Paul A. Chindemi

Rat aglycotransferrin and human monoglycotransferrin: production and metabolic properties

Rat transferrin (rTf), containing one complex glycan, and human transferrin (hTf), containing two complex glycans, were treated with peptide:N-glycosidase F (PNGase) under nondenaturing conditions. Apo rTf with a nonfucosylated standard biantennary glycan, but not its diferric counterpart, yielded satisfactory amounts (approximately 55% in 7 h) of aglyco Tf (AgTf). The presence of a chitobiose core fucose reduced yields to approximately 30%, whereas an additional NeuAc on the GlcNAc in the Man(alpha 1-3) branch had no adverse effect. Triton X-100 impaired deglycosylation. The main product (approximately 65%) obtained from apo hTf was monoglyco Tf (MgTf). Analysis of the cyanogen bromide fragments of MgTf revealed that PNGase did not discriminate between the two glycosylation sites of hTf. A negligible portion (2-4%) of AgTf, that was also obtained during the reaction, probably resulted from PNGase action on denatured hTf molecules. Modified Tfs were separated by affinity chromatography, radiolabeled, mixed with another preparation of interest, and injected in rats. Total-body radiation measurements showed that the half-life of rat AgTf was 19-20% shorter than that of rTf but 9% longer than that of asialo Tf. This suggests that close to 76% of the change in the degradative rate observed after desialylating rTf is referable to charge loss rather than the exposure of Gal residues. Human MgTf was catabolized by rats 8-9% faster than the parent protein, while human AgTf behaved in vivo like a denatured protein. It is concluded that sialyl carboxyls are a codeterminant of the normal lifetime of transferrins.

Reduced hepatic iron uptake from rat aglycotransferrin.

SummaryRat aglycotransferrin (rAgTf) was produced from the disialosyl diantennary fraction of rat transferrin (rTf) by treatment with peptide:N-glycosidase F. Following removal of the enzyme by gel filtration and isolation of the deglycosylated protein by lectin chromatography, rAgTf was compared to rTf both in vitro and in vivo. No significant differences were found between the two proteins with respect to affinity for iron and kinetics of Fe release from the N-lobe and C-lobe. The fluorescence emission spectrum of apo-rTf was red-shfited by approximately 3 nm relative to diferric rTf; however, no spectral difference was detected between rTf and rAgTf when the analogous forms (apo or diferric) were compared. Plasma clearance of radioactive iron administered to rats as either rTf or rAgTf was comparable. Reticulocytes took up iron from rAgTf slightly faster than from rTf. In contrast, Fe acquisition by the liver from rAgTf was significantly reduced relative to rTf. This finding contrasts sharply with earlier observations with asialotransferrin (rAgTf) and provides a basis for discounting charge loss as the mechanism of enhanced hepatic Fe uptake from rAgTf. It is suggested that the glycan complement of rTf, while unimportant for interaction of the protein with specific receptors, probably plays a role in the interaction with low-affinity hepatic binding sites.

The effects of cytotropic compounds on the resialylation of human asialotransferrin type 3 in the rat

Effects of chloroquine, colchicine, leupeptin, taxol and vinblastine on the resialylation and degradation of human [125I]asialotransferrin type 3 were studied in rats. An improved experimental technique was applied that permitted the quantification of resialylated ligand produced by individual animals over 3 h by using deconvolution. All three microtubule inhibitors increased the proportion of the dose undergoing resialylation by 35-39%. In addition, colchicine, and, especially, vinblastine enhanced the overall recovery of the dose as protein-bound 125I. The dose recovery was also augmented by leupeptin without any concomitant change in resialylation. Chloroquine suppressed resialylation and this effect could only be partially lifted by the administration of colchicine. The blood of colchicine-treated rats possessed no resialylating activity toward the ligand even when supplemented with additional alkaloid in vitro. The observations support the view that the respective fractions of the ligand destined for resialylation and degradation can, to a certain extent, be varied independently of each other. The effects of short-term starvation (20 h) and refeeding (4 h) on these processes are also presented.

The Roles of Secondary Binding Sites for Transferrin in the Liver and on Macrophages

As reviewed by several authors, 1,2 ample evidence has been gathered over the past two decades to postulate a central role for specific receptors in transferrin-mediated iron metabolism of eukaryotic cells. These trypsin-sensitive3 glycoprotein dimers attract diferric transferrin (2Fe-Tf) from the extracellular fluid for endocytosis, followed by the release of iron in an intracellular location and return of both receptor and iron-free Tf (apo Tf) to the plasmalemma. Many observations, mainly on various hematopoietic cell lines, support the existence of an iron acquisition mechanism as just outlined.

Metabolic stability of the fucose in rat transferrin

The metabolic behaviour of the chitobiose core fucose that is a natural constituent of a large proportion of rat transferrin molecules was studied in rats comparatively to that of the polypeptide portion of the glyco‐protein by using appropriate labels ([3H]fucose and 125I) and affinity chromatographic techniques (lentil‐Sepharose). No evidence was obtained to suggest that this residue is cleaved from the glycan in significant amounts before removal of the entire glycoprotein for catabolism. Similarly, [14C]fucose linked to GlcNAc residues in the antennae of human asialotransferrin was being eliminated in pigeons at the same rate as the polypeptide itself. It is concluded that in spite of transferrins exposure to the cellular milieu, the fate of its fucose is distinctly different from that of the same in plasma membrane glycoproteins.