Hepatic iron uptake from and degradation of rat asialotransferrin prepared from the least anionic (major) component of rat transferrin were studied in intact rats. In experiments lasting 60-90 min, rat asialotransferrin delivered a three to four times larger fraction of the Fe dose to the liver than rat transferrin. Variations in the concentration of endogenous circulating rat 2Fe-transferrin by up to 300% failed to affect the enhanced hepatic delivery of Fe from rat asialotransferrin. However, pretreating the animals with a large dose of asialomucin, or fully sialylated human transferrin, or a combination of both did affect the delivery. In all cases, rat asialotransferrin delivered Fe to the liver at rates comparable with those seen with rat transferrin. The reason for the efficacy of human transferrin was clarified in competitive binding studies on rat hepatocytes and reticulocytes, which showed that human transferrin possessed an approximately sevenfold higher affinity for rat transferrin receptors than the homologous protein. These findings suggest that the enhanced hepatic uptake of Fe from rat asialotransferrin is mediated by simultaneous binding of the ligand both through its glycan and transferrin receptor affinity site. Pretreatment with asialomucin and human transferrin had no suppressing effect on basal hepatic delivery of iron from rat 2Fe-transferrin. The data suggest that deposition of a significant fraction of Fe in rat liver from rat transferrin is likely to take place by a mechanism not involving transferrin receptors. Desialylation shortened the metabolic half-life of rat transferrin from 33 to 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

(1987) Biochem. Cell Biol. 65,948 -9541 was used to isolate from three DEAE-cellulose chromatographic fractions of diferric rat serotransferrin (rTf) subpopulations having discernible affinities for concanavalin A (ConA). These entities are designated rTf-1 (not retarded by ConA column), rTf-2 (retarded) and rTf-3 (bound). Each rTf type was found to be endowed with carbohydrate sufficient to account for a single diantennary glycan/protein molecule. Glycan structures were determined on the glycopeptides by employing GLC/MS and 400-MHz 'H-NMR spectroscopy.All glycans possessed a common, trimannosyl-N,N-diacetylchitobiose core with or without one L-fucose a-l,6-linked to the Asn-linked GlcNAc. However, there were differences in the antennae. Thus, in rTf-3, both antennae were of the disialylated diantennary N-acetyllactosamine type which is frequently encountered in other plasma glycoproteins. However, the a-1,3-Man-linked antenna in rTf-1 as well as rTf-2 had the sequence: NeuSAc(ct2-3)Gal(fl1-3)[Neu5Ac(ct2-6)]C1cNAc(fll-2)Man.In addition, the a-l,&Man-linked antenna deviated in rTf-2 from the standard structure by having the sequence: Neu5Ac(a2-3)Gal(fl1-3)GlcNAc(P1-2)Man. The possible relevance of the above structures to the ConA binding of rTf is discussed. A further preparation, obtained from the most anionic DEAE-cellulose fraction (peak V) of rTf contained several tetrasialylated diantennary glycans whose precise structures remain to be established in future studies.We have shown recently that the glycan heterogeneity of rat serotransferrin (rTf) is more extensive than previously thought [l]. Evidence for this was obtained by the resolution of iron-saturated rTf (2Fe-rTf) into several components during anion-exchange chromatography as well as affinity chromatography on concanavalin A (ConA) and lentil lectin. Sequential application of these techniques revealed that at least six variants of rTf exist with respect to glycan. They were not an artifact of protein purification, for their presence could be demonstrated after tracing rTf in fresh plasma with radioiron.We have now purified these rTf components in sufficient quantities to enable us to determine their glycan structures. Our results are reported below. MATERIALS AND METHODS Preparation of i+Tf and designation of its componentsPurification of rTf from freshly pooled plasma was undertaken by the three-step procedure previously described [l]. The last step, which consisted of gradient elution from a column of DEAE-cellulose (Balston), yielded five peaks of rTf, referred to as DEAE peak I, 11, etc. in the order of their appearance. The present work concerns rTf in DEAE peak 111 (60-63% of total rTf), DEAE peak IV (30-32%), and DEAE peak V (3-4%). The protein obtained in peaks I11 and IV was used without further purification. However, rTf in DEAE peak V was contaminated with peak IV material. This was removed in two subsequent steps, namely, (a) by chromatofocusing on a PBE 94 column (Pharmacia) that was equilibrated with 10 mM histidine/HCl pH 6.2 and ...

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.

The content and distribution of transferrin receptors (Tf-R) in suspended adult rat hepatocytes were studied using 125I-protein A in combination with either a monoclonal (MRC OX-26) or a polyclonal antibody to Tf-R. Internal receptors were made accessible by permeabilization with digitonin. The number of Tf-R detected depended on the batch of collagenase used for liver perfusion. By using the monoclonal reagent in conjunction with the less damaging of two batches of the enzyme, 129,000 receptors were found per cell, with 47,000 (37%) of these on the surface. The polyclonal reagent yielded Tf-R numbers which were consistently higher than those obtained with MRC OX-26. This difference is interpreted as being due to the binding of several (on the average 5-6) molecules of polyclonal IgG per molecule of Tf-R. Remarkably, transferrin binding by Tf-R was not affected by this cluster of associated IgG and the overlayer of protein A. Parallel studies with 131I-transferrin in a simplified binding assay system yielded surface Tf-R estimates which, in most cases, were close to the values obtained with MRC OX-26. After prolonged exposure to collagenase, the ligand-binding capacity of Tf-R was more affected than its immunoreactivity. In preliminary studies, monensin (10 microM) produced a 32%-50% shift of Tf-R from the surface to the inside, whereas short-term incubation with epidermal growth factor (0.17 mM) brought about no clear-cut Tf-R redistribution.