The iron-binding prcpertios of melanotransferrin, the tumour-associated antigen also known as ~97, have been investigated by UWvisible and Ruorescencc spectroscopy, amino acid sequence comparison, and modelling. These show that, in contrast to other vansferrins. mclanotransferrin binds only one Fe'+ ion per molecule. The binding properties of its N-terminal site arc similar to other transrcrrins, but its C-terminal site does not bind iron at all. The differences ca:l be related to spe;i!ic amino acid changes in the C-terminal site.Iron binding; Melanotransfcrrin; Transferrin; Melanoma cell antigen
I, INTRODUCTIONMelanotransferrin, (MT& also known as the tumour-associated antigen, p97, is a monomeric glycoprotein, Ii/f, 97,000, which is expressed by human melanoma cells and certain other tissues [l-3], but is present only in trace amounts in normal adult tissues [4]. Its amino acid sequence [S] shows a high level of identity with proteins of the transferrin family (-40% identity with human transferrin or lactoferrin), it binds iron [6] and it has been proposed to play a role in iron translocation [5].MTf does, however, differ significantly from other transferrins. It is membrane-bound, and it differs in certain key amino acid residues which in other transferrins are involved in iron binding. Examination of the MTf sequence in relation td the three-dimenqional structures of human lactoferrin [7,83 and rabbit serum transferrin [9] has Ied to the proposal that it has an intact transferrin-type iron binding site in its N-terminal half but a possibly defective site in its C-terminal half [lo]. We have tested this proposal by investigation of the iron-binding properties of MTf, and discuss the results in the light of its presumed three-dimensional structure.
MATERIALS AND METHODS I. isolation of MT/MTf was purified from cell culture supcrnatanls of transfected mouse melanoma clone 2a cells contained in ten IO-shelf cell factories. The cultures were allowed to incubate at 37°C for 10 days at which time the cells had grown to confluence and were showing signs of deterioration. Cell debris was removed by filtration and the filtrate was concentrated IO-to 1%fold with a Pellicon ultrafiltration system (Millipore) using a 5 sq. ft. 3O,ooO NMWL casctte. The retentate (approx. 2 I) was 0.2 pm filtered and applied overnight to a column (1.6 x 16 cm) of immobilize4 96.5 mAb [3] that had been previously washed with 0. I M citricacid. pH 2.2, and equilibrated in PBS, pH 7.2. The column was washed thoroughly with PBS and the bound MTf was cluted with 0.1 M citrate bulTer, pH 4.0, followed by immediate neutralization with Tris. The purified MTf (approx. IO mg) was dinlysed into PBS and sterile filtered. Batch analysis consisted of gel filtration HPLC, SDS-PAGE, isoelectric focusing and double-determinant binding ELISA,
Mcamrawtrs of irott bittdingIron was added as ferric nitrilotriacetatc (FcNTA) to solutions of MTf in 0.025 M Tris-HCI, pH 7.8, containing 0.01 M NaHCO, and 0. I M NaCI. The protein concentration was estimated usin...