Changes in milk carbohydrates during lactation in the eastern quoll, Dasyurus viverrinus (Marsupialia)

1. Milk samples were obtained at various stages of lactation from 16 captive eastern quolls. 2. The mean milk carbohydrate concentration was 7.4% (w/v; total hexose) at 8 weeks post partum, decreasing to 5.2% at 17 weeks and to less than 2% at the end of lactation. 3. The predominant monosaccharide constituent of the carbohydrates was galactose, followed by glucosamine, glucose and sialic acid. 4. Thin layer and gel chromatography showed that the milk carbohydrates consisted of lactose and a variety of higher oligosaccharides, some of which appeared to be identical to oligosaccharides of known structure previously isolated from milk of the tammar wallaby, Macropus eugenii. 5. In general, both the quantitative and qualitative changes observed in the milk carbohydrates of the eastern quoll were similar to those documented for macropodids.     

Exploring the structural diversity of mammalian carbohydrates ("glycospace") by statistical databank analysis.

The diversity of three major classes of mammalian carbohydrates, mainly glycolipids and O- and N-linked glycans, deposited in the databank GLYCOSCIENCES.de was subjected to statistical analyses. Size, chain length, and branching complexity were accessed and revealed that the average oligosaccharide is composed of about eight monosaccharide units. About a quarter of all oligosaccharides are strictly linear, and the remainder are branched at least once. Glucosamine, galactose, and mannose are dominating and comprise ~75% of the monosaccharides within mammalian oligosaccharide frameworks. alpha-Linked sialic acid, alpha-linked fucose, and beta-linked galactose decorate the majority of reducing termini. Glucose as the most abundant carbohydrate in mammals plays only a very minor role within these structures. Particular emphasis was placed on analyzing the way the monosaccharide units are linked within the oligomeric framework. Just 11 monosaccharide connections account for >75% of all linkages. Thus, the number of structural combinations found in nature, the part of the occupied mammalian glycospace, is much smaller than expected. As a result, a potential set of building blocks for oligosaccharide assembly is presented. This potential building block set was correlated with the accessible 3299 mammalian carbohydrate structures in the GLYCOSCIENCES.de databank. Only 36 building blocks are required to construct 75% of the 3299 mammalian oligosaccharides.     

Changes in milk protein composition during acute involution at different phases of tammar wallaby (Macropus eugenii) lactation

This study exploited the unusual lactation cycle of the tammar wallaby (Macropus eugenii) to characterise milk composition during acute involution, a time when the mammary gland is subjected to increased risk of infection. In early-lactation, tammar milk contains elevated levels of complex oligosaccharides and low protein and lipid content. Later in lactation, protein and lipid concentrations increase significantly, whereas carbohydrate content is reduced dramatically and changes to monosaccharides. Following initiation of involution at early-lactation, the carbohydrate concentration greatly decreased, while lipid and protein concentrations were elevated, suggesting that complex oligosaccharides are the major osmole in milk at this time. In contrast, involution at late lactation, when carbohydrate concentration was very low, led to an increase in the lipid concentration, but the concentration of protein was not significantly altered. This indicates that protein synthesis during acute involution at late lactation in the tammar may be down-regulated much more rapidly than during early-lactation. Analysis of milk at day 3 after the onset of involution at early-lactation identified a number of potential antimicrobials secreted at high concentrations, including lysozyme, dermcidin, polymeric immunoglobulin receptor and fragments of beta-lactoglobulin. These proteins may protect the mammary gland by minimising the risk of potential infection during involution.     

Changes in glycosylation of human bile-salt-stimulated lipase during lactation

Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption-ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.     

Changes in alpha-lactalbumin, total lactose, UDP-galactose hydrolase and other factors in tammar wallaby (Macropus eugenii) milk during lactation

alpha-Lactalbumin was isolated from milk of M. eugenii and its concentration in milk samples taken at various times during lactation (0-40 weeks post partum) was determined by single radial immunodiffusion using rabbit antiserum to the purified protein. The alpha-lactalbumin concentration remained almost constant throughout lactation even though the concentration of total lactose (free lactose plus lactose contained in oligosaccharides) fell to zero after 34 weeks post partum. This fall in lactose was accompanied by a rise in the free galactose and glucose concentrations and marked increases in UDP-galactose hydrolase, nucleotide pyrophosphatase, alkaline phosphatase and acid beta-galactosidase activities. It is suggested that the in vitro hydrolysis of UDP-galactose was due to nucleotide pyrophosphatase and that this enzyme may also play a role in vivo late in lactation by making UDP-galactose unavailable for the synthesis of lactose. Alternatively, lactose and lactose-containing oligosaccharides might be degraded by the acid beta-galactosidase during or after secretion.     

