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PMID: 7492299 Published · ppublish English Journal Article

Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis.

The Biochemical journal ·Vol. 312 ( Pt 1) ·1995-11-15 ·Pages 107-14

van Berkel PH, Geerts ME, van Veen HA, Kooiman PM, Pieper FR, de Boer HA, Nuijens JH

Abstract

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 reactivity towards various antibodies of rhLF that had been expressed in the absence or presence of tunicamycin (which blocks N-linked glycosylation) did not differ from that of natural (human milk-derived) hLF. Cation-exchange chromatography and N-terminal protein sequencing showed identical cationic properties and an intact N-terminal sequence for rhLF and natural hLF. SDS/PAGE of rhLF expressed in the presence of tunicamycin revealed a protein with the same M(r) as that of enzymically deglycosylated natural hLF. Both glycosylated and unglycosylated rhLF appeared to be completely saturated with iron. The affinity of natural hLF, glycosylated and non-glycosylated rhLF for both human lysozyme (Kd 4.5 x 10(-8) M) and bacterial lipopolysaccharide did not differ. SDS/PAGE of hLF species subjected to trypsin indicated that unglycosylated rhLF was much more susceptible to degradation. Furthermore, this analysis suggests that N-glycosylation heterogeneity in natural hLF and rhLF resides in the C-lobe. Thus our results provide no argument for differential antibacterial and/or anti-inflammatory activity of natural and (glycosylated) rhLF and suggest that a major function of glycosylation in hLF is to protect it against proteolysis.

MeSH Terms
Amino Acid Sequence Anti-Inflammatory Agents/chemistry,metabolism Cell Line Chromatography, Agarose Glycosylation Humans Iron/metabolism Kidney/metabolism Lactoferrin/chemistry,genetics,metabolism Lipopolysaccharides/metabolism Molecular Sequence Data Muramidase/metabolism Protein Binding Radioimmunoassay Recombinant Proteins/chemistry,genetics,metabolism Trypsin/metabolism Tunicamycin/pharmacology
Chemicals
Anti-Inflammatory Agents Lipopolysaccharides Recombinant Proteins Tunicamycin Iron Muramidase Lactoferrin Trypsin
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
van Berkel P H
Leiden Institute of Chemistry, Medical Biotechnology Department, Gorlaeus Laboratories, Leiden University, The Netherlands.
Geerts M E
van Veen H A
Kooiman P M
Pieper F R
de Boer H A
Nuijens J H
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj3120107
Published
1995-11-15
Pages
107-14
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1136233
Subset
IM
External Links
PubMed source DOI: 10.1042/bj3120107 PMC Full Text View journal details
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