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Enzymatic synthesis and purification of galactosylated chitosan oligosaccharides reducing adhesion of enterotoxigenic Escherichia coli K88 Ya Lu Yan, Ying Hu, David J Simpson, and Michael G. Gänzle J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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Journal of Agricultural and Food Chemistry

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Enzymatic synthesis and purification of galactosylated chitosan oligosaccharides

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reducing adhesion of enterotoxigenic Escherichia coli K88

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Ya Lu Yana), Ying Hua), David J. Simpsona), Michael G. Gänzlea,b)*

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a)

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Canada.

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b)

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P.R. China.

University of Alberta, Dept. of Agricultural, Food and Nutritional Science, Edmonton,

Hubei University of Technology, College of Bioengineering and Food Science, Wuhan,

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*Corresponding author footnote

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University of Alberta, Dept. of Agricultural, Food and Nutritional Science, 4-10 Ag/For

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Centre, Edmonton, AB T6E2P5, Canada

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Phone:+1 780 492 3634

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Email: [email protected]

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15

16

1 ACS Paragon Plus Environment


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Abstract

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Enterotoxigenic Escherichia coli (ETEC) K88 cause diarrhea in weaned piglets, and

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represent a suitable model system for ETEC causing childhood diarrhea. This study

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aimed to evaluate the effects of oligosaccharides against ETEC K88 adhesion to porcine

21

erythrocytes with two bioassays. Galactosylated chitosan-oligosaccharides (Gal-COS)

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were synthesized through transgalactosylation by β-galactosidase. Fractions 2 – 5 of Gal-

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COS were obtained through cation exchange and size exclusion chromatography.

24

Fractions 2 – 5 of acetylated Gal-COS were obtained through chemical acetylation

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followed by size exclusion chromatography. Gal-COS F2 containing the largest

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oligosaccharides had the highest anti-adhesion activity with the minimum inhibitory

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concentration of 0.22 g/L, followed by F3 and F4. Acetylation of Gal-COS decreased

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their ability to reduce ETEC K88 adhesion. The composition of active oligosaccharides

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was determined with LC-MS. Galactosylation of COS produces oligosaccharides which

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reduce ETEC K88 adhesion; moreover, resulting oligosaccharides match the composition

31

of human milk oligosaccharides, which prevent adhesion of multiple pathogens.

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Keywords:

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Galacto-oligosaccharides, enterotoxigenic Escherichia coli, beta-galactosidase, pathogen

34

adhesion.

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Introduction

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Indigestible dietary oligosaccharides including galacto-oligosaccharides (GOS) have

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several biological activities that improve host health by modulating composition and

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activity of intestinal microbiota, by preventing the adhesion of pathogens to intestinal

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tissues, and by specific interactions with the immune system.1, The conceptual template

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for the food applications of oligosaccharides with specific health benefits are human milk

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oligosaccharides (HMOs). HMO comprise over 150 different oligosaccharides and their

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concentration in human milk ranges from 5 to 20 g/L. 2 HMOs consist of five

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monosaccharides, galactose, glucose, N-acetyl-glucosamine (GlcNAc), fucose, and N-

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acetyl-neuraminic acid (sialic acid).2 HMOs shape the development of infant

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microbiome, 3 block adhesion of pathogens including Escherichia coli 4 , Salmonella

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Fyris,4 and Campylobacter jejuni5 to host cells, and thus prevent infections.

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In food and feed applications, bifidogenic properties of HMO are currently substituted by

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other non-digestible oligosaccharides including galacto-oligosaccharides (GOS), fructo-

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oligosaccharides (FOS), inulin, and lactulose, which stimulate growth and activity of

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intestinal microbiota.6,7 The ability of oligosaccharides to inhibit adhesion of pathogen,

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however, is highly specific for the target organism and the oligosaccharide structure1,8

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and HMO are thus not readily replaced by other glycans. The structural diversity of

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HMOs, a prerequisite for their interference with adhesion of multiple pathogens, also

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impedes the production at the commercial scale. A commercial GOS preparation also

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inhibited the adherence of enteropathogenic Escherichia coli (EPEC) to Hep-2 and Caco-

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2 cells.9 However, the use of GOS to reduce adhesion of enterotoxigenic Escherichia coli,

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a major cause of childhood diarrhea, has not been reported. 3 ACS Paragon Plus Environment


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GOS are synthesized by transgalactosylation of lactose with β-galactosidase. 10 In

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response to the demand for improved HMOs substitutes, transgalactosylation with β-

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galactosidase was extended to acceptor carbohydrates other than lactose to synthesize a

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diversity of oligosaccharides.1 Fructose, mannose, sucrose, N-acetyl-glucosamine, and

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fucose have been used in transgalactosylation reactions as acceptor sugars to synthesize

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diverse galactosylated oligosaccharides.10,11,12 Of these acceptor carbohydrates, N-acetyl-

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glucosamine (GlcNAc) has received particular attention because it is a constituent of

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HMO,2 binds to fimbriae of E. coli that mediate adhesion to the intestinal mucosa,13 and

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occurs in nature as the constituent monosaccharide of chitin, an abundant polysaccharide

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occurring in the shell of arthropods and the cell wall of fungi. Chitin and chitin-

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oligosaccharides are poorly soluble in water; commercial conversion of chitin from

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shrimp generally yields the deacetylated chitosan or chitosan-oligosaccharides (COS).

