Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants

摘要

pHuman 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 (Asnsup138/sup, Asnsup479/sup and Asnsup624/sup). 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 Asnsup138/sup and Asnsup479/sup) in approx. 85% of all hLF molecules. Glycosylation at a single site (Asnsup479/sup) or at all three sites occurs in approx. 5% and 9% of hLF respectively. The extent of glycosylation at Asnsup624/sup was increased to approx. 29% and 40% of Asnsup479/sup and Asnsup138/479/sup mutant molecules respectively, which indicates that glycosylation at Asnsup624/sup in natural hLF might be limited by glycosylation at Asnsup479/sup. The presence in supernatant of unglycosylated hLF (approx. 60% of the total) after mutations of Asnsup138/sup and Asnsup479/sup 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 (Asnsup138/479/624/sup mutant) but not after mutation at both Asnsup138/sup and Asnsup479/sup suggests that an altered conformation rather than the lack of glycosylation has rendered the Asnsup138/479/624/sup mutant susceptible to intra- and/or extra-cellular degradation./p