Heterogeneity in utilization of N-glycosylation sites Asn(624) and Asn(138) in human lactoferrin: A study with glycosylation-site mutants

Human lactoferrin (hLF) is a glycoprotein involved in the host defence against infection and excessive inflammation. Our objective was to determine to what extent each of the three sequons for N-linked glycosylation in hLF is actually used. Human kidney-derived 293(S) cell lines expressing recombinant hLF (rhLF) or glycosylation-site mutants were produced. The mutations involved replacement of asparagine residues with glutamine at one or more sequons for N-glycosylation (Asn(138), Asn(479) and Asn(624)). Comparative SDS/PAGE analyses of rhLF, mutated rhLF and human-milk-derived (natural) hLF led us to propose that glycosylation of hLF occurs at two sites (at Asn(138) and Asn(479)) in approx. 85% of all hLF molecules. Glycosylation at a single site (Asn(479)) or at all three sites occurs in approx. 5% and 9% of hLF respectively. The extent of glycosylation at Asn(624) was increased to approx. 29% and 40% of Asn(479) and Asn(138/479) mutant molecules respectively, which indicates that glycosylation at Asn(624) in natural hLF might be limited by glycosylation at Asn(479). The presence in supernatant of unglycosylated hLF (approx. 60% of the total) after mutations of Asn(138) and Asn(479) suggests that glycosylation of hLF is not an absolute requirement for its secretion. The pronounced degradation of unglycosylated hLF in supernatant after mutation at all three glycosylation sites (Asn(138/479/624) mutant) but not after mutation at both Asn(138) and Asn(479) suggests that an altered conformation rather than the lack of glycosylation has rendered the Asn(138/479/624) mutant susceptible to intra- and/or extra-cellular degradation.