Occurrence of tetra- and pentasaccharides with the sialyl-Le(a) structure in human milk

The occurrence of two novel oligosaccharides in human milk was investigated. These oligosaccharides were purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, indicated the structures of these compounds to be NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNAc and NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNac beta 1----3Gal. This constitutes the first evidence for the occurrence of N-acetylglucosamine or galactose as the reducing-end residue of human milk oligosaccharides. These two oligosaccharides bound MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but to another sialyl-Le(a) structure-directed monoclonal antibody, NS-19-9, only weakly.     

Characterization of mucin-type oligosaccharides with the sialyl-Le(a) structure from human colorectal adenocarcinoma cells

Oligosaccharides with the sialyl-Le(a) structure have been isolated on an affinity column of a monoclonal antibody, MSW 113, from mucin-type glycoproteins derived from the surfaces of SW 1116 and LS 180 cells, and their secretions. The oligosaccharides were polydisperse with respect to molecular size, the oligosaccharides derived from glycoproteins in culture media being larger than those in cell lysates, as assessed by gel filtration. Some of the oligosaccharides were susceptible to degradation by endo-beta-galactosidase (E. freundii), as judged from the change in the gel filtration pattern. These results indicate that oligosaccharides with the sialyl-Le(a) structure derived from mucin-type glycoproteins produced by human colonic cancer cells are extremely large in size and complex in structure, and that some of them contain the poly-N-acetyllactosamine structure.     

Recombinant E-selectin-protein mediates tumor cell adhesion via sialyl-Le(a) and sialyl-Le(x).

E-selectin (previously known as ELAM-1) is one of the adhesion molecules expressed on activated endothelium. Here we show that HL-60 cells express sialyl-Le(x), but not Sialyl-Le(a) on their surface, a colon carcinoma cell line COLO 205 express both these epitopes and another colon carcinoma COLO 320 does not express either one of them. HL-60 and COLO 205 cell adhere strongly to E-selectin coated microwells, whereas COLO 320 does not adhere at all to E-selectin. Finally we provide evidence that monoclonal anti-sialyl-Le(x) can abolish part of the adherence of HL-60 cells to recombinant E-selectin. The adherence of COLO 205 cells can be decreased by either monoclonal anti-sialyl-Le(a) or anti-sialyl-Le(x) antibodies. These results indicate that cell-associated sialylated carbohydrate moieties can act as ligands for recombinant E-selectin.     

Microbial production of human milk oligosaccharide lactodifucotetraose

Human milk oligosaccharides (HMOs) are potent bioactive compounds that modulate neonatal health and are of interest for development as potential drug treatments for adult diseases. The potential of these molecules, their limited access from natural sources, and difficulty in large-scale isolation of individual HMOs for studies and applications have motivated the development of chemical syntheses and in vitro enzymatic catalysis strategies. Whole cell biocatalysts are emerging as alternative self-regulating production platforms that have the potential to reduce the cost for enzymatic synthesis of HMOs. Whole cell biocatalysts for the production of short-chained, linear and small monofucosylated HMOs have been reported but those for fucosylated structures with higher complexity have not been explored. In this study, we established a strategy for producing a difucosylated HMO, lactodifucotetraose (LDFT), from lactose and L-fucose in Escherichia coli. We used two bacterial fucosyltransferases with narrow acceptor selectivity to drive the sequential fucosylation of lactose and intermediate 2′-fucosyllactose (2′-FL) to produce LDFT. Deletion of substrate degradation pathways that decoupled cellular growth from LDFT production, enhanced expression of native substrate transporters and modular induction of the genes in the LDFT biosynthetic pathway allowed complete conversion of lactose into LDFT and minor quantities of the side product 3-fucosyllactose (3-FL). Overall, 5.1 g/L of LDFT was produced from 3 g/L lactose and 3 g/L L-fucose in 24 h. Our results demonstrate promising applications of engineered microbial biosystems for the production of multi-fucosylated HMOs for biochemical studies.     

Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography

A gel permeation chromatographic method for quantifying neutral oligosaccharide fractions from human milk has been developed. Oligosaccharides from monofucosyllactoses to trifucosyllacto-N-hexaoses were separated according to size on a Fractogel TSK HW 40 (S) column. Refractive index detection of monofucosyllactoses to difucosyllacto-N-tetraoses yielded a constant mass response factor of ca. 1 relative to glucose. After the addition of glucose as an internal standard, oligosaccharides were isolated from human milk by ethanol precipitation or two ultrafiltration procedures. The oligosaccharide concentrations found by the ultrafiltration procedures were significantly lower (significance level 0.05) than those determined by the ethanol precipitation procedure.     

Recent advances on human milk oligosaccharide antimicrobial activity

Over the past century, human health has been enhanced by antimicrobial development. Following the deployment of the first antibiotics in the 1940s, bacterial resistance evolved and has increasingly outmaneuvered even the most promising antimicrobial agents. Accordingly, increased interest has been placed on alternative methods to circumvent antimicrobial resistance evolution. In the enclosed short review, we discuss the antimicrobial properties of human breast milk with a special emphasis on human milk oligosaccharides (HMOs). We recount studies across gram-negative and gram-positive pathogens, highlighting the usage of HMOs in promoting human health and wellness.     

Lactic acid bacteria fermentation of human milk oligosaccharide components, human milk oligosaccharides and galactooligosaccharides

Human milk contains about 7% lactose and 1% human milk oligosaccharides (HMOs) consisting of lactose with linked fucose, N-acetylglucosamine and sialic acid. In infant formula, galactooligosaccharides (GOSs) are added to replace HMOs. This study investigated the ability of six strains of lactic acid bacteria (LAB), Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Streptococcus thermophilus and Leuconostoc mesenteroides subsp. cremoris, to digest HMO components, defined HMOs, and GOSs. All strains grew on lactose and glucose. N-acetylglucosamine utilization varied between strains and was maximal in L. plantarum; fucose utilization was low or absent in all strains. Both hetero- and homofermentative LAB utilized N-acetylglucosamine via the Embden-Meyerhof pathway. Lactobacillus acidophilus and L. plantarum were the most versatile in hydrolysing pNP analogues and the only strains releasing mono- and disaccharides from defined HMOs. Whole cells of all six LAB hydrolysed oNP-galactoside and pNP-galactoside indicating β-galactosidase activity. High β-galactosidase activity of L. reuteri, L. fermentum, S. thermophilus and L. mesenteroides subsp. cremoris whole cells correlated to lactose and GOS hydrolysis. Hydrolysis of lactose and GOSs by heterologously expressed β-galactosidases confirmed that LAB β-galactosidases are involved in GOS digestion. In summary, the strains of LAB used were not capable of utilizing complex HMOs but metabolized HMO components and GOSs.     

Immunoaffinity isolation of apolipoprotein E-containing lipoproteins

Discrete apolipoprotein E-containing lipoproteins can be identified when EDTA plasma is fractionated on columns of 4% agarose. The present study has demonstrated, by physical and metabolic criteria, that these apolipoprotein E-containing lipoprotein subclasses may be further isolated by immunoaffinity chromatography. Whole plasma was first bound to an anti-apolipoprotein E immunoadsorbent prior to gel filtration on 4% agarose. After elution from the affinity column and dialysis, the bound fraction was chromatographed on 4% agarose. Discrete subfractions of apolipoprotein E could be demonstrated within elution volumes similar to those observed in the original plasma. When whole plasma was first submitted to gel filtration and the apolipoprotein E-containing lipoproteins of either intermediate- or of high-density lipoprotein (HDL) size were subsequently bound to anti-apolipoprotein E columns, the bound eluted fractions maintained their size and physical properties as shown by electron microscopy and by rechromatography on columns of 4% agarose. The metabolic integrity of apolipoprotein E-containing very-low-density lipoproteins (VLDL) was examined by coinjection into a cynomolgus monkey of 125I-labeled apolipoprotein E-rich and 131I-labeled apolipoprotein E-deficient human VLDL which had been separated by immunoaffinity chromatography. The plasma specific activity time curves of the apolipoprotein B in VLDL, intermediate-density (IDL) and low-density (LDL) lipoproteins demonstrated rates of decay and precursor-product relationships similar to those obtained after injection of whole labeled VLDL, supporting the metabolic integrity of VLDL isolated by immunoaffinity chromatography.     

