Lectin-based analysis of fucosylated glycoproteins of human skim milk during 47 days of lactation

Abstract

Glycoproteins of human milk are multifunctional molecules, and their fucosylated variants are potentially active molecules in immunological events ensuring breastfed infants optimal development and protection against infection diseases. The expression of fucosylated glycotopes may correspond to milk maturation stages. The relative amounts of fucosylated glycotopes of human skim milk glycoproteins over the course of lactation from the 2(nd) day to the 47(th) day were analyzed in colostrums, transitional and mature milk samples of 43 healthy mothers by lectin-blotting using α1-2-, α1-6-, and α1-3-fucose specific biotinylated Ulex europaeus (UEA), Lens culinaris (LCA), and Lotus tetragonolobus (LTA) lectins, respectively. The reactivities of UEA and LCA with the milk glycoproteins showed the highest expression of α1-2- and α1-6-fucosylated glycotopes on colostrum glycoproteins. The level of UEA-reactive glycoproteins from the beginning of lactation to the 14(th) day was high and relatively stable in contrast to LCA-reactive glycoproteins, the level of which significantly decreased from 2-3 to 7-8 days then remained almost unchanged until the 12(th)-14(th) days. Next, during the progression of lactation the reactivities with both lectins declined significantly. Eighty percent of α1-2- and/or α1-6-fucosylated glycoproteins showed a high negative correlation with milk maturation. In contrast, most of the analyzed milk glycoproteins were not recognized or weakly recognized by LTA and remained at a low unchanged level over lactation. Only a 30-kDa milk glycoprotein was evidently LTA-reactive, showing a negative correlation with milk maturation. The gradual decline of high expression of α1-2- and α1-6-, but not α1-3-, fucoses on human milk glycoproteins of healthy mothers over lactation was associated with milk maturation.

Keywords: Fucose; Fucosylated glycotopes; Human milk glycoproteins; Lactation; Lectins.

Fig. 1

Representative patterns of human skim milk proteins over lactation. A human skim milk sample containing 30 μg of proteins was loaded per lane of SDS-PAGE. After separation the proteins were transferred onto nitrocellulose, and the membrane was stained with a colloidal silver [44]. For experimental details see Material and methods

Fig. 2

Representative patterns of human milk glycoprotein reactivities with fucose-specific lectins over lactation. For analysis of fucosylation of milk glycoproteins, the samples were collected only from mothers who have secretor status. The pooled milk sample containing 30 μg of proteins was loaded per lane of SDS-PAGE. After separation, the glycoproteins were transferred onto nitrocellulose, and the membrane was subjected to the reaction with (a) Ulex europaeus, (b) Lens culinaris, and (c) Lotus tetragonolobus biotinylated lectin (Vector Laboratories Inc. Burlingame. USA), respectively. The formed lectin-glycoprotein complex was detected by the reaction with phosphatase-labeled ExtrAvidin (Sigma. St. Louis. MO. USA). All blots were done in duplicate. For experimental details see Material and methods

Fig. 3

General pattern of fucose-specific lectin- reactivity of human milk glycoproteins over lactation. The mean value of the relative amounts of the lectin-reactive milk glycoproteins was calculated for those showing (a) a significant correlation and (b) no significant correlation with milk maturation. For the calculation, the following SDS-PAGE bands (Table 3) were selected in: (a): reactive with UEA - 220–225 kDa, 160–175 kDa, 35–50 kDa;, LCA - 260–310 kDa, 160–175 kDa, 133 kDa, 78–86 kDa, 70–75 kDa, 35–50 kDa, 30 kDa; and LTA - 30 kDa; and in (b): reactive with UEA - 260–310 kDa, 133 kDa, 78–86 kDa, 70–75 kDa, 63–67 kDa, 30 kDa; LCA - 220–225 kDa, 63–67 kDa;, and LTA - 260–310 kDa, 220–225 kDa, 160–175 kDa, 133 kDa, 78–86 kDa, 70–75 kDa, 63–67 kDa, 35–50 kDa. For other details see under Table 3