Free oligosaccharides and glycosylation of bile salt-stimulated lipase in human milk

• Bile salt-stimulated lipase (BSSL) is a glycosylated protein present in milk at a concentration of 100-200 mg/L. It is an enzyme important for fat digestion in the newborn infant. The protein backbone contains one possible site for N-glyeosylation and several sites for 0-glyeosylation. The glycosylation of BSSL may be important for protection against proteolytic degradation and/or secretion of BSSL. The oligosaeeharides bound to BSSL may also, together with other protein-bound oligosacchatides and free oligosaecharides in milk, play an important role in the defense against pathogenic microorganisms. Human milk contains approximately 5-20 g/L of free milk oligosacchatides, and more than 100 different structures have been identified. There are individual differences in the content of milk oligosaeeharides depending on Lewis and seeretor status.Milk samples were collected from healthy donors at different times during lactation. BSSL was purified from the milk of five donors. Structural characterization of BSSL glyeosylation was performed by high-performance anionexchange chromatography (HP AEC), Bio-Gel P-4 chromatography, lectin affinity chromatography, gas chromatography coupled to mass-spectrometry (MS) and mattix assisted laser desorption-ionization time-of-flight MS. Certain carbohydrate epitopes were detected by monoclonal antibodies and lectins. Some of the methods above were used in combination with ptior derivatization, desialylation or digestion with different exo- and endoglycosidases. Thirteen major free oligosaccharides were quantified in milk from five individuals. Free milk oligosacchatides were purified by P4-Gel chromatography and analyzed by HPAEC.HP AEC coupled to pulsed amperometric detection is extensively used for analysis and quantification of oligosaccharides. Separation is achieved using highly alkaline conditions that lead to ionization of some of the hydroxyl groups, which can then interact with the anion-exchange matrix. The effect of colunm temperature was examined in a range of 13 to 40 oC. A large variation in retention times was found depending on small differences in colunm temperature. Moreover, individual oligosaccharides did not show the same temperature dependence. By use of different column temperatures, HP AEC could be optimized for analysis of milk oligosaccharides.BSSL was found to contain approximately one N-linked and nine O-linked oligosaccharides. The 0-glycans were stmcturally heterogeneous and contained· fucose and/or sialic acid. Each 0-glycan contained an average of eight monosaccharide units. The major N-linked oligosaccharides on human BSSL were mono-sialylated biantennary complex type structures with or without one, two or three fucose residues.Recombinant human BSSL expressed in CHO and C-127 cells were analyzed and found to be differently glycosylated than native BSSL. In contrast to native BSSL, recombinant BSSL did not contain fucose. On BSSL expressed in C-127 cells, the O-glycans were shorter and more extensively sialylated than O-glycans on native BSSL. The majority of N-linked oligosaccharides on recombinant BSSL had the same core structure (biantennary complex type) as native BSSL.Glycosylation of BSSL changed during lactation. BSSL had a higher carbohydrate and sialic acid content in the first lactation month. There was also a shift from preferentially α2-6 to α2-3 linked sialic acid on the protein-bound oligosaccharides during lactation. This shift was also found for free sialylated milk oligosaccharides, and suggests a change in the activity of certain sialyltransferases during lactation.A gradual increase in the expression of the fucosylated carbohydrate epitope Lewis x (Galß1-4[Fucal-3]GlcNAc-) was found on BSSL during the whole lactation period. This was reflected in a higher relative amount of fucosylated N-linked oligosaccharides present on BSSL later in lactation. A similar increase in fucosylation was indicated by analysis of free milk oligosaccharides. One of the major milk oligosaccharides, 3-fucosyllactose (3-FL), also increased in concentration during lactation. However, lacto-N-fucopentaose (LNFIII), the only free milk oligosaccharide containing the Lewis x epitope, showed a constant concentration. This finding does not exclude the possibility that the same fucosyltransferase is involved in the synthesis of Lewis x on BSSL, 3-FL and LNFIII. The precursor of LNFIII, lacto-N-neotetraose (LNnT) showed a marked decrease during lactation, which may explain the different pattem found for LNFIIl. The increase of 3-FL and Lewis x on BSSL was found for all individuals. The other free oligosaccharides studied decreased during lactation, except for lacto-N-fucopentaose li (LNFII), lacto-di-fucotetraose (LDFT) and 3-sialyllactose (3-SL), which showed constant concentrations.Total fucosyltransferase activity decreased during lactation in milk from both secretors and non-secretors. The specific α1-3 fucosyl transferase activity toward lactose also decreased during lactation, which indicated that fucosyltransferase activity in milk does not reflect the activity in the mammary epithelial cells.In conclusion, there are changes in glycosylation during lactation, which involves both protein-bound and free milk oligosaccharides. The different patterns for individual oligosaccharides indicate both down and up regulation of certain glycosyltransferases in the mammary gland during lactation. The importance of these changes for the infant's adaptation to the environment remains to be elucidated.

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