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COS exhibit high solubility in water, however, they are also highly reactive as they

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contain aldehyde and amino groups as reactants in the Maillard reaction, which occurs

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even during incubation at ambient temperature.14

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Chitobiose, chitotriose, and COS are acceptor sugars for β-galactosidase to produce di-,

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tri-, tetra-oligosaccharides with galactose and GlcNAc / glucosamine.15 GOS as well as

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COS reduce adhesion of pathogenic E. coli to intestinal mucosal cells,9,16 however, the

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activity of galactosylated COS remains unknown. Therefore, this study aimed to

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determine whether galactosylated COS inhibit adhesion of enterotoxigenic E. coli

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expressing K88 fimbriae to porcine cells. 17 , 18 ETEC K88 is an important cause for

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diarrhea in weaning piglets;19 different from human ETEC strains, is a model system that

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readily allows validation of in vitro results in vivo.17,18 To determine whether


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galactosylation influences the biological activity, oligosaccharides were fractionated by

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cation exchange and size exclusion chromatography. The influence of acetylation was

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determined by chemical acetylation.

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Material and methods

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Preparation of β-galactosidase. Lactococcus lactis MG1363 expressing the LacLM type

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β-galactosidase of L. plantarum.11,15 were streaked onto modified M17 (mM17) agar

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plates that were supplemented with 5% glucose and 5 mg/L erythromycin and incubated

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at 30 ºC under anaerobic condition for 48 h. Crude cell extract (CCE) was prepared as

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previously described.15 Cells were harvested by centrifugation, washed and resuspended

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in 50 mM phosphate buffer (PB) (pH 6.5) with 10% glycerol and 1 mM magnesium

92

chloride. Cells were disrupted with a Mini Beadbeater (model 693, Biospec, Bartlesville,

93

OK) for 2 min and cell debris was removed by centrifugation at 15,300 × g for 10 min at

94

4 ºC. Protein content and β-galactosidase activity of CCE were measured by Bradford

95

protein assay and o-nitrophenyl-β-galactoside respectively. When necessary, CCE was

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diluted with PB containing 10% glycerol and MgCl2 to standardize the activity to 25-30

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µmol/(min × mg protein).12

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Transgalactosylation reaction of β-galactosidase with lactose and COS. COS with a

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degree of polymerisation (DP) ranging from 2 to 6 and a degree of deacetylation of > 95%

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were provided by Glycobio (Dalian, China). The size distribution of the COS was

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confirmed by size exclusion chromatography as described below (data not shown).

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Transgalactosylation reactions were conducted by the addition of 20% (v/v) of

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standardized CCE into 180 g/L of COS and 180 g/L of lactose, followed by incubation



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for 16 h at 45 ºC. Reactions were terminated by addition of perchloric acid to a

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concentration of 3.5% (v/v). Control reactions contained 360 g/L lactose with 20% CCE

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(control without COS); 360 g/L COS with 20% CCE (v/v) (control without GOS); or 180

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g/L COS and 180 g/L lactose without CCE (control without CCE). The

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transgalactosylation and control reactions were monitored by measuring the UV-Vis

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absorbance in the range of 200-500 nm for 16 h with 1 h intervals. Oligosaccharide

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samples were lyophilized prior to analyses described below.

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Preparation of acetylated COS and acetylated galactosylated COS. The acetylation of

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COS and Gal-COS was performed as described20 with some modifications. COS or Gal-

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COS were dissolved in 50:50 (v/v) methanol: water at the concentration of 40 g/L. Acetic

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anhydride (0.5 equiv/glucosamine unit of COS) was added to COS or Gal-COS with

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continuous stirring at room temperature for 4 h. The acetylation was terminated by 40-

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fold dilution with water. Oligosaccharides were lyophilized. The degree of acetylation of

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acetylated oligosaccharides was measured by titration with 0.1 M of NaOH by dissolving

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acetylated oligosaccharides into 0.1 M

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conducted in triplicates.

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Lyophilized

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chromatography as described above. Five fractions were collected. Acetylated COS,

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acetylated Gal-COS, and five collected fractions were lyophilized and re-dissolved to a

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concentration of 10 g / L prior to analysis by HPAEC-PAD and LC-MS, and tested for

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anti-adhesive activities with the methods described below.

acetylated

Gal-COS

were

HCl as described. 21 , 22 All reactions were

further

separated

by

size

exclusion


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High Performance Anion Exchange Chromatography with Pulsed Amperometric

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Detection (HPAEC-PAD). Oligosaccharides were diluted to 1 g/L prior to analysis on a

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HPAEC-PAD ICS-3000 system (Dionex, Oakville, Canada). Samples (10 µL) were

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separated on a CarboPac PA-20 Dionex carbohydrates column using water (A), 200 mM

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NaOH (B), and 1 M sodium acetate (C) as eluent at flow rate of 0.25 mL/min with

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following gradient: 0 min 6% B, 20 min 100% B. GOS were further analyzed using

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gradient starting at 30.4% B, 1.3% C and increasing to 30.4% B, 11.34% C at 22 min.

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Hemagglutination assay to determine the effect of oligosaccharides on ETEC K88

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adhesion to porcine erythrocytes. All samples used in the assay were lyophilized and

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re-dissolved in phosphate–buffer saline (PBS) (pH 7.2). The hemagglutination assay was

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performed with the porcine ETEC ECL 13795 with K88 fimbriae as described18 with

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some modifications. E. coli ECL13795 were cultivated on Minca agar overnight and

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washed with 1 mL PBS. Twenty-five-microliter E. coli cell suspensions with an OD600nm

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of 5 – 10 were diluted 2-fold in V-bottom 96-well polystyrene microtiter plates (Corning).

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The same volume of PBS or tested oligosaccharides with different concentrations was

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added, and the suspensions were incubated for 5 min before the addition of 25 µL of

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erythrocyte suspension into each well. Erythrocytes were prepared by 2-fold dilution of

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10% porcine red blood cells (Innovative Research, Novi, MI, USA). The plate was

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incubated at 4 ºC overnight prior to visual inspection. Activity was noted when the

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sample carbohydrate increased the cell density of ETEC presenting hemagglutination and

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lack of a defined pellet of erythrocytes at least four fold. All samples were diluted from

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10 g/L to exhaustion of the biological activity, or to 0.1 g/L.



