Binding and uptake of transcobalamin II by human fibroblasts.
We have used purified, (125)I-labeled human transcobalamin II (TC II), saturated with cobalamin (Cbl), to study the uptake process for the TC II-Cbl complex by intact normal cultured human skin fibroblasts. We have also investigated the possibility that a defect in one step of this process underlies that inborn error of Cbl metabolism-designated cbl C-in which mutant cells are unable to retain Cbl intracellularly or convert it to its coenzyme forms. TC II-Cbl binding at 4 degrees C reached a plateau after 3-4 hr; 95% of the bound (125)I was releasable with trypsin. Binding of TC II-Cbl at 4 degrees C could be inhibited by human and rabbit TC II-Cbl and human TC II devoid of Cbl but not by other Cbl-binding proteins, albumin, or free Cbl. Specific binding reached saturation at congruent with5 ng TC II/ml (0.13 nM) and could be inhibited by ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'- tetraacetic acid. At 37 degrees C, the TC II-Cbl complex was internalized as shown by a progressive decrease in the trypsin-releasable fraction of bound (125)I. After 2 h at 37 degrees C, increasing amounts of acid-soluble (125)I were found in the incubation medium indicating that the labeled TC II was being degraded. Chloroquine, an inhibitor of lysosomal proteolysis, prevented this degradation. The binding, internalization, and degradation of TC II-Cbl by cbl C cells was indistingusihable from that by control cells. Our studies provide additional support for the concepts: (a) that the TC II-Cbl complex binds to a specific cell surface receptor through a site on the TC II; (b) that the interaction between the receptor and TC II is calcium dependent; (c) that the TC II-Cbl is internalized via endocytosis; (d) that the degradation of TC II and release of Cbl from the complex occurs in lysosomes. We also conclude that the defect in cbl C must reside at some step beyond this receptor-mediated uptake process.
D001798:Blood Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D003037:Cobalt Radioisotopes; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D011485:Protein Binding; D012867:Skin / Q000166:cytology / Q000378:metabolism; D014155:Transcobalamins / Q000378:metabolism*
D001798:Blood Proteins; D003037:Cobalt Radioisotopes; D007457:Iodine Radioisotopes; D014155:Transcobalamins; D002738:Chloroquine; D002118:Calcium