Studying Lactoferrin N-Glycosylation

Abstract

Lactoferrin is a multifunctional glycoprotein found in the milk of most mammals. In addition to its well-known role of binding iron, lactoferrin carries many important biological functions, including the promotion of cell proliferation and differentiation, and as an anti-bacterial, anti-viral, and anti-parasitic protein. These functions differ among lactoferrin homologs in mammals. Although considerable attention has been given to the many functions of lactoferrin, its primary nutritional contribution is presumed to be related to its iron-binding characteristics, whereas the role of glycosylation has been neglected. Given the critical role of glycan binding in many biological processes, the glycan moieties in lactoferrin are likely to contribute significantly to the biological roles of lactoferrin. Despite the high amino acid sequence homology in different lactoferrins (up to 99%), each exhibits a unique glycosylation pattern that may be responsible for heterogeneity of the biological properties of lactoferrins. An important task for the production of biotherapeutics and medical foods containing bioactive glycoproteins is the assessment of the contributions of individual glycans to the observed bioactivities. This review examines how the study of lactoferrin glycosylation patterns can increase our understanding of lactoferrin functionality.

Keywords: N-glycans; bioinfomatic libraries; deglycosylating enzymes; lactoferrin; mass spectrophotometry; structure-activity studies.

Figure 1

Biological roles of lactoferrin (adapted from Brock) [42].

Figure 2

Potential and actual N-glycosylation sites of bovine and human lactoferrin (adapted from Van Veen et al. [30]).

Figure 3

Three types of N-glycans (Adapted from Essentials of Glycobiology, 2nd edition, 2009) [56].

Figure 4

Examples of unique glycans for human, bovine, and goat lactoferrins. Green circles, yellow circles, blue squares, red triangles, purple diamonds, and gray diamonds represent mannose, galactose, N-acetlyglucosamine, Fucose, sialic acid, and N-glycolylneuraminic acid residues, respectively.

Figure 5

Endoglycosidases and their specificity on N-glycans.

Figure 6

Components of a mass spectrometer.

Figure 7

MS/MS spectra of a neutral lactoferrin N-glycan. Deconvoluted MS/MS spectrum of the neutral N-glycan 5Hex-2HexNAc. Green circles and blue squares represent mannose and HexNAc, respectively.