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Enzyme-linked immunosorbent assay (ELISA) to determine the effect of

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oligosaccharides on ETEC K88 adhesion to porcine erythrocytes. The ability of

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oligosaccharides to inhibit E. coli K88 adhesion to porcine erythrocytes was also assayed

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by ELISA. Each step of the following assay was separated by washing the wells three

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times with PBS. A 96-well high bind microtiter plate (Corning) was coated overnight

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with 5% porcine red blood cells (Innovative Research, Novi, MI, USA) and blocked with

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3% bovine serum albumin for 1 h at 4 ºC. Oligosaccharides were dissolved in PBS at the

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concentration of 10 g/L. ETEC were mixed with PBS or oligosaccharides and then added

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into plates with 1 h incubation at 4 ºC. Red blood cells without ETEC addition, red blood

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cells with only ETEC addition, and no red blood cells but with ETEC addition were used

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as controls. The wells were then treated with mouse anti E. coli K88A antibody (Biorad,

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USA) at a dilution of 1:2000. After 1 h incubation, the wells were treated with a goat

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anti-mouse IgG (H+L) secondary antibody conjugated to horseradish peroxidase

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(Invitrogen, Fisher Scientific, CA) for 1h. After addition of TMB substrates and

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incubated for 30 min, the reaction was stopped with 2 M sulphuric acid, and the

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absorption at 450 nm was measured on a Varioscan Flash Microplate reader (Thermo

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Scientific, CA).

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Separation of charged oligosaccharides by cation exchange chromatography and

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size exclusion chromatography. To separate charged oligosaccharides from lactose and

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GOS, reaction mixtures obtained with COS, GOS and galactosylated COS (Gal-COS)

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were separated through solid phase extraction method with cation exchange column

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Strata SCX (55 µm, 70A, Phenomenex, USA). The SCX column was conditioned with 3

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mL of methanol prior to extraction. Column was equilibrated with 0.1% trifluoroacetic


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acid (TFA). Sugar samples were diluted with 0.1% (TFA) to a concentration of 10 g/L

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and loaded onto the column. Charged oligosaccharides, containing COS and Gal-COS,

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were eluted with 0.2% triethylamine. Fractions collected during loading of the column

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were referred as Gal-COS flow through (Gal-COS FT) and fractions collected after

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elution were referred to as Gal-COS. The collected fractions were lyophilized and re-

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dissolved to a concentration of 10 g/L. All fractions were analysed by HPAEC-PAD and

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hemagglutination assay.

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Lyophilized COS, GOS, and Gal-COS fractions obtained by cation exchange

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chromatography were further separated by size exclusion chromatography on a Superdex

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peptide 10/300 GL column (10x240 mm, 13 µm, GE healthcare life sciences, USA)

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eluted with 0.2M ammonium acetate at 0.3 mL/min. Separations were carried out on a

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Agilent 1200 HPLC system coupled to refractive index (RI) and multiple wavelength

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detector. Glucose (Fisher), lactose (Sigma), raffinose (Sigma), stachyose (Sigma), inulin

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from chicory (Sigma), and dextran (100,000-200,000 Da, Sigma) were used as external

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standards for molecular weight calibration. Five fractions were collected for each sample.

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All collected fractions were lyophilized and re-dissolved to a concentration of 10 g/L

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prior to analysis by HPAEC-PAD, LC/MS and anti-adhesive activities.

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Liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS).

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LC-ESI-MS analysis of oligosaccharides was conducted by Mass Spectrometry Facility

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of University of Alberta. LC-MS was performed using an Agilent 1200 SL HPLC system

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with GlycanPac AXH-1 column (2.1x150 mm, 1.9 µm, Thermo Scientific, Sunnyvale,

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USA), thermostated at 40 ºC, with a buffer gradient system composed of 96:4 (v/v)

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acetonitrile (ACN): water as mobile phase A and 80 mM ammonium formate in water, 9 ACS Paragon Plus Environment


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pH 4.4 as mobile phase B. Oligosaccharides were separated using the following gradient:

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10% B for 2 min, 10% to 30% B over a period of 10 min, 30% to 75% B over of 9 min,

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75% to 80% B over a period of 1 min, 80% to 10% B over a period of 2 min and held at

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10% B for 3 min.

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Mass spectra were acquired in positive mode ionization using an Agilent 6220 Accurate-

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Mass TOF HPLC/MS system (Santa Clara, CA, USA) equipped with a dual sprayer

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electrospray ionization source with the second sprayer providing a reference mass

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solution. Mass spectrometric conditions were drying gas 10 L/min at 300 ºC, nebulizer 30

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psi, and mass range 100-3200 Da. Analysis of the HPLC-MS data was done using the

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Agilent Mass Hunter Qualitative Analysis software (ver.B.07.00).

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Stability of COS, acetylated-COS, Gal-COS, and acetylated Gal-COS during

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storage. Freeze-dried COS, acetylated-COS, Gal-COS, and acetylated Gal-COS (0.01-

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0.05g/ vial) were placed into individual HPLC vials and stored in closed containers at 37

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ºC and 6 ºC for 7 d. The water activity (aW) in the containers was maintained by addition

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of saturated sodium chloride, saturated sodium bromide, and silica gel beads to achieve a

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constant aW of 0.75, 0.55, and 0.1, respectively. UV-vis absorbance spectra of tested

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oligosaccharides were measured before and after 7 d storage. The absorbance was

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recorded in the range of 200-600 nm. Tested sugars were dissolved to a concentration of

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10 g/L prior to analysis. The compositions of tested sugar before and after storage were

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analyzed by HPAEC-PAD with the method described above.


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Statistical analysis. Purification and fractionation was performed from three independent

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enzymatic reactions. Bioassays were performed in triplicate technical repeats. Results of

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the bioassay-guided fractionation are presented as means ± standard error of the mean.

