The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis.

Lactoferrin (LF) is an iron-binding glycoprotein of the innate host defence system. To elucidate the role of N-linked glycosylation in protection of LF against proteolysis, we compared the tryptic susceptibility of human LF (hLF) variants from human milk, expressed in human 293(S) cells or in the milk of transgenic mice and cows. The analysis revealed that recombinant hLF (rhLF) with mutations Ile130-->Thr and Gly404-->Cys was about twofold more susceptible than glycosylated and unglycosylated variants with the naturally occurring Ile130 and Gly404. Hence, N-linked glycosylation is not involved in protection of hLF against tryptic proteolysis. Apparently, the previously reported protection by N-linked glycosylation of hLF [van Berkel, P.H.C., Geerts, M.E.J., van Veen, H.A., Kooiman, P.M., Pieper, F., de Boer, H.A. & Nuijens, J.H. (1995) Biochem. J. 312, 107-114] is restricted to rhLF containing the Thr130 and Cys404. Comparison of the tryptic proteolysis of hLF and bovine LF (bLF) revealed that hLF is about 100-fold more resistant than bLF. Glycosylation variants A and B of bLF differed by about 10-fold in susceptibility to trypsin. This difference is due to glycosylation at Asn281 in bLF-A. Hence, glycosylation at Asn281 protects bLF against cleavage by trypsin at Lys282.

Analytical cation-exchange chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand. An analytical method, employing cation-exchange chromatography on Mono S, was developed to assess the N-terminal integrity of hLF preparations. The method, which separates N-terminally intact hLF from hLF species lacking two (Gly(1)-Arg(2)) or three (Gly(1)-Arg(2)-Arg(3)) residues, showed that 5-58% of total hLF in commercially obtained preparations was N-terminally degraded. The elution profile of hLF on Mono S unequivocally differed from lactoferrins from other species as well as homologous and other whey proteins. Analysis of fresh human whey samples revealed two variants of N-terminally intact hLF, but not limitedly proteolyzed hLF. Mono S chromatography of 2 out of 26 individual human whey samples showed a rare polymorphic hLF variant with three N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Ser(5)-) instead of the usual variant with four N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)-Ser(6)-). In conclusion, Mono S cation-exchange chromatography appeared a robust method to assess the identity, purity, N-terminal integrity, and the presence of polymorphic and intact hLF variants.

Characterization of monoclonal antibodies against human lactoferrin.

The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties.