Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines.

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.

Expression of cell surface GPI-anchored human p97 in baculovirus-infected insect cells.

The baculovirus/insect cell system (Autographa californica multiple nuclear polyhedrosis virus/Spodoptera frugiperda Sf9 cells) was used to express the GPI-anchored human melanoma tumor antigen, melanotransferrin or p97. This system served to study the expression and productivity of recombinant GPI-anchored p97 by insect cells. The Sf9 cells expressed a cell surface GPI-anchored form of p97 as well as a soluble form of p97 that did not appear to be derived from the GPI-anchored form of p97. Both recombinant forms, although Endo H resistant, migrated slightly faster ( approximately 88 kDa) than the native p97 ( approximately 95-97 kDa). The insect GPI-anchored p97 was sensitive to PI-PLC, which exposed a detectable cross-reacting determinant. The Sf9 cell surface p97 expression was similar to that of human melanoma (SK-MEL-28) cells, whereas the Sf9 cell specific secretion rate was 10-fold higher. Also Sf9 cells retained considerably higher levels of p97 within the cell. The Sf9 cell surface expression of p97 varied with time after infection, with the maximum expression, which appeared independent of multiplicities of infection greater than 1, occurring at 48 h. After 48 h, levels of cell surface and secreted p97 fell whereas p97 retained within the cell increased, which possibly reflected the lytic nature of the expression system. The successful expression of GPI-anchored human p97 by the baculovirus/insect cell system not only provides a source of p97 for further research but also is the basis of an alternative method for the commercial production of GPI-anchored proteins.

Increasing GPI-anchored protein harvest concentrations from suspension and porous microcarrier CHO cell cultures.

Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for the production of recombinant glycosyl-phosphatidylinositol (GPI) anchored proteins. The cells were cultured either in suspension or immobilized on porous microcarriers and p97 was selectively cleaved from the cell surface by the bacterial enzyme, phosphatidylinositol-phospholipase C (PI-PLC). The kinetics of p97 cleavage from the cell surface by PI-PLC was shown to be approximated by Michaelis-Menten kinetics. The recovered p97 concentrations were increased by reusing the PI-PLC enzyme solution to harvest multiple batches of cells. A convenient PI-PLC assay was developed to monitor the harvesting process and to determine the stability of PI-PLC under harvesting conditions. Although the Pl-PLC was stable under harvesting conditions, it rapidly adsorbed to the cell surface and was depleted from the reused enzyme solution. In order to maintain PI-PLC activity, it was necessary to add fresh PI-PLC to the reused enzyme solution before harvesting a fresh batch of cells. The maximum p97 concentration that could be obtained from harvesting CHO cells cultured on porous microcarriers was limited by the dilution effects of sample removal, adding fresh PI-PLC and liquid associated with settled microcarriers. A model was developed that adequately predicted the p97 concentration after each harvest and the maximum p97 concentration that could be achieved by this harvesting method. The dilution effects were minimized by harvesting from centrifuged suspension culture cells and the harvested p97 concentration was increased by over sixfold to 0.64 mg/mL.

Serum levels of the iron binding protein p97 are elevated in Alzheimer's disease.

Alzheimer's disease is a progressive and incurable disease whose prevalence increases dramatically with age. A biochemical marker for monitoring the onset and progression of the disease would be a valuable tool for disease management. In addition, such a marker might be used as an end point in clinical intervention protocols. Here we provide evidence that the soluble form of the iron binding protein p97 is found in elevated amounts in the serum of Alzheimer's patients compared with healthy controls. This biochemical marker has the potential for identifying subjects afflicted with the disease and possibly for monitoring the onset and longitudinal progression of the disease.

Pumping iron in the '90s.

The role o f iron in cell division, cell death and human disease has recently gained increased attention. The best studied process for iron uptake into mammalian cells involves traps ferrin and its receptor. This review discusses evidence supporting the existence of other routes by which iron can enter mammalian cells. Specifically, iron uptake by the cell-surface GPI-linked traps ferrin homologue, melanotransferrin or p97, is described and possible functions of this traps ferrin-independent pathway are proposed.

Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium.

One method of iron transport across the blood brain barrier (BBB) involves the transferrin receptor (TR), which is localized to the specialized brain capillary endothelium. The melanotransferrin (MTf) molecule, also called p97, has been widely described as a melanoma specific molecule, however, its expression in brain tissues has not been addressed. MTf has a high level of sequence homology to transferrin (Tf) and lactoferrin, but is unusual because it predominantly occurs as a membrane bound, glycosylphosphatidylinositol (GPI) anchored molecule, but can also occur as a soluble form. We have recently demonstrated that GPI-anchored MTf provides a novel route for cellular iron uptake which is independent of Tf and its receptor. Here we consider whether MTf may have a role in the transport of iron across the BBB. The distributions of MTf, Tf and the TR were studied immunohistochemically in human brain tissues. The distributions of MTf and TR were remarkably similar, and quite different from that of Tf. In all brain tissues examined, MTf and the TR were highly localized to capillary endothelium, while Tf itself was mainly localized to glial cells. These data suggest that MTf may play a role in iron transport within the human brain.

Membrane anchored protein production from spheroid, porous, and solid microcarrier chinese hamster ovary cell cultures.

The influence of the microcarrier type on the performance of a controlled release process used to produce a recombinant glycosyl-phosphatidylinositol anchored protein was investigated. Chinese hamster ovary (CHO) cells expressing the human melanoma tumor antigen (p97) were cultured in 10% serum on Cultispher-GH porous microcarriers and then, for protein production, maintained in 2% serum. Cells were harvested every 48 h and p97 was recovered at 90 mug/mL and 40% purity. Harvested p97 concentrations were increased by harvestingfrom spheroid (241 mug/mL) and smaller porous microcarrier, Cultispher-G (167 mug/mL) cultures. The low total cell specific p97 production of cells cultured on Cultispher-GH was due to necrosis of cells within the beads, decreased p97 expression of the immobilized cells, dilution by the liquid (up to 40% volume) associated with settled beads, and incomplete recovery of p97 from within the beads. Cells cultured on solid microcarriers, Cytodex-1, had the highest cell viability and cell specific p97 production, It is recommended that a two-stage cyclic harvesting process of cells cultured on small Cultispher-G or on Cytodex-1 beads would minimize protein loss and maximize cell specific protein recovery. (c) 1995 John Wiley & Sons Inc.

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.

Glycolipid membrane anchored recombinant protein production from CHO cells cultGred on porous microcarriers.

Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano-transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl-phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T-flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate-buffered saline (PBS) containing 10 mU/mL phosphatidylinositol-phospholipase C (PI-PLC). The GPI anchor was selectively cleaved by PI-PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 microg/mL due to difficulties in maintaining stable confluent T-flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 10(7) cell/mL. The p97 was harvested at up to 100 microg/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum.

Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells.

A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins.