Auswahl der wissenschaftlichen Literatur zum Thema „Melanotransferrin“

Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimer's disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimer's disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimer's disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G–Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimer's disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimer's disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimer's disease.

✓ A case of melanotic neuroectodermal tumor of infancy arising from the transverse sinus is presented. The tumor was located on the outer surface of the dura and extended extracranially through the occipitomastoid suture. Two cell populations were observed: pigmented melanocyte-like cells and small neuroblast-like cells. Ultrastructural analysis revealed epithelial tumor cells and melanosomes at various stages. Expression of melanotransferrin messenger RNA transcripts within the tumor tissue was observed using a reverse transcriptase—polymerase chain reaction method in addition to immunohistological studies. The positive expression of melanotransferrin confirmed that this melanotic neuroectodermal tumor was derived from neural crest cells.

The EOS47 antigen is a 100-kD cell surface glycoprotein selectively expressed by avian retrovirus-transformed eosinophils and their precursors. We have purified the EOS47 protein to homogeneity and used peptide sequence information to clone EOS47-encoding cDNAs. The open reading frames from these cDNAs predict a 738 amino acid protein with homology to human melanotransferrin, a membrane-found, transferrin-like protein that is expressed at high levels by a subset of melanomas, tumor cell lines, fetal intestine, and liver, but not by most normal adult tissues. The predicted protein sequence of EOS47 displays a 61% sequence identity with melanotransferrin and conservation of all 28 cysteine residues, indicating a similar tertiary structure. The finding that EOS47 lacks several of the iron-coordinating amino acids present in all transferrins suggests that it may be impaired in its ability to bind iron. In nonhematopoietic tissues, EOS47 is expressed at high levels by epithelial brush borders of small intestine and kidney and at lower levels by cells lining the sinusoids of the liver. Within hematopoietic tissues, EOS47 is restricted to a subpopulation of cells (1% to 5%) in bone marrow and early spleen and fluorescence-activated cell sorting of EOS47+ cells leads to a dramatic ( > 30-fold) enrichment of peroxidase+ eosinophils. In contrast, peripheral blood eosinophils are EOS47-, suggesting that the antigen is expressed by newly formed eosinophils and that expression ceases shortly before these cells emigrate from the bone marrow into the peripheral blood. Our results show that melanotransferrin is a stage-specific marker of eosinophils and should be useful for their isolation and further characterization.

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 × 107m–1at 25 °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 °C, iron binding occurs in a 1:1 ratio with aKappof 4.4 × 1017m–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.

Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Doctor of Philosophy(PhD)
Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Melanoma, one of the deadliest forms of skin cancers, is a malignant tumor derived from abnormal proliferation of epidermal melanocytes, and its formation involves a series of events leading to altered cellular properties and rapid proliferation. High cellular iron content has been connected to the development of cancers in human. One way iron may affect cancer development and progression is by altering cell growth and proliferation. Iron has been proposed to promote progression from G₁ to S phase of the cell cycle through activation of ribonucleotide reductase during DNA synthesis. Iron is normally absorbed by the enterocyte of the duodenum where it is transported throughout the body via serum transferrin. However, the existence of transferrin-transferrin receptor independent iron transport pathways has been shown. Such pathways are thought to play critical roles in non-transferrin bound iron overload in diseases such as hemochromatosis and Alzheimer’ s disease, though the molecular details of these pathways remain obscure. Many genes are up-regulated to aid the abnormal proliferation of cancer cells and subsequent invasion of tissues. In the case of melanoma, the expression of melanotransferrin (p97), an iron binding molecule, is elevated. In this thesis, the cellular and functional role of glycosyiphosphatidylinositol (GPI)-anchored p97 in melanoma was investigated. The first aim of this thesis was to perform detailed investigation of the internalization pathway of GPI-anchored p97 in melanoma cells. Although many GPI anchored proteins have been studied previously, there is still an on-going debate whether caveolae-dependent or clathrin-dependent pathways are involved. Using confocal immunofluorescence microscopy and quantitative immunoelectron microscopy, iron bound GPI-anchored p97 was shown to be internalized via a caveolae vesicles dependent endocytotic pathway. In addition, endosomal disruption studies demonstrated that the intracellular trafficking of the GPI-anchored p97 in melanoma cells is endosomal dependent. The studies performed in this thesis demonstrate that GPI-anchored p97 protein can mediate the iron uptake in melanoma cells. As GPI-anchored p97 is highly expressed in melanoma cells, this thesis also examined the functional role of GPI anchored p97 in melanoma cells and tumors. The expression of GPI-anchored p97 in melanoma cells through over-expression and down-regulation techniques. The results demonstrated that the GPI-anchored p97 promote melanoma cell proliferation, melanin production and secretion in vitro and melanoma tumor growth in vivo. Taken together, this thesis sheds new light on the relationship between GPI-anchored p97, melanoma development and cellular iron uptake.

