring the possibility of using recombinant vaccinia virus to express a specific tumor-associated antigen as a vaccine against human cancer. To this end, we constructed a recombinant virus, v-p97NY, which contains the entire coding sequence for p97 under the control of the vaccinia virus 7.5K promoter. Upon infection of tissue culture cells, v-p97NY expressed high levels of a membrane-bound glycoprotein immunoreactive with a p97-specific monoclonal antibody. Immunization of mice with this recombinant elicited high-titered antibodies against p97. Spleen cells isolated from these mice proliferated in vitro when stimulated either with purified p97 protein or with syngeneic cells expressing p97 antigen. Delayed-type hypersensitivity was also observed in immunized mice after challenge with p97-expressing cells. These findings indicate the potential usefulness of v-p97NY and similar recombinants in tumor immunotherapy.     

Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.     

Identification,expression and tissue distribution of a renalase homologue from mouse

FAD (flavin adenine dinucleotide)-dependent monoamine oxidases play very important roles in many biological processes. A novel monoamine oxidase, named renalase, has been identified in human kidney recently and is found to be markedly reduced in patients with end-stage renal disease (ESRD). Here, we reported the identification of a renalase homologue from mouse, termed mMAO-C (mouse monoamine oxidase-C) after the monoamine oxidase-A and -B (MAO-A and -B). This gene locates on the mouse chromosome 19C1 and its coding region spans 7 exons. The deuced amino acid sequences were predicted to contain a typical secretive signal peptide and a conserved amine oxidase domain. Phylogenetic analysis and multiple sequences alignment indicated that mMAO-C-like sequences exist in all examined species and share significant similarities. This gene has been submitted to the NCBI GenBank database (Accession number: DQ788834). With expression vectors generated from the cloned mMAO-C gene, exogenous protein was effectively expressed in both prokaryotic and eukaryotic cells. Recombinant mMAO-C protein was secreted out of human cell lines, indicating the biological function of its signal peptide. Moreover, tissue expression pattern analysis revealed that mMAO-C gene is predominantly expressed in the mouse kidney and testicle, which implies that kidney and testicle are the main sources of renalase secretion. Shortly, this study provides an insight into understanding the physiological and biological functions of mMAO-C and its homologues in endocrine.     

Purification and partial amino acid sequence analysis of the cellular tumour antigen, p53, from mouse SV40-transformed cells

The cellular tumour antigen p53 is implicated in the transformation process. To compare p53 from transformed cells and their normal counterparts in detail, and so to identify any structural differences that might alter p53 function, requires information on the primary structure of the protein. By making use of immunochemical techniques we have been able to purify nanomole amounts of p53. This was sufficient, using high sensitivity automated gas-phase sequencing techniques to determine the amino acid sequence of two tryptic peptides from p53. Their sequences agree completely with the predicted polypeptide sequence derived from a cloned cDNA for p53 mRNA and provide the first data on the amino acid sequence of p53. A combination of the high sensitivity amino acid sequencing procedures used here and cDNA sequence analysis should provide the complete amino acid sequence of p53.     

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)     

A molecular model for the tumour-associated antigen, p97, suggests a Zn-binding function.

The primary structure of p97 (melanotransferrin) has been compared with other members of the transferrin superfamily. A molecular structure of p97 has been modelled based on the crystal structure of diferric rabbit serum transferrin. The most significant amino acid substitutions in p97 are almost exclusively limited to only two regions; the C-lobe iron-binding cleft and the interlobe contact region. The latter includes within the N-terminal lobe a Zn-binding consensus sequence found in metallopeptidases, and in the C-terminal lobe a glutamic acid residue (Glu-394) capable of completing a potential thermolysin-like Zn-binding site. Thus, p97 may have a Zn-binding potential, unique amongst the transferrin superfamily.