人乳寡糖2′-岩藻糖基乳糖的功能及其全细胞生物合成

母乳是新生儿最理想的营养剂。其中,人乳寡糖作为母乳的第三大固体组分,对新生儿的生长、发育及健康状况有重要影响,被应用于婴幼儿配方食品中。2′-岩藻糖基乳糖(2′-fucosyllactose,2′-FL),是分泌型母乳中含量最高的人乳寡糖,约占人乳寡糖总量的30%,具有重要的营养和医学价值。2′-FL能够促进婴幼儿生长发育、提高其认知能力、增强免疫力、抗过敏、抗病毒,以及调节肠道菌群,是极具潜力的新型营养强化剂。但是,2′-FL来源于母乳,依靠分离、提取获得大量2′-FL并不现实,因此亟需进行人工合成。人工合成2′-FL的方法有3种,包括：化学合成法、酶催化合成法和全细胞生物合成法。全细胞生物合成法因其成本相对低廉,且易于规模化扩大,而引起了国内外的广泛关注和研究。目前,国外很多跨国公司均开始布局2′-FL的工业化生产和应用,而我国在该领域尚处于研发阶段,因此,了解和掌握2′-FL的合成方法对我国开展2′-FL规模化生产具有重要的意义。本文旨在介绍2′-FL的功能特性,系统阐述其全细胞生物合成的关键技术和最新进展,讨论了针对限速步骤进一步提高产量的策略,旨在为2′-FL的合成和商业化生...     

Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate

Abstract The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti- M. leprae hyperimmune serum coupled to CNBr-Sepharose 4B. A high molecular weigh ( M _r) M. leprae protein (MLP) with a subunit M _r of 22000 was isolated. MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M. leprae . The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M. paratuberculosis . It showed 44% homology with N-terminal end of E. coli bacterioferritin. In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera. The role of bacterioferritin in M. leprae and the importance of MLP as an immunogen has been discussed.     

The oligosaccharide units of a human type L immunoglobulin M (macroglobulin)

1. The carbohydrate composition of the monomeric unit of a type L macroglobulin (immunoglobulin M) was determined as 6 residues of fucose, 35 of mannose, 11 of galactose, 27 of N-acetylglucosamine and 9 of sialic acid. 2. Two types of oligosaccharide unit were present in the protein, one of which (Ca type) contained fucose, mannose, galactose, N-acetylglucosamine and sialic acid in the molar proportions 1:3-4:2:3-5:0-2, and the other (Cb type) contained mannose and N-acetylglucosamine in the proportions 6-8:2-3. 3. A tentative structure is proposed for the Cb type unit. 4. An S-carboxymethylcysteine-containing glycopeptide with a Ca-type unit was isolated after reduction, alkylation and tryptic digestion of the protein. 5. The immunoglobulin monomer appears to contain six oligosaccharide units of the Ca type and two of the Cb type.     

Fucose-containing oligosaccharides from human milk from a donor of blood group 0 Le(a) nonsecretor

The neutral oligosaccharides from the milk of a single donor with blood group 0, Lewis(a+b-) nonsecretor were separated into 18 fractions essentially according to the number of carbohydrate constituents using gel permeation chromatography on Biogel P-4 and Fractogel TSK HW-40. Further separation was achieved by HPLC and by HPTLC after reduction and peracetylation. The fractions obtained were analysed by FAB mass spectrometry with and without derivatization and by one- and two-dimensional proton NMR. Besides the already described 3-fucosidolactose, fucopentaose II (2), fucopentaose III (6) and difucohexaose II (4) the following fucosylated oligosaccharides could be identified. Among the higher oligosaccharides a branched lacto-N-decaose (12) was obtained in pure form after removal of the fucose residues by mild acid treatment.     