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Results

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Transgalactosylation and separation of COS. Transgalactosylation of COS and lactose

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with β-galactosidase were analyzed by HPAEC-PAD (Fig. 1) Transgalactosylation

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activity of β-galactosidase was apparent but galactosylated oligosaccharides were

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partially obscured by the presence of glucose, galactose, COS, and galacto-

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oligosaccharides (Fig 1A and 1B). Previously, Gal-COS were synthesized via

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transgalactosylation of galactose from lactose to the non-reducing end of COS by β-

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galactosidase.15 Charged oligosaccharides including COS and Gal-COS were separated

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from the reaction mixture by cation exchange chromatography. In the separation of

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transgalactosylation reactions with COS as acceptor, COS and Gal-COS were bound to

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the column and eluted with triethylamine (Fig. 1B); glucose, galactose, lactose and

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galacto-oligosaccharides did not bind to the columns (data not shown).

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The enzymatic reaction mixture and all fractions obtained by separation with cation

229

exchange chromatography were tested for anti-adhesion activity against ETEC K88. All

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samples were adjusted to 10 g/L and diluted to exhaustion of the biological activity.

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Enzymatic reactions without further purification had the strongest anti-adhesive activities,

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followed by Gal-COS purified by cation exchange columns (Table 1). Glucose, galactose,

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lactose, COS and GOS had no effect on agglutination of ETEC to porcine erythrocytes.

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Monitoring the transgalactosylation reactions with lactose or lactose and COS by



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measuring UV absorbance and comparison to reactions with COS and CCE suggested

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that the presence of COS leads to formation of Maillard compounds. Oligosaccharide

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mixtures generated by enzymatic synthesis in presence of COS thus require further

238

separation before conclusions on their biological activity can be drawn. COS reduced

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K88 adhesion after separation on cation exchange columns, however, the effective

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concentration was twice as high as the effective concentration of Gal-COS (Table 1). The

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results indicate that Gal-COS have higher anti-adhesion activity than COS or GOS.

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Fractionation of oligosaccharides by SEC. In order to determine the effect of molecular

243

weight of oligosaccharides on anti-adhesion activities against ETEC K88, COS, GOS and

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Gal-COS were separated based on their molecular weight. Five different fractions were

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collected for each sample and designated F1 to F5 in order of decreasing molecular

246

weight. Most Gal-COS eluted in F4 and F3, followed by F2 and F5 (Fig 2, upper trace).

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The carbohydrate concentration in Gal-COS F1 was below the detection limit of the RI

248

detector. The molecular weight range of fractions was estimated as follows: F2, MW

249

1500-2500 Da, corresponding to DP8 and higher; F3, MW 900-1500 Da, corresponding to

250

DP6 – 8; F4, MW 500-1200 Da, corresponding to DP 3 – 6, F5, less than 500 Da and DP

251

of less than 3. All fractions were tested for anti-adhesive activities against ETEC K88

252

(Table 2). Fractions containing Gal-COS were two to 8 times more active when

253

compared to the corresponding factions containing COS only. The activity of Gal-COS

254

decreased with decreasing molecular weight (Table 2).

255

In addition to carbohydrates, F5 contained compounds with UV absorbance at 254 and

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280 nm. This confirmed that Maillard reaction products influenced the results of the

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hemagglutination assay as Gal-COS elution fraction 5 (Table 2) and COS-containing 12 ACS Paragon Plus Environment

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control reactions (data not shown) inhibited E. coli adhesion to porcine red blood cells at

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10 g/L.

260

F1 and F5 were excluded from further analysis owing to the low yield, the low activity,

261

and the presence of UV-absorbing compounds in F5. F2, F3 and F4 of Gal-COS were

262

analyzed by HPAEC-PAD. Different patterns of oligosaccharides were observed in each

263

fractions, however, the lack of galactosylated COS standards prevented identification

264

(data not shown).

265

The effect of Gal-COS on adhesion of E. coli was confirmed with an additional bioassay

266

based on ELISA detection of K88 fimbriae (Fig. 4). Consistent with results obtained with

267

the hemagglutination assay, the highest anti-adhesive activity was observed with Gal-

268

COS after purification with cation exchange columns; Gal-COS F2 and Gal-COS F3 also

269

exhibited strong anti-adhesion effects (Fig. 4). Lactose and COS did not reduce ETEC

270

K88 attachment onto porcine red blood cells. Different from the hemagglutination assay,

271

however, the Gal-COS reaction mixture had no anti-adhesive activity (Fig. 4), further

272

indicating that Maillard reaction products interfere with the hemagglutination assay.

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Taken together, ELISA confirmed that the Gal-COS and the Gal-COS fractions F2, F3

274

and F4 inhibit adherence of ETEC K88 to porcine red blood cells, and that Maillard

275

reaction products interfere with the hemagglutination assay.

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Identification of oligosaccharides by LC-ESI-MS. Active fractions were analyzed by

277

LC-MS to achieve an accurate determination of Gal-COS. The high resolution MS

278

provided information on the chemical formula; the presence of Gal-COS was deduced

279

from the enzymatic method of synthesis and the absence of the corresponding products in



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the control reactions. Galactosylated COS were present in all active fractions but absent

281

in the corresponding fractions containing fractionated COS (Table 3 and data not shown).

282

The predominant degree of polymerization in the respective fractions corresponded to the

283

separation range of the SEC column used for fractionation (Table 3 and Figure 2).

284

Galactosylated COS were the major compounds in each of the fractions tested (Table 3);

285

Gal-GOS were composed of glucosamine and GlcNAc with one to four galactosyl-

286

residues.