Milk composition of three free-ranging African elephant (Loxodonta africana africana) cows during mid lactation

Data are presented that indicate the dynamic changes of nutrients in milk from three free ranging African elephant (Loxodonta africana africana) cows during lactation. At the respective collection times of 12, 14 and 18 months of lactation the nutrient content was 47.3, 52.0 and 68.6 g protein; 60.7, 87.4 and 170.8 g fat; 1.6, 2.1 0.5 g lactose and 20.9, 21.5 and 8.6 g oligosaccharides per kg milk. The protein fraction respectively consisted of 18.0, 31.7 and 45.9 g caseins/kg milk and of 29.3, 20.3 and 22.7 g whey proteins/kg milk. Electrophoresis and identification of protein bands showed that polymorphs of one whey protein may be present in elephant's milk similar to polymorphs of alpha-lactalbumin found in cow's milk. From the middle of the lactation time lactose was replaced by oligosaccharides as major carbohydrate, and the major compound of these was identified as isoglobotriose by 1H NMR spectroscopy. The lipid fraction contains a high content, of capric and lauric acids, approximately 70% of the total fatty acids, and low content of myristic, palmitic and oleic acids. During these lactation times the content of short chain fatty acids, capric and caprylic acids increased, while fatty acids lauric acid and longer decreased.     

Characterization of oligosaccharides in milk of a mink, Mustela vison

Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.     

Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as alpha-2,6-linkage and no alpha-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.     

Qualitative and quantitative changes in milk fat during lactation in the tammar wallaby (Macropus eugenii)

There are major quantitative and qualitative changes in the milk lipids during lactation in the tammar wallaby, Macropus eugenii. The crude lipid content of the milk is relatively low during the first 10 weeks of lactation; between 10 and 26 weeks post partum the lipid content increases gradually but after that it increases rapidly. The triglyceride fraction of the lipid at early stages of lactation contains a large amount of palmitic acid and relatively little oleic acid whereas mature milk exhibits little palmitic and much oleic acid. In the early stages of lactation fat represents 15% of the total solids and carbohydrate 55%; around 26-30 weeks post partum the carbohydrate moiety falls sharply to a level less than 2% of the solids while lipids increase to c. 60% of the solids. These changes coincide with increases in milk solids, emergence of the young from the pouch, ingestion of herbage, and fermentation of cellulose in the stomach.     

Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II

The carbohydrate binding specificity of Allomyrina dichotoma lectin II was investigated by analyzing the behavior of various complex type oligosaccharides and human milk oligosaccharides on an A. dichotoma lectin II-agarose column. Basically, the lectin interacts with the Gal beta 1----4GlcNAc group. Substitution of their terminal galactose residues by Neu5Ac alpha 2----6 will enhance their affinity to the lectin. By contraries, substitution at the C-2 or C-3 position of their terminal galactose with other sugars including sialic acid deprives their affinity to the lectin. With this characteristic, the immobilized lectin column can be used to separate complex type oligosaccharides with the Neu5Ac alpha 2----6Gal beta 1----4GlcNAc group from their isomeric oligosaccharides with the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc group, where Neu5Ac is N-acetylneuraminic acid.     

Evidence for the presence of complex carbohydrates in Candida albicans cell wall glycoproteins

In order to test the hypothesis that cell wall glycoproteins of Candida albicans contained non-mannan oligosaccharides, the sugar composition of cell wall extracts and fractions of cell wall extracts was examined by means of fluorophore-assisted carbohydrate electrophoresis (FACE). In addition to the expected mannose, glucose, and N-acetyl-glucosamine, this analysis showed the presence of galactose, N-acetyl-galactosamine, fucose, and sialic acid and two unknown sugars. These sugars are also associated with complex oligosaccharides of mammalian glycoproteins. Presence of fucosylated cell wall components was further demonstrated by lectin-blotting analysis of cell wall extracts. Besides their structural role, complex carbohydrate structures on the surface of C. albicans may represent additional motifs through which interactions of this fungus with host cells and tissues could be established.     

