Lactoferrin (Lf) is an iron binding glycoprotein of the transferrin family that is expressed in most biological fluids and is a major component of mammals' innate immune system. Its protective effect ranges from direct antimicrobial activities against a large panel of microorganisms, including bacteria, viruses, fungi, and parasites, to anti-inflammatory and anticancer activities. This plethora of activities is made possible by mechanisms of action implementing not only the capacity of Lf to bind iron but also interactions of Lf with molecular and cellular components of both host and pathogens. This chapter summarizes our current understanding of the Lf structure-function relationships that explain the roles of Lf in host defense.
The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.
Recent methodological developments in metabolic oligosaccharide engineering (MOE) pave the way for tremendous advances in glycobiology. Herein, we propose a Sequential Bioorthogonal Dual Strategy (SBDS) combining the use of two unprotected alkyne-tagged monosaccharide reporters (ManNAl and SiaNAl) with the bioligation of fluorescent probes by copper-catalysed azide-alkyne cycloaddition (CuAAC). With SBDS, we are able to shed light on trafficking and cellular uptake mechanisms of sialic acid. Using their corresponding analogues, we visualized that SiaNAl enters via endocytosis, whereas its biosynthetic intermediate ManNAl uptake is mediated by a yet unknown but specific plasma membrane transporter. Sialin, a lysosomal protein, is shown to be crucial for the export of exogenous sialic acid from lysosomes to the cytosol. Metabolic labeling with alkyne-tagged derivatives of N-acetylneuraminic acid (Neu5Ac) or N-acetylmannosamine (ManNAc) could thus be used to follow endocytosis in physiological vs. pathological conditions.
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