287

Preparation of acetylated COS and acetylated galactosylated COS. Gal-COS

288

contained both glucosamine and GlcNAc as constituting monosaccharides. To determine

289

whether the degree of acetylation influences activity, Gal-COS were separated by cation

290

exchange chromatography and acetylated chemically. Titration of Gal-COS before and

291

after acetylation estimated the degree of acetylation as 50%. Analysis of acetylated Gal-

292

COS by LC-MS confirmed the synthesis of N-acetyl-glucosamine containing

293

oligosaccharides. LC-MS results (Table 4) showed galactosylated oligosaccharides with

294

different degree of acetylation. Acetylated Gal-COS fractions differed in their anti-

295

adhesive activity (Table 5). The largest fraction (Fig. 4) with molecular weight from 1700

296

to 2500 Da exhibited the strongest activity, followed by the fractions with molecular

297

range of 1000 to 2000 Da and 700 to 1300 Da. However, acetylation decreased the anti-

298

adhesive activity 3 – 10 fold when compared to the corresponding fractions containing

299

Gal-COS (Table 2 and 5).

300

Stability of Gal-COS and acetylated Gal-COS during storage. Past studies and

301

observations during purification of Gal-COS in this study indicated that handling of COS

302

at ambient temperature may result in formation of Maillard products which affect their 14 ACS Paragon Plus Environment

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activity.14 To determine whether acetylation or galactosylation influences the reactivity

304

during dry storage, the degradation of Gal-COS was monitored after lyophilisation. Gal-

305

COS were compared to COS, acetylated COS and acetylated Gal-COS. Samples were

306

stored at different temperatures and water activities. The change in UV-Vis absorbance

307

after 7 days of storage was used as a simple tool to monitor major changes. The

308

differences in the UV-Vis spectra are plotted in a heat map to monitor the stability over

309

time. An increased absorption was observed for COS storage under 37 ºC for all three

310

water activities conditions as indicated with lighter colour area in heat map (Figure 5). A

311

minor increase in absorbance was observed in acetylated COS, Gal-COS and acetylated

312

Gal-COS under all conditions. No changes were observed in COS stored at 6 ºC with all

313

tested water activities. An increased UV absorption between 340-360 nm was observed

314

for Gal-COS maybe because the Mallard reaction products formed during enzymatic

315

reaction. COS were not stable when stored at 37ºC, however, galactosylation, acetylation,

316

or both would increase their stability. The lower UV absorption of acetylated Gal-COS

317

after 7 d storage when compared to Gal-COS indicated a lower reactivity.

318

Discussion

319

This study demonstrates that Gal-COS inhibit adhesion of ETEC K88 to porcine blood

320

cells, and may thus be used to inhibit pathogen adhesion in vivo. The activity was

321

confirmed by two bioassays, a hemagglutination assay, and an ELISA assay targeting

322

K88 fimbriae. Transgalactosylation strongly increased the anti-adhesive activity of COS;

323

moreover, high molecular weight oligosaccharides had higher biological activity while

324

acetylation decreased the effect on pathogen adhesion.



Page 16 of 37

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Transgalactosylation of COS with β-galactosidase as catalyst and lactose as galactosyl-

326

donor provides an inexpensive approach to synthesize oligosaccharides that contain

327

GlcNAc- or glucosamine and exhibit biological activity.1 The production of glucosamine

328

or COS currently employs acid hydrolysis of chitin from crab and shrimp shell 23 or

329

enzymatic hydrolysis of chitosan or chitin using chitosanase24 or chitinase,25 respectively.

330

COS inhibited adhesion of EPEC16 and ETEC (this study) to eukaryotic cells, however,

331

COS are highly reactive and form Maillard products during storage or in enzymatic

332

reactions. These products interfere with their biological activity. Cation exchange and

333

size exclusion purification excluded the effects of Maillard products on hemagglutination

334

assay. ELISA assay confirmed the inhibition of ETEC adhesion by Gal-COS. Moreover,

335

Gal-COS with a DP ranging from 8 – 12 exhibited the highest activity; these compounds

336

have a lower reactivity in the Maillard reaction when compared to short-chain COS.

337

Fimbriae mediated pathogen-host interactions are highly specific for host species, body

338

site, and in several cases, also the age of the animals.8 For example, host adaptation of

339

Salmonella enterica Typhimurium was related to allelic variations in the glycan binding

340

domain of FimH. 26 Likewise, host specificity of ETEC relates to the fimbriae that

341

mediate adhesion. ETEC expressing class 1b and class 5 fimbria cause childhood

342

diarrhea, 27 while ETEC with fimbria F4 (K88) cause disease in animals.19 Porcine

343

aminopeptidase N, a membrane bound glycoprotein, was recently identified as receptor

344

for F4 fimbriae. Binding of F4 fimbriae to aminopeptidase N was dependent on

345

decoration of the protein with sialic acid. 28 ETEC K88 fimbriae bind to GlcNAc, N-

346

acetyl-galactosamine, N-acetylmannosamine, and D-galactosamine

347

oligosaccharides consisting of GlcNAc and galactose residues at the reducing end were

29

and β-linked


Page 17 of 37


348

suggested to be essential for adherence of K88 fimbriae to host cells.29, 30 , 31 , 32 In

349

agreement with the GlcNAc and galactose-containing target glycans of K88 fimbriae, the

350

effect of Gal-COS containing β-linked galactosyl and GlcNAc or glucosamine residues

351

on ETEC K88 attachment to porcine red blood cells was stronger when compared to non-

352

galactosylated COS (Table 2). However, acetylation to match the acetylation in porcine

353

glycans recognized by K88 fimbriae reduced activity.

