GLYCOSYLATED AND UNGLYCOSYLATED HUMAN LACTOFERRINS BOTH BIND IRON ANDSHOW IDENTICAL AFFINITIES TOWARDS HUMAN LYSOZYME AND BACTERIAL LIPOPOLYSACCHARIDE, BUT DIFFER IN THEIR SUSCEPTIBILITIES TOWARDS TRYPTIC PROTEOLYSIS

We studied the role of N-glycosylation of human lactoferrin (hLF) with respect to properties that are relevant to its antibacterial and anti -inflammatory activities. A human kidney-derived 293(S) cell line that constitutively expresses recombinant hLF (rhLF) was produced. The rea ctivity towards various antibodies of rhLF that had been expressed in the absence or presence of tunicamycin (which blocks N-linked glycosyl ation) did not differ from that of natural (human milk-derived) hLF. C ation-exchange chromatography and N-terminal protein sequencing showed identical cationic properties and an intact N-terminal sequence for r hLF and natural hLF. SDS/PAGE of rhLF expressed in the presence of tun icamycin revealed a protein with the same 2M, as that of enzymically d eglycosylated natural hLF. Both glycosylated and unglycosylated rhLF a ppeared to be completely saturated with iron. The affinity of natural hLF, glycosylated and non-glycosylated rhLF for both human lysozyme (K -d 4.5 x 10(-8) M) and bacterial lipopolysaccharide did not differ. SD S/PAGE of hLF species subjected to trypsin indicated that unglycosylat ed rhLF was much more susceptible to degradation. Furthermore, this an alysis suggests that N-glycosylation heterogeneity in natural hLF and rhLF resides in the C-lobe. Thus our results provide no argument for d ifferential antibacterial and/or anti-inflammatory activity of natural and (glycosylated) rhLF and suggest that a major function of glycosyl ation in hLF is to protect it against proteolysis.