Screening for complex carbohydrates inhibiting hemagglutinations by CFA/I- and CFA/II-expressing enterotoxigenic Escherichia coli strains

Abstract Numerous structural families of naturally occurring glycopeptides and oligosaccharides have been evaluated as potential inhibitors of hemagglutinations mediated by CFA/I- and CFA/II-positive enterotoxigenic Escherichia coli strains. Among the preparations tested were glycopeptides with short O-linked (mucin-type) chains, various mixtures containing N-linked glycans (either oligomannoside-, hybrid- or complex-type), three fractions of human milk oligosaccharides, and glycopeptides derived from either pooled new-born meconiums or pooled human red blood cell membranes. In almost all cases, the same inhibitory preparations were active toward all E. coli strains. This emphasizes the close analogy between the carbohydrate specificities of the colonization factors concerned. Such inhibitors always contained lactosamine units in their oligosaccharide backbones, but this structural requirement alone was not sufficient for activity. The glycopeptide mixture derived from human erythrocyte membranes (known to contain blood group-related carbohydrate antigens carried by a lactosaminoglycan backbone) behaved as a potent hemagglutination inhibitor, especially towards CFA/II-expressing strains. This last result clearly indicates the structural family in which complex carbohydrates should be selected to establish precisely the specificity of these CFA/II adhesins.     

Structure--function studies of oligosaccharides of recombinant human thyrotrophin by sequential deglycosylation and resialylation

Recombinant human thyrotrophin (rhTSH) contains oligosaccharidesterminating in -galactose-sialic acid, and had lower metabolicclearance and higher in vivo bioactivity compared to pituitaryhTSH, which has oligosaccharides terminating predominantly in-N-acetylgalactosamine-sulphate. Previous studies using completeremoval of the oligosaccharide chains showed an important rolefor the carbohydrate in the biological activity of the hormone.In the present study, we have determined the contribution ofthe individual monosaccharides to hormonal activity by sequentialdeglycosylation of rhTSH using exoglycosidases. We have alsoinvestigated the effect of resialylation of desialylated rhTSHusing sialyltransferases. Sequential removal of sialic acid,galactose or N-acetylglucosamine resulted in a > 10-foldincrease in the in vitro bioactivity of rhTSH. The metabolicclearance of the derivatives was faster than that of intacthormone, but agalacto-rhTSH was cleared slower than asialo-rhTSH.However, the in vivo bioactivity decreased progressively witheach monosaccharide removal. The increased cyclic AMP-stimulatingactivity, increased metabolic clearance and the decreased invivo biologic activity were all reversed by resialylation ofthe terminal galactose residues. These results indicate thatthe in vitro, as well as the in vivo, bioactivities of rhTSHare modulated by terminal sialylation. The effects of sequentialdeglycosylation on the in vitro activity of rhTSH are differentfrom those reported earlier for human chorionic gonadotrophin.Thus, modification of the oligosaccharides by glycosidases andglycosyltransferases can be used as a powerful tool to delineatethe function of carbohydrate in glycoproteins and to engineermore potent hormone analogues with a longer half-life and/orhigher bioactivity. deglycosylation exoglycosidases oligosaccharides recombinant thyrotrophin sialyltransferases     

Transcriptome profiling of bovine milk oligosaccharide metabolism genes using RNA-sequencing

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing. A list was created of 121 glycosylation-related genes involved in oligosaccharide metabolism pathways in bovine by analyzing the oligosaccharide profiles and performing an extensive literature search. No significant differences were observed in either oligosaccharide profiles or expressions of glycosylation-related genes between Holstein and Jersey cows. The highest concentrations of free oligosaccharides were observed in the colostrum samples and a sharp decrease was observed in the concentration of free oligosaccharides on day 15, followed by progressive decrease on days 90 and 250. Ninety-two glycosylation-related genes were expressed in milk somatic cells. Most of these genes exhibited higher expression in day 250 samples indicating increases in net glycosylation-related metabolism in spite of decreases in free milk oligosaccharides in late lactation milk. Even though fucosylated free oligosaccharides were not identified, gene expression indicated the likely presence of fucosylated oligosaccharides in bovine milk. Fucosidase genes were expressed in milk and a possible explanation for not detecting fucosylated free oligosaccharides is the degradation of large fucosylated free oligosaccharides by the fucosidases. Detailed characterization of enzymes encoded by the 92 glycosylation-related genes identified in this study will provide the basic knowledge for metabolic network analysis of oligosaccharides in mammalian milk. These candidate genes will guide the design of a targeted breeding strategy to optimize the content of beneficial oligosaccharides in bovine milk.     

Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme

The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.     

Carbohydrates of the milk of the platypus

Twelve samples of milk of the platypus, Ornithorhynchus anatinus, had a mean content of 3 X 3% hexose. Of this, almost half was L-fucose. Of the total monosaccharides present in acid hydrolysates of the water-soluble carbohydrates, L-fucose constituted 33%, D-galactose 29%, glucosamine 20%, D-glucose 11% and sialic acid 7%. Free lactose was found in only trace amounts. In all samples, the major oligosaccharide was difucosyllactose, which represented 39-52% of the total hexose. Five higher neutral oligosaccharides (from penta- to nonasaccharides) were isolated and their monosaccharide compositions determined. Each contained one or more residues of fucose, glucosamine and galactose and one residue of glucose. The presence in the milk of 4-O-acetyl-N-acetylneuraminlactose was detected by thin-layer chromatography. All milk samples examined contained protein material (probably glycoprotein), which was not precipitated by chloroform-methanol extraction. No evidence was obtained for quantitative or qualitative changes in carbohydrates during the course of the lactation season except for a small decline in total hexose towards the end of the season.     

Milk composition of free-ranging springbok (Antidorcas marsupialis)

Milk was obtained from three free-ranging springbok ewes of the Karoo, South Africa. The nutrient content was 74.4+/-13.8 g protein; 145.2+/-4.5 g fat; and 42.3+/-16.4 g lactose/kg milk. Small amounts of glucose, galactose and fucose were noted, and 0.3+/-0.4 g oligosaccharides. The protein fraction respectively consisted of 60.0+/-13.7 g caseins/kg milk and of 14.1+/-4.5 g whey proteins/kg milk. The lactation stage of the springbok ewes was not known, but variation in milk composition among individuals indicates that they were at different stages. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in caprine milk. The lipid fraction contains 604.0+/-26.5 g saturated fatty acids/kg milk fat, and 278.2+/-20.5 and 45.2+/-3.6 g/kg mono and poly-unsaturated fatty acids respectively. Compared to domesticated dairy species, a low content of short chain length fatty acids was observed, while stearic acid was at higher, and arachidonic acid at lower levels. Substantial levels of uneven carbon chain fatty acids were also observed. Springbok milk is much more concentrated than the milks of most ruminants, with higher fat and oligosaccharide contents.     

Epithelial basement membrane of bovine renal tubuli. Isolation and analysis of the carbohydrate chains.

The carbohydrate chains present in the tubular basement membrane of bovine kidney were studied. Digestion with collagenase followed with pronase resulted in a complete solubilization of the basement membrane. The different glycopeptides were purified by gel filtration and ion-exchange chromatography. Two kinds of carbohydrate chains could be characterized: oligosaccharides composed of glucosamine, mannose, galactose, fucose and sialic acid, and glucosylgalactose disaccharides. A very small portion of the oligosaccharide chains (ca. 4%) appeared to be free of sialic acid. The bulk of these chains contained sialic acid and fucose, although in small amounts. Only traces of galactosamine were found.     

C-reactive protein binds to phosphorylated carbohydrates

C-reactive protein (CRP) is a major acute phase protein in man. In order to more fully understand the physiological role of this serum protein, we have demonstrated high avidity binding for a defined chemically synthesized carbo-hydrate ligand which represents the repeating disaccharide of lipophosphoglycan, the major surface glycoconjugate of the unicellular parasite Leishmania donovani. Increasing the number of phosphorylated disaccharides in a molecule from one up to seven did not increase the avidity for CRP, however increasing this to 10 potential CRP binding sites did. In order to define the important features of this complex and variable structure for CRP binding we competed CRP binding to whole Leishmania parasites with amino, sulfated, phosphorylated, and unsubstituted monosaccharides, of which only phosphorylated monosaccharides were able to inhibit. Both the carbohydrate and the position of phosphorylation influenced the avidity for CRP. Synthetic oligosaccharides and phospho-oligosaccharides of various lengths and conformations were used to define the structural requirements for CRP recognition. The optimum structure for recognition of a single phosphate group was between two monosaccharide pyranose rings, and within a linear rather than a cyclic molecule. This stresses the importance of the interaction of the CRP binding site with both the carbohydrate and the phosphate group. CRP function may be mediated via the recognition of large arrays of phosphorylated carbohydrates as are characteristic of the surface of microorganisms.