354

ETEC induced diarrhea in suckling and post-weaning piglets cause significant economic

355

losses in swine production. ETEC initiate host colonization through receptor-fimbriae

356

interaction; diarrhea is caused by heat-labile or heat-stable enterotoxins secreted after

357

colonization29. ETEC with fimbriae K88 (F4) and F18 infect suckling and post-weaning

358

piglets, respectively.19,

359

processing, reduced K88 attachment to swine epithelia in vivo and in vitro. 33

360

Exopolysaccharides extracted form Lactobacillus reuteri also reduced ETEC K88

361

adherence in vitro18 and in vivo.17 Other food- or feed-derived glycans including wheat

362

bran, galactomannan or phenolic compounds from locust bean, or glycopeptides derived

363

from ovomucin interfered with adhesion of ETEC K88,34 however, the active compounds

364

have not been identified. Bioassay-guided separation and fractionation of Gal-COS as

365

performed in this study allowed to relate molecular weight and degree of acetylation of

366

Gal-COS to their anti-adhesion activity. Moreover, the use of two complementary

367

bioassays confirmed that the oligosaccharides target K8 fimbriae (Table 2 and Figure 4).

368

The activity of Gal-COS decreased with decreasing molecular weight and acetylation

369

decreased activity. This result matches observations on the anti-adhesive activity of COS

370

against EPEC, which also decreased with reduced DP and increased acetylation.16

30

Casein glycomacropetide, a glycoprotein obtained from cheese



Page 18 of 37

371

Remarkably, the galactosylation of COS increased anti-adhesion activity when compared

372

to COS. Gal-GOS are promising alternatives to antibiotic therapy for the swine industry.

373

Bacterial pathogens other than ETEC K88 also recognize β-linked galactosylated

374

oligosaccharides,8 and Gal-COS may thus target additional human or animal pathogens.

375

The concept of using oligosaccharides for therapeutic applications is increasingly

376

validated by in vivo studies.17 Oligosaccharide synthesis with glycosyl hydrolases

377

requires high concentrations of both donor and acceptor sugars, and generates

378

oligosaccharide mixtures differing in the site of glycosylation, the linkage type, and the

379

degree

380

oligosaccharides were produced with other retaining glycosyl hydrolases including ɑ-

381

galactosidase, 35 fucosidase, trans-sialidase 36 and N-acetyl-glucosaminidase.1 Metabolic

382

engineering of microorganisms to synthesise oligosaccharides with glycosyl transferases

383

provides an alternative approach for oligosaccharide production. Fucosyllactose has

384

recently become commercially available through fermentation of fucose and lactose with

385

E. coli harbouring fucosyltransferase. 37 Oligosaccharide synthesis with intracellular

386

glycosyl transferases generates lower yields when compared to glycosyl hydrolases but

387

produces defined oligosaccharides.

388

transferase activity and the preference for the linkage type of retaining glycosyl

389

hydrolases.39,40,41 Relating the selectivity of oligosaccharide synthesis to the specificity of

390

glycan recognition of pathogens, however, makes the use of oligosaccharide mixtures

391

preferential as these may target multiple pathogens. Human milk oligosaccharides are a

392

mixture of more than 150 oligosaccharides targeting adhesion of multiple pathogens.4

of

polymerisation15,17

(this

38

paper).

In

addition

to

β-galactosidases,

Site-directed mutagenesis can improve the


Page 19 of 37


393

In conclusion, this study demonstrated that partially purified galactosylated chitosan-

394

oligosaccharides strongly interfered with adhesion of ETEC K88 to porcine erythrocytes.

395

In addition, the influence of the molecular weight and degree of acetylation was

396

elucidated, which is necessary to address the low solubility of chitin oligosaccharides,

397

and the high reactivity of chitosan oligosaccharides. Acetylation of COS or Gal-COS

398

increases stability but decreases anti-adhesive activity.

399

Acknowledgements

400

The Alberta Livestock and Meat Agency is acknowledged for funding (Grant No.

401

2015B009R).

402

Supporting Information Available: Figure S1. HPAEC-PAD chromatography of COS

403

before and after storage at 37 ºC;

404

Reference

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oligosaccharides are associated with protection against diarrhea in breast-fed infants. J. Pediatr. 2004, 145, 297-303. 6 Ackerman, D. L.; Craft, K. M.; Townsend, S. D. Infant food applications of complex carbohydrates: Structure, synthesis, and function. Carbohydr. Res. 2017, 437, 16-27. 7 Davis, L. M. G.; Martinez, I.; Walter, J.; Goin, C.; Hutkins, R. W. Barcoded Pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans. Plos One 2011, 6, e25200. 8 Shoaf-Sweeney, K. D.; Hutkins, R. W. Adherence, anti-adherence, and oligosaccharides preventing pathogens from sticking to the host. Adv. Food Nutr. Res. 2009, 55, 101e161. 9 Shoaf, K.; Mulvey, G. L.; Armstrong, G. D.; Hutkins, R. W. Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells. Infect. Immun. 2006, 74, 6920-6928. 10 Gänzle, M. G. Enzymatic synthesis of galacto-oligosaccharides and other lactose derivatives (hetero-oligosaccharides) from lactose. Int. Dairy J. 2012, 22, 116-122. 11 Schwab, C.; Sorensen, K. I.; Gänzle, M. G. Heterologous expression of glycoside hydrolase family 2 and 42 βgalactosidases of lactic acid bacteria in Lactococcus lactis. Syst. Appl. Microbiol. 2010, 33, 300-307. 12 Schwab, C.; Lee, V.; Sorensen, K. I.; Gänzle, M. G. Production of galactooligosaccharides and heterooligosaccharides with disrupted cell extracts and whole cells of lactic acid bacteria and bifidobacteria. Int. Dairy J. 2011, 21, 748-754. 13 Rhen, M., Klemm, P., Korhonen, T.K. Identification of two new hemagglutinins of Escherichia coli, N-acetyl-D-glucosamine-specific fimbriae and a blood group Mspecific agglutinin, by cloning the corresponding genes in Escherichia coli K-12. J. Bacteriol. 1986, 168, 1234-1242. 14 Hrynets, Y.; Ndagijimana, M.; Betti, M. Studies on the formation of Maillard and caramelization products from glucosamine incubated at 37 °C. J. Agric. Food Chem. 2015, 63, 6249-6261. 15 Black, B. A.; Yan, Y.; Galle, S.; Hu, Y.; Curtis, J. M.; Gänzle, M. G. Characterization of novel galactosylated chitin-oligosaccharides and chitosan-oligosaccharides. Int. Dairy J. 2014, 39, 330-335. 16 Quintero-Villegas, M. I.; Aam, B. B.; Rupnow, J.; Sorlie, M.; Eijsink, V. G.; Hutkins, R. W. Adherence inhibition of enteropathogenic Escherichia coli by


