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Abstract

Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, beta-galactosidase or N-Acetyl-beta-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions.

A New Method for Obtaining Human Transferrin C-Lobe in the Native Conformation: Preparation and Properties

“…In contrast, expression of transferrin C-lobes has been less satisfactory, with much poorer yields and limited evidence of structural integrity (8,10). A previous study of recombinant human transferrin C-lobe, expressed by E. coli as inclusion bodies requiring solubilization and renaturation in urea and reducing solutions, reported spectroscopic and iron-binding verification of the native conformation but small yield (11). As indicated, we have obtained expression of immunoreactive human C-lobes in insect cells and in mammalian cells, but also in poor yield of a product unable to bind iron or copper in a specific manner.…”

“…Expression of wild-type and mutated holotransferrins and their N-lobes has been accomplished in several laboratories, but expression of C-lobes in the verified native conformation has been more problematic (8)(9)(10). Recombinant human C-lobe has been produced in Escherichia coli in the only report verifying native conformation, but renaturation of the product was necessary to achieve a yield of 5% of the total C-lobe protein (11). A possible explanation of the difficulty is that proper folding of the C-lobe, with its 11 disulfide bonds and 231 combinations of ways they might be formed, is normally guided by prior synthesis and folding of the N-lobe which then acts as a kind of chaperone for construction of the C-lobe.…”

Known and potential roles of transferrin in iron biology

“…While not a state of TF deficiency per se , aberrant levels of TF glycosylation are used as indicators of chronic alcohol consumption (Golka and Wiese 2004) and congenital disorders of glycosylation, a group of inherited diseases characterized by defective glycan biosynthesis (Jaeken 2010); while an absence of glycosylation does not impair binding of TF to TF receptor or Fe, it does impair cellular Fe uptake in vitro and in vivo (Hu et al 1991; Mason et al 1993; Hoefkens et al 1997). …”

X-ray Crystallography and Mass Spectroscopy Reveal that the N-lobe of Human Transferrin Expressed inPichia pastorisIs Folded Correctly but Is Glycosylated on Serine-32,

“…A prokaryotic system such as E. coli allows the easy production of large amounts of biological material, but transferrin can only be produced in an unfolded form having little biological activity (19)(20)(21)(22). A refolding step allows the production of transferrins that can bind iron and have appropriate absorption and EPR spectra, but this step reduces the yield to only ∼3 mg/L (53). At the other extreme, mammalian tissue culture systems such as BHK cells offer authentic folding of complex, disulfidecontaining proteins.…”