The chromatographic heterogeneity of rat transferrin on immobilized concanavalin A and lentil lectin

Abstract

A procedure was developed for the isolation of the microheterogeneous forms of rat transferrin consisting of anion-exchange and serial lectin affinity chromatographies. By deploying this technique, four to five different anionic species of the protein were detected in plasma. The two major components obtained, which encompassed 92-94% of the plasma transferrin, were further studied by sequential lectin chromatography. The larger of the two, representing 60-63% of plasma transferrin, was bound by concanavalin A - Sepharose, while the smaller one (30-32% of plasma transferrin) resolved into an unbound (25-27% of plasma transferrin) and a retarded (4-5% of plasma transferrin) fraction. The latter eluted from the column in a volume which was 1.9 times larger than that required for the passage of nonretarded transferrin. In accordance with their fucose contents, each of these three concanavalin A fractions resolved into a bound (20-29%) and an unbound (71-80%) subfraction by chromatography on lentil-Sepharose. It is concluded that there exist two kinds of glycan microheterogeneity in rat transferrin and that they are unrelated to each other. Consequently, at least six different forms of rat transferrin are available with respect to glycosylation. Epididymal fucosidase cleaved fucose from apotransferrin slowly and from the tryptic glycopeptide rapidly. Exploratory studies performed in vivo failed thus far to identify the significance of fucose in rat transferrin.

Expression of glycosylated and nonglycosylated human transferrin in mammalian cells. Characterization of the recombinant proteins with comparison to three commercially available transferrins

“…The detailed studies by Regoeczi and his co-workers on glycan variants of rat Tf showed that certain changes including deletion of the glycan alter its half-life in the circulation and affect iron transport in vivo (Marz et al, 1982;Regoeczi et al, 1987;Hu et al, 1992a,b). Future studies must include assessment of the in vivo behavior of the nonglycosylated hTf and use of radiolabeled iron to assess the ability of the recombinant transferrins to donate iron to cells.…”

Structural Investigations of Glycoconjugates at High Sensitivity

“…Next, the approach was utilized to compare the glycosylation of the two polypeptide chains of murine Ia antigens. 286 Fractionations and characterization were performed on the N-glycan structures derived from human immunoglobulin G, 287 human von Willebrand factor, 288 equine chromnicgonadotropin and lutropin, 289 natural and recombinant blood coagulation factor VIII, 290 rat liver β-glu-curonidase, 291 human urinary kallikrein, 292,293 rat transferrin, 294 human serum transferrin, 199 human placental fibronectin, 295 human and rabbit testosterone-binding globulin, 296 natural and recombinant human interferon-β1, 297 recombinant human interleukin V, 298 recombinant human prorenin, 299 human leukocyte common antigen CD45, 300 human pancreatic bile-salt-dependent lipase, 301 zona pellucida 2 and zona pellucida 3 glycoproteins from mouse, 302 and human intercellular adhesion molecule-3 (CD50). 303 Differences among rat alkaline phosphatases from various organs were established by the serial lectin affinity technique.…”

Interaction of transferrin and its iron-binding fragments with heparin.

“…The principal component of rat transferrin (rTf), containing a standard diantennary disialosyl glycan, was prepared as before [5]. It was used either in the diferric or the iron-free (apo) form.…”