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chitooligosaccharides with specific degrees of acetylation and polymerization. J. Agric. Food Chem. 2013, 61, 2748-2754. 17 Chen, X. Y.; Woodward, A.; Zijlstra, R. T.; Gänzle, M. G. Exopolysaccharides synthesized by Lactobacillus reuteri protect against enterotoxigenic Escherichia coli in piglets. Appl. Environ. Microbiol. 2014, 80, 5752-5760. 18 Wang, Y.; Gänzle, M. G.; Schwab, C. Exopolysaccharide synthesized by Lactobacillus reuteri decreases the ability of enterotoxigenic Escherichia coli to bind to porcine erythrocytes. Appl. Environ. Microbiol. 2010, 76, 4863-4866. 19 Xia, P.; Zou, Y.; Wang, Y.; Song, Y.; Liu, W.; Francis, D. H.; Zhu, G. Receptor for the F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). Appl. Microbiol. Biotechnol. 2015, 99, 4953-4959. 20 Hu, Y.; Du, Y.; Wang, X.; Feng, T. Self-aggregation of water-soluble chitosan and solubilization of thymol as an antimicrobial agent. Journal of Biomedical Materials Research Part a 2009, 90A, 874-881. 21 Liu, D.; Wei, Y.; Yao, P.; Jiang, L. Determination of the degree of acetylation of chitosan by UV spectrophotometry using dual standards. Carbohydr. Res. 2006, 341, 782-785. 22 Zhang, Y. Q.; Xue, C. H.; Xue, Y.; Gao, R. C.; Zhang, X. L. Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction. Carbohydr. Res. 2005, 340, 1914-1917. 23 Hossain, G. S.; Shin, H. D.; Li, J.; Wang, M.; Du, G.; Chen, J.; Liu, L. Metabolic engineering for amino-, oligo-, and polysugar production in microbes. Appl. Microbiol. Biotechnol. 2016, 100, 2523-2533. 24 Thadathil, N.; Velappan, S. P. Recent developments in chitosanase research and its biotechnological applications: A review. Food Chem. 2014, 150, 392-399. 25 Liu, L.; Liu, Y.; Shin, H.; Chen, R.; Li, J.; Du, G.; Chen, J. Microbial production of glucosamine and N-acetylglucosamine: advances and perspectives. Appl. Microbiol. Biotechnol. 2013, 97, 6149-6158. 26 Yue, M.; Han, X.; De Masi, L.; Zhu, C.; Ma, X.; Zhang, J.; Wu, R.; Schmieder, R.; Kaushik, R. S.; Fraser, G. P.; Zhao, S.; McDermott, P. F.; Weill, F. X.; Mainil, J. G.; Arze, C.; Fricke, W. F.; Edwards, R. A.; Brisson, D.; Zhang, N. R.; Rankin, S. C.; Schifferli, D. M. Allelic variation contributes to bacterial host specificity. Nat. Commun. 2015, 6, 8754



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27 Mortezaei, N.; Epler, C. R.; Shao, P. P.; Shirdel, M.; Singh, B.; McVeigh, A.; Uhlin, B. E.; Savarino, S. J.; Andersson, M.; Bullitt, E. Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways. Mol. Microbiol. 2015, 95, 116-126. 28 Melkebeek, V.; Rasschaert, K.; Bellot, P.; Tilleman, K.; Favoreel, H.; Deforce, D.; De Geest, B. G.; Goddeeris, B. M.; Cox, E. Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response. Mucosal Immunology 2012, 5, 635-645. 29 Jin, L. Z.; Zhao, X. Intestinal receptors for adhesive fimbriae of enterotoxigenic Escherichia coli (ETEC) K88 in swine - a review. Appl. Microbiol. Biotechnol. 2000, 54, 311-318. 30 Moonens, K.; Van den Broeck, I.; De Kerpel, M.; Deboeck, F.; Raymaekers, H.; Remaut, H.; De Greve, H. Structural and functional insight into the carbohydrate receptor binding of F4 fimbriae-producing enterotoxigenic Escherichia coli. J. Biol. Chem. 2015, 290, 8409-8419. 31 Grange, P. A.; Mouricout, M. A.; Levery, S. B.; Francis, D. H.; Erickson, A. K. Evaluation of receptor binding specificity of Escherichia coli K88 (F4) fimbrial adhesin variants using porcine serum transferrin and glycosphingolipids as model receptors. Infect. Immun. 2002, 70, 2336-2343. 32 Sarabia-Sainz, A.; Ramos-Clamont, G.; del Carmen Candia-Plata, Ma Maria; Vazquez-Moreno, L. Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin. Int. J. Biol. Macromol. 2009, 44, 175-181. 33 Gustavo Hermes, R.; Molist, F.; Francisco Perez, J.; Gomez de Segura, A.; Ywazaki, M.; Davin, R.; Nofrarias, M.; Korhonen, T. K.; Virkola, R.; Martin-Orue, S. M. Casein glycomacropeptide in the diet may reduce Escherichia coli attachment to the intestinal mucosa and increase the intestinal lactobacilli of early weaned piglets after an enterotoxigenic E. coli K88 challenge. Br. J. Nutr. 2013, 109, 1001-1012. 34 Gonzalez-Ortiz, G.; Francisco Perez, J.; Gustavo Hermes, R.; Molist, F.; JimenezDiaz, R.; Maria Martin-Orue, S. Screening the ability of natural feed ingredients to interfere with the adherence of enterotoxigenic Escherichia coli ( ETEC) K88 to the porcine intestinal mucus. Br. J. Nutr. 2014, 111, 633-642. 35 Wang, Y.; Black, B. A.; Curtis, J. M.; Gänzle, M. G. Characterization of alphagalacto-oligosaccharides formed via heterologous expression of alpha-galactosidases from Lactobacillus reuteri in Lactococcus lactis. Appl. Microbiol. Biotechnol. 2014, 98, 2507-2517


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36 Holck, J.; Larsen, D. M.; Michalak, M.; Li, H.; Kjaerulff, L.; Kirpekar, F.; Gotfredsen, C. H.; Forssten, S.; Ouwehand, A. C.; Mikkelsen, J. D.; Meyer, A. S. Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase. New Biotechnology 2014, 31, 156-165 37 Weichert, S.; Jennewein, S.; Huefner, E.; Weiss, C.; Borkowski, J.; Putze, J.; Schroten, H. Bioengineered 2 '-fucosyllactose and 3-fucosyllactose inhibit the adhesion of Pseudomonas aeruginosa and enteric pathogens to human intestinal and respiratory cell lines. Nutr. Res. 2013, 33, 831-838. 38 Crout, D. H.; Vic, G. Glycosidases and glycosyl transferases in glycoside and oligosaccharide synthesis. Current Opinion in Chemical Biology 1998, 2, 98-111 39 Zakariassen, H.; Hansen, M. C.; Joranli, M.; Eijsink, V. G. H.; Sorlie, M. Mutational Effects on Transglycosylating Activity of Family 18 Chitinases and Construction of a Hypertransglycosylating Mutant. Biochemistry (N. Y. ) 2011, 50, 5693-5703 40 Jorgensen, F.; Hansen, O. C.; Stougaard, P. High-efficiency synthesis of oligosaccharides with a truncated beta-galactosidase from Bifidobacterium bifidum. Appl. Microbiol. Biotechnol. 2001, 57, 647-652 41 Meng, X.; Dobruchowska, J. M.; Pijning, T.; Gerwig, G. J.; Kamerling, J. P.; Dijkhuizen, L. Truncation of domain V of the multidomain glucansucrase GTF180 of Lactobacillus reuteri 180 heavily impairs its polysaccharide-synthesizing ability. Appl. Microbiol. Biotechnol. 2015, 99, 5885-5894 405



Page 24 of 37

Table 1. Inhibition of erythrocyte agglutination by ETEC K88 strain with oligosaccharides at various concentrations. Hemagglutination was determined with E. coli K88 ECL13795 (O149, virotype STb:LT:EAST1:F4), and results were based on three independent assays. Different letters indicate statistical differences (P 10

COS elutiona)

6.67 ± 0.96a

COS FTb)

9.44 ± 0.00b

Glucose

> 10

Galactose

> 10

Glucosamine

> 10

Sample

Lactose Gal-COS mixture

a)

> 10 c)

0.69 ± 0.00c

Gal-COS FT b)

5.00 ± 0.00a

Gal-COSa)

2.92 ± 0.48d

GOS > 10 charged oligosaccharide fraction after purification by cation exchange chromatography.

b)

uncharged fraction of enzymatic reaction products that did not bind to cation exchange column.

c)

unpurified enzymatic reaction products or equivalent incubation of enzyme and COS


Page 25 of 37


Table 2. Inhibition of erythrocyte agglutination by ETEC K88 strain with Gal-COS purified by cation exchange chromatography at various concentrations. The corresponding COS fractions were used for comparison. Hemagglutination was determined with E. coli K88 ECL13795 (O149, virotype STb:LT:EAST1:F4), and results were based on three independent assays. Values in the same column that do not share a common superscript are significantly different (P 10

COS F2

8.33 ± 0.96a

COS F3

2.78 ± 0.48b

COS F4

6.11 ± 0.96a

COS F5

> 10

Gal-COS F2

0.22 ± 0.00c

Gal-COS F3

1.88 ± 0.24d

Gal-COS F4

3.06 ± 0.48b

Gal-COS F5

> 10

GOS fractions

> 10 in all tested fractions

Sample

a)

Fractions numbers 1 – 5 correspond to fractions with decreasing molecular weight after separation with size exclusion chromatography.



Page 26 of 37

Table 3. List of compounds identified in Gal-COS elution fractions and their mass accuracy Oligosaccharides fractions

Gal-COS F2

Gal-COS F3

Gal-Gal-(GlcN)4(GlcNAc)6 Gal-(GlcN)6(GlcNAc)5 Gal-(GlcN)7(GlcNAc)4 Gal-(GlcN)8(GlcNAc)3 Gal-(GlcN)5(GlcNAc)5 Gal-Gal-(GlcN)6(GlcNAc)3 Gal-(GlcN)8(GlcNAc)2 Gal-Gal-(GlcN)6(GlcNAc)2 Gal-Gal-(GlcN)7(GlcNAc) Gal-Gal-Gal-Gal-(GlcN)5 Gal-Gal-Gal-(GlcN)6 Gal-Gal-(GlcN)7 Gal-Gal-Gal-Gal-(GlcN)4 Gal-Gal-Gal-(GlcN)5 Gal-Gal-(GlcN)6 Gal-Gal-Gal-(GlcN)4 Gal-(GlcN)6 (GlcN)7 Gal-Gal-(GlcN)4 Gal-(GlcN)5 (GlcN)6 Gal-Gal-Gal-(GlcN)4 