Glycoconjugate stability in human milk: glycosidase activities and sugar release.

Many human milk glycoconjugates (glycolipids, glycoproteins, mucins, glycosaminoglycans) and oligosaccharides are biologically active, and their activity depends on the precise structure of the glycan. The sugars on the terminus of the glycan are vulnerable to cleavage by glycosidases. Because glycoconjugates incubate together with endogenous glycosidases in the breast between feedings, and in expressed milk during storage, the presence and activity of glycosidases in human milk was investigated. alpha-L-Fucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, N-acetyl-beta-hexosaminidase, beta-D-glucuronidase, and neuraminidase were measured in milk samples from 4 donors by use of synthetic fluorogenic glycosides; fucosidase and hexosaminidase displayed the highest activity. The catabolic activity of the major glycosidases was confirmed by measuring the corresponding free sugars in milk. Free fucose, N-acetylneuraminic acid, and N-acetylhexosamines were measured and their identities were confirmed by high-performance liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry. Incubation of samples for 16 hr at 37 degrees C and 20 degrees C, but not at 4 degrees C, resulted in time-dependent increases in the amount of free fucose, N-acetylneuraminic acid, and N-acetylhexosamines, consistent with their enzymatic release by the endogenous glycosidases. Stored frozen milk had the same levels of these sugars as did samples analyzed immediately after collection, indicating that glycosidases are inactive in the frozen milk. Samples analyzed immediately after collection contained only small amounts of free sugars, suggesting that glycoconjugate degradation during the typical residence time of milk in the breast is modest.

alpha1,3 Fucosyltransferase, alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase, and Le(x) glycoconjugates in developing rat brain.

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Le(x)) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15-P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10-P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an alpha1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5-10) with different types of acceptors, and the differential expression of Le(x) containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, alpha-L-fucosidase, beta-D-galactosidase and alpha-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases.

Compartmentalization of fucosyltransferase and alpha-L-fucosidase in human milk.

Several pathogenic agents of pediatric gastroenteritis are inhibited by fucosylated oligosaccharides of human milk. Biosynthesis and degradation of fucosyloligosaccharides is controlled by fucosyltransferase and alpha-L-fucosidase. The activity of these enzymes varies reciprocally over the course of lactation. We hypothesized that differences in the specific organization of these enzymes in compartments of human milk might contribute to such differences in activity. Therefore, the distribution of these enzymes in various compartments of human milk was investigated. After ultracentrifugation at 120,000g for 2h, the fucosyltransferase activity distributes evenly between the supernatant and the membranous pellet. Ultracentrifugation at 180,000g for 17 h further fractionated the milk into a clear supernatant, a fluff layer from the supernatant, and a pellet. The fluff was visualized by electron microscopy. The distribution of fucosyltransferase activity in colostrum was compared with that of mature milk from the same donor. In mature milk from Day 30 of lactation, most fucosyltransferase activity was in the membranous fluff fraction, while in colostrum from Day 5 of lactation, most of the fucosyltransferase activity was in the supernatant. In contrast to fucosyltransferase, fucosidase activity was found only in the soluble milk fraction; upon prolonged ultracentrifugation, most of this was membrane associated. The nature of human milk fucosidase was studied. This enzyme is glycosylated and exhibits several characteristics common to other fucosidases. Under the conditions found in human milk, it exhibits almost full activity. The variation in compartmentalization of fucosyltransferase activity during lactation may reflect variations in metabolism of the fucosyloligosaccharides of human milk that protect against disease.

[Alpha-L-fucosidase activity in human blood]. / Al'fa - L-Fukozidaznaia aktivnost' cheloveka.

The alpha-L-fucosidase activity was studied in blood plasma and cells. In leucocytes and blood plasma of healthy persons the fucosidase activity varied considerably. The fucosidase activity in blood was not altered under pathological conditions other than fucosidosis. The fucosidase activity did not depend on sex and age of the persons studied. No correlation was observed between the values of the fucosidase activity. Two forms of fucosidase (alpha-L-fucosidase I and alpha-L-fucosidase II) were found in human leucocytes and blood plasma. Isoelectric focusing experiments indicates multiple forms alpha-L-fucosidase from human serum. The possibility is discussed of the use of human blood plasma and cells not only for estimation of the total fucosidase activity but also for studies of different forms of the enzyme.