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Structural determination of two N-linked glycans isolated from recombinant human lactoferrin expressed in BHK cells
D Legrand1, V Salmon, B Coddeville
1Laboratoire de Chimie Biologique, UMR CNRS n.111, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Insights
Recombinant human lactoferrin (rHLF) was analyzed for its glycan structures. The rHLF contains N-acetyllactosaminic biantennary glycans, with specific sialylation and fucosylation patterns relevant for biological studies.
Area of Science:
- Biochemistry
- Glycobiology
- Protein Chemistry
Background:
- Human lactoferrin is a key iron-binding glycoprotein with antimicrobial and immunomodulatory functions.
- Understanding the glycosylation of recombinant lactoferrin is crucial for its therapeutic applications.
- Previous studies have characterized native lactoferrin glycosylation from various sources.
Purpose of the Study:
- To characterize the N-linked glycan structures of recombinant human lactoferrin (rHLF) expressed in BHK cells.
- To compare the glycan profile of rHLF with native lactoferrin.
- To provide structural insights for the interpretation of rHLF biological activity.
Main Methods:
- Isolation of full-length cDNA for human lactoferrin.
- Expression of recombinant protein in BHK cells.
- Hydrazinolysis of N-linked glycans.
- Analysis of released glycans using 400-MHz 1H-NMR spectroscopy.
Main Results:
- Identified N-acetyllactosaminic biantennary glycans.
- Determined sialylation patterns: 80% alpha-2,3-disialylated and 20% alpha-2,3-monosialylated.
- Found 70% of glycans were alpha-1,6-fucosylated at the asparagine-linked GlcNAc residue.
- rHLF glycan structure is similar but has specific features compared to native lactoferrins.
Conclusions:
- The recombinant human lactoferrin produced in BHK cells possesses complex N-linked glycans.
- The specific sialylation and fucosylation patterns of rHLF are characterized.
- These structural features should be considered in the interpretation of in vivo and in vitro biological studies of rHLF.
Abstract:
A full-length cDNA coding for human lactoferrin was isolated from a mammary gland library and the recombinant protein was expressed in BHK cells as described by Stowell K. M. et al. [1991, Biochem. J. 276, 349-355]. Two N-linked glycans from purified recombinant lactoferrin were released by hydrazinolysis and analyzed by 400-MHz 1H-NMR spectroscopy. The identified structures corresponded to N-acetyllactosaminic biantennary glycans and were alpha-2,3-disialylated forms (80%) or alpha-2,3-monosialylated (20%) forms. Moreover, 70% of total glycans were alpha-1,6-fucosylated at the GlcNAc residue linked to asparagine. In regard to its glycan moiety, the recombinant glycoprotein is close to native lactoferrins from milk or leucocytes but shows specific structural features which should be taken into account prior to in vivo and in vitro biological studies.