Date of Award

11-1988

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Dr. E. Regoeczi

Abstract

Rat transferrin (RTf) was used to study the interaction of transferrin' (Tf) with the liver, with special reference to the çlycan of Tf.

Iron uptake from Tf by cells is believed to occur by Tf receptor (TfR)-mediated endocytosis. Results from the present studies revealed that over a large range of competing diferric RTf concentrations, a constant percentage of iron is uptaken from RTf by the liver in vivo, and by hepatocytes in vitro. Hepatocytes were found to contain ~129,000 TfR/cell with ~40% expressed on the surface as estimated by both a polyclonal anti-TfR antiserum, produced as part of this thesis work, and a monoclonal anti-TfR antibody. On the basis of this estimate it was calculated that TfRs are not present in sufficient quantities to account for the observed uptake of iron. Studies of uptake and release of polyvinyl-pyrrolidone (PVP), RTf, and iron (as diferric Tf) were carried out in suspended hepatocytes. These studies demonstrated that the most likely mechanism to account for the results and to explain the iron uptake is "mixed-type" pinocytosis. The uptake of iron was found to be modulated by the type of glycan on RTf.

On the basis of glycan microheterogeneity, at least six subforms of RTf are found to exist in rat plasma. These subforms are RTf-1, RTf-2·and RTf-3 as resolved by concanavalin A; ~20% of each is fucosylated and ~80% non-fucosylated. (The presence of fucose was found to have no measurable effect on catabolic rate, plasma iron disappearance or iron donation to liver in vivo or hepatocytes in vitro.) The slalylated subforms have different half-lives (RTf-1>RTf-2>RTf-3) with RTf-1 being significantly longer than RTf-3. Comparison of plasma iron disappearance and rates of iron donation to liver in suggested a trend (RTf-l>RTf-3>RTf-2) which was reproduced and found to be significant in studies with hepatocytes: iron uptake by hepatocytes from RTf-1 and RTf-3 could be competitively inhibited by an excess of the homologous subform. Desialylation of the subforms (RAsTfs) significantly reduced the half-lives and altered the order (RAsTf-3>RAsTf-1>RAsTf-2) with RAsTf-3 being significantly longer than RAsTf-2. The desialylated subforms were superior donors of iron to the liver in vivo. Studies to explain the enhanced rate of iron delivery by RAsTf, discounted the possibility of differing rates of iron release, but allowed postulation of a synergistic dual receptor mechanism. Results from experiments with hepatocytes in vitro supported the proposed mechanism. It is concluded that subtle differences in glycan structure can result in functional differences between Tf subforms.

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