Difference in Binding and Fate of Lactotransferrin in Jurkat Human Lymphoblastic T-Cells and in T-47D Human Breast Cancer Cells

Summary

A 95-kDa lactotransferrin-binding protein has been isolated from the cell culture medium of the human Jurkat lymphoblastic T-cell. It corresponds to the soluble form of the 105-kDa lymphocyte lactotransferrin receptor. A monoclonal antibody has been raised against the soluble lactotransferrin binding protein; it recognized both the soluble lactotransferrin-binding protein and the membrane lymphocyte lactotransferrin receptor after SDS-PAGE in the presence of 2-mercaptoethanol and electrotransfer on nitrocellulose. The 105-kDa lymphocyte receptor has been localized in Jurkat cells, at the cell surface in coated-pit-like domains. The binding of either iron-saturated or iron-free lactotransferrin to the Jurkat human lymphoblastic T-cell line was saturable with a dissociation constant (K _d ) of 40 nM, and the total number of binding sites was estimated to be ~300,000. Jurkat cells internalize lactotransferrin; after binding at 4°C, a shift to 37°C causes cells to internalize lactotransferrin, with the maximum intracellular concentration found at 3–8 and 12–15 min for iron-saturated and iron-free forms, respectively. In contrast, epithelial breast cancer cell T-47D, which expresses low amounts of 105-kDa receptor and high amounts of proteoglycans, binds [¹²⁵I]-labeled lactotransferrin with a dissociation constant of 120 nM and a high number of sites/cell (1.1 × 10⁶), and does not internalize lactotransferrin. Moreover, as demonstrated using [¹²⁵I]-labeled lactotransferrin, after 30 min incubation at 37°C, about 40% of lactotransferrin was still associated with the plasma membrane, 60% being released in an undegraded form into the medium. Furthermore, iron was not taken up by T-47D cells but partially released in the external medium (45% of total lactotransferrin-bound iron).