Influence of labeling on the glycan affinities and specificities of glycan-binding proteins. A case study involving a C-terminal fragment of human galectin-3

Glycan interactions with glycan-binding proteins (GBPs) play essential roles in a wide variety of cellular processes. Currently, the glycan specificities of GBPs are most often inferred from binding data generated using glycan arrays, wherein the GBP is incubated with oligosaccharides immobilized on a glass surface. Detection of glycan–GBP binding is typically fluorescence-based, involving the labeling of the GBP with a fluorophore or with biotin, which binds to fluorophore-labeled streptavidin, or using a fluorophore-labeled antibody that recognizes the GBP. While it is known that covalent labeling of a GBP may influence its binding properties, these effects have not been well studied and are usually overlooked when analyzing glycan array data. In the present study, electrospray ionization mass spectrometry (ESI-MS) was used to quantitatively evaluate the impact of GBP labeling on oligosaccharide affinities and specificities. The influence of three common labeling approaches, biotinylation, labeling with a fluorescent dye and introducing an iodination reagent, on the affinities of a series of human milk and blood group oligosaccharides for a C-terminal fragment of human galectin-3 was evaluated. In all cases labeling resulted in a measurable decrease in oligosaccharide affinity, by as much as 90%, and the magnitude of the change was sensitive to the nature of the ligand. These findings demonstrate that GBP labeling may affect both the absolute and relative affinities and, thereby, obscure the true glycan binding properties. These results also serve to illustrate the utility of the direct ESI-MS assay for quantitatively evaluating the effects of protein labeling on ligand binding.

Chemerin and Dermcidin in Human Milk and Their Alteration in Gestational Diabetes

Background: Chemerin and dermcidin, which have antimicrobial properties, are molecules that are also related to insulin resistance and inflammation. Research aims: The aims were to determine the amounts of chemerin and dermcidin in the milk and blood of mothers with gestational diabetes, and to compare the amounts of chemerin and dermcidin in the milk and blood of mothers with and without diabetes. Methods: This was a two-group nonrandomized longitudinal study with a convenience sampling of mothers without gestational diabetes ( n = 27) and mothers with gestational diabetes ( n = 26). Human milk and blood samples were obtained from these mothers during colostrum, transitional, and mature milk periods. The amount of chemerin and dermcidin in these samples was measured by enzyme-linked immunosorbent assay. Results: The presence of chemerin and dermcidin was first detected in human milk. The amounts of chemerin and dermcidin in the blood of all the mothers were greater in the colostrum period and lowest in the mature period. The amount of chemerin and dermcidin in the milk of all the mothers was greater than that in the blood. The amounts of chemerin and dermcidin were significantly increased in both blood and human milk within the gestational age samples. Conclusions: Chemerin and dermcidin may contribute to the protection of infants from infections during infancy. Increased amounts of these molecules found within the gestational diabetes group may also prevent adverse maternal and fetal outcomes.