It has been hypothesised that MTf may participate in melanoma progression due to the fact that the MTf plays a vital role in proliferation and tumorigenesis. This suggestion has been supported by the high levels of MTf expression in melanoma cells and the fact that previous studies demonstrated silencing MTf decreased melanoma tumour xenograft growth in vivo. In contrast, N-myc downstream regulated gene 1 (NDRG1), is a potent metastasis suppressor and acts to inhibit several oncogenic pathways, such as WNT, PI3K/AKT, etc. This thesis sought to elucidate the mechanism of MTf activity as a novel pro-oncogenic signalling protein involved in melanoma pathogenesis and whose molecular mechanism(s) of action remain unclear. Since metastasis accounts for most cancer deaths and is a major problem in melanoma, it was critical to further discover the molecular mechanisms that underlie NDRG1’s ability to inhibit progression and metastasis of melanoma. Furthermore, this thesis examined a promising anti-metastatic therapeutic strategy by assessing the novel, clinically trialled, anti-cancer agent, namely di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which potently up-regulates NDRG1 in a variety of tumour cells. This dissertation consists of 6 chapters: A comprehensive literature review in Chapter 1 and a general materials and methods chapter (Chapter 2). This is followed by 3 results chapters, namely: Chapter 3 examining the inter-relationship between NDRG1 and MTf in vitro and in human melanoma samples; Chapter 4: exploring the effect of MTf on the crucial upstream receptors of WNT- and PI3K signalling, including LRP6, c-Met, VEGFR2 and FGFR1; and Chapter 5 understanding how MTf drives proliferation and activates these oncogenic pathways when c-Myc is silenced. These chapters are then followed by Chapter 6, which is a general discussion of findings and future directions.

P97, encore appele melanotransferrine de part son homologie avec la transferrine, est un antigene tumoral des cellules de melanomes. Ces dernieres sont des cellules tumorales qui derivent des melanocytes, cellules de la peau specialisees dans la synthese de la melanine. L'etude de la region 5' du gene de la melanotransferrine (mtf) a permis l'identification d'un enhancer localise a 2 kpb en amont du promoteur et compose de deux regions qui agissent en synergie pour diriger la forte expression du gene dans les cellules de melanomes. Cet element de regulation est associe a une region accessible de la chromatine ou les nucleosomes semblent positionnes. L'analyse des facteurs de transcription interagissant avec l'enhancer a permis de mettre en evidence deux sites de fixation pour des membres de la famille ap1 et un site de fixation pour le facteur usf. Un des sites ap1 est la cible d'un complexe ternaire, specifique des cellules de melanomes, entre l'adn de cette region, ap1 et un autre facteur qui pourrait appartenir a la famille ets et dont l'expression serait plus restreinte a la lignee melanocytaire. La formation de ce complexe sur l'adn est liee a une forte activite transactivatrice. Par ailleurs, l'expression de l'arn messager de la mtf varie en fonction du temps de culture. Elle est correlee a une expression parallele des arns messagers des membres de la famille ap1 et a une augmentation de l'activite de liaison a l'adn des membres de cette famille. Parallelement, l'etude de l'accessibilite de la chromatine a permis de mettre en evidence deux sites hypersensibles a la dnasei, localises dans les introns, specifiques des cellules de melanomes. Ces sites hypersensibles pourraient correspondre a des regions de regulation qui agiraient avec l'enhancer pour diriger l'expression du gene dans les cellules de melanomes

When producing recombinant mammalian proteins in an expression system, the successful completion of posttranslational modifications is an area of concern. One such modification is the attachment of a protein to a glycosyl-phosphatidylinositol (GPI)-anchor in the membrane. In order to investigate this, the baculovirus/insect cell system (Autographa californica multiple nuclear polyhedrosis virus/'Spodoptera frugiperda Sf9 cells) was used to express the human melanoma-associated antigen, p97 or melanotransferrin. An unusual feature of this protein is its attachment to the cell surface by a GPI-anchor. The expression of p97 at the surface of recombinant virus infected Sf9 cells was shown by FACS analysis using monoclonal antibodies that recognize two different epitopes. Immunoprecipitation of p97 from [35S]-methionine metabolically labelled p97 B-2-I infected Sf9 cells revealed that recombinant p97 is slightly smaller than p97 expressed by the SK-MEL-28 human melanoma cell line and that a soluble form of p97 was present in the spent medium of the infected Sf9 cells. While the GPI-anchored form of recombinant p97 partitioned into the detergent phase upon Triton X-114 extraction, the form found in the spent medium partitioned into the aqueous phase, suggesting that it may be analogous to the secreted form of p97 produced by SK-MEL-28 cells. The glycosylation of recombinant p97 from virus infected Sf9 cells was also analyzed. Although an Endoglycosidase H-resistant form of p97 was detected, it is likely that the processing of N-linked oligosaccharides to the complex-type was incomplete. Finally, the GPI-llinkage of p97 expressed in Sf9 cells was demonstrated by phosphatidylinositol-specific phospholipase C sensitivity and Triton X- 114 extraction. The ability to express GPI-linked proteins in this system will be useful for the bioengineering and commercial production of proteins.