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

Abstract

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 (Asn138, Asn479 and Asn624). 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 Asn138 and Asn479) in approx. 85% of all hLF molecules. Glycosylation at a single site (Asn479) or at all three sites occurs in approx, 5% and 9% of hLF respectively. The extent of glycosylation at Asn624 was increased to approx. 29% and 40% of Asn479 and Asn138/479 mutant molecules respectively, which indicates that glycosylation at Asn624 in natural hLF might be limited by glycosylation at Asn479. The presence in supernatant of unglycosylated hLF (approx. 60% of the total) after mutations of Asn138 and Asn479 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 (Asn138/479/624 mutant) but not after mutation at both Asn138 and Asn479 suggests that an altered conformation rather than the lack of glycosylation has rendered the Asn138/479/624 mutant susceptible to intra- and/or extra-cellular degradation.

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

“…Human lactoferrin (hLF) is a glycoprotein that has mostly core-fucosylated glycans at the two N-glycosylation sites (48,49); this was confirmed by MS analysis (Fig. 5A and supplemental Table S2).…”

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

“…It has been demonstrated that Asn138 and Asn479 are preferentially Nglycosylated in LF produced in human kidney-derived 293(S) cell lines, indicating site-specific heterogeneity [35]. However, all three N-linked sites of rhLF from our glycoengineered P. pastoris are predominantly occupied as characterized by peptide mapping and LC-MS (Fig.…”

“…Glycosylation at a single site (N479) or at all three sites (N138, N479, and N624) occurs in only approximately 5% and 9% of hLF, respectively [35]. In order to determine Nglycosylation occupancy of each site, rhLF was digested with trypsin.…”

“…The key to understanding the molecular basis of LF various activities is thought to reside in part according to patterns of glycosylation [37]. There are three possible N-linked glycosylation sites in hLF, one at Asn138, a second site at Asn479, and a third site at Asn624; differential utilization of these sites results in distinct glycosylation variants [30,35]. Human LF glycans are the N-acetyllactosaminic type, α1-3-fucosylated on the Nacetylglucosamine residue linked to the peptide chain.…”

Transgenic Cows That Produce Recombinant Human Lactoferrin in Milk Are Not Protected from Experimental Escherichia coli Intramammary Infection

“…Quantitative recombinant human and natural bovine lactoferrin analyses were conducted by enzyme-linked immunosorbent assays (ELISAs). hLf was measured by an rhLf-specific ELISA according to the procedure recommended by Pharmingen, and anti-hLf was adsorbed with Sepharose to remove cross-reacting antibodies (54). bLf levels were measured using a bovine lactoferrin ELISA quantitation kit (Bethyl Laboratories, Inc.).…”