A comparison of 3 methods of lipoprotein (a) isolation from human plasma]

Three methods for purification of lipoprotein (a) [Lp(a)] from human plasma were compared. Method I: two-stage ultracentrifugation with subsequent gel-filtration of Lp(a) containing fractions (1.063-1.090 g/ml) on Sepharose CL-4B. Method II: ultracentrifugation followed by affinity chromatography of plasma fraction (1.063 g/ml) on anti-apoB sorbent. Method III: affinity chromatography of the whole plasma on anti-apo(a) sorbent. The Lp(a) yield of these methods is 35, 54 and 41%, respectively. The method III is preferable of these three because it permitted high purification of a large amount of Lp(a) by single-step chromatography.     

Bifidobacterium-selective isolation and enumeration from chicken caeca by a modified oligosaccharide antibiotic-selective agar medium

AIMS: To determine the efficacy and selectivity of an acidified, antibiotic-selective, oligosaccharide-containing media for enumerating Bifidobacterium spp. from chicken caeca samples. METHODS AND RESULTS: Transoligosaccharide propionate agar medium (TOS) modified by addition of mupirocin (50 microg ml-1) and glacial acetic acid (1%, v/v), did not inhibit the growth of bifidobacteria compared with the control media yet inhibited the growth of Lactobacillus acidophilus, Lactobacillus gallinarum, Lactobacillus helveticus and Streptococcus gordonii. CONCLUSIONS: Addition of mupirocin (50 microg ml-1) and glacial acetic acid (1%, v/v) to TOS (TOS-AM50), is an effective selective medium for isolation and enumeration of Bifidobacterium spp. from chicken caeca samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of an intestinal bifidobacteria-selective media contributes to the study of probiotics and prebiotics in poultry and potentially other species.     

Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds.

The potential of immunoaffinity chromatography as a means of purifying legumin from a wide range of Pisum (pea) types was assessed. The method required small amounts of highly purified legumin from a single Pisum type, and this was obtained by salting out with (NH4)2SO4 followed by zonal isoelectric precipitation, ion-exchange chromatography on DEAE-cellulose and sucrose-density-gradient centrifugation. Some physiocochemical properties of purified legumin were determined, a number of which (Strokes radius, subunit molecular weights, subunit N-terminal residues and subunit molar ratios) have not previously been reported for Pisum legumin. Examination of Pisum legumin by two-dimensional gel isoelectric focusing/electrophoresis indicated the existence of extensive subunit heterogeneity, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate showed apparent variation in the nature of this heterogeneity from one Pisum variety to another. Despite this variation, immunoaffinity chromatography on immobilized anti-legumin (which was prepared by affinity chromatography on the immubolized purified legumin from the single Pisum type) was shown to be a generally applicable method for the purification of undegraded legumin from a range of pisum types, including two primate lines.     

A novel pentasaccharide from immunostimulant oligosaccharide fraction of buffalo milk.

A processed oligosaccharide mixture of buffalo milk induced significant stimulation of antibody, delayed-type hypersensitivity response to sheep red blood cells in BALB/c mice. This also stimulated non-specific immune response of the animals measured in terms of macrophage migration index. A novel pentasaccharide has been isolated from the oligosaccharide containing fraction having immunostimulant activity of buffalo milk. This compound was isolated by a combination of gel filtration chromatography, silica gel column chromatography of derivatised oligosaccharides while the homogeneity was confirmed by high performance liquid chromatography. The results of structural analyses, i.e. proton nuclear magnetic resonance, fast atom bombardment mass spectrometry, chemical transformations and degradations are consistent with the following structure: GlcNAcbeta(1-->3)Galbeta(1-->4)GlcNAcbeta(1-->3)Gal beta(1-->4)Glc