Valérie Salmon

Structural Determination of Two N-Linked Glycans Isolated from Recombinant Human Lactoferrin Expressed in BHK Cells

A full‐length cDNA coding for human lactoferrin was isolated from a mammary gland library and the recombinant protein was expressed in BHK cells as described by Stowell K.M. et al. [1991, Biochem. J. 276, 349–355]. Two N‐linked glycans from purified recombinant lactoferrin were released by hydrazinolysis and analyzed by 400‐MHz 1H‐NMR spectroscopy. The identified structures corresponded to N‐acetyllactosaminic biantennary glycans and were α‐2,3‐disialylated forms (80%) or α‐2,3‐monosialylated (20%) forms. Moreover, 70% of total glycans were α‐1,6‐fucosylated at the GlcNAc residue linked to asparagine. In regard to its glycan moiety, the recombinant glycoprotein is close to native lactoferrins from milk or leucocytes but shows specific structural features which should be taken into account prior to in vivo and in vitro biological studies.

Role of the First N-Terminal Basic Cluster of Human Lactoferrin (R2R3R4R5) in the Interactions with the Jurkat Human Lymphoblastic T-Cells

We previously characterized a receptor of Mr 105,000 for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of R2R3R4R5 of hLf in the interaction with cells, we studied the binding of hLf variants obtained either by tryptic proteolysis (hLf-2N, hLF-3N and hLf-4N) or by mutagenesis (rhLf-5N). Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. The binding parameters of bovine Lf and native hLf did not differ, whereas the binding parameters of murine Lf resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulfation, reduced the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N indicating that the hLf binding sites include sulfated molecules. The results suggest that the interaction of hLf with about 80,000 binding sites per Jurkat cell, mainly sulfated molecules, is dependent on R2R3R4, but not on R5. Interaction with about 20,000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. We conclude that the deletion of R2-R5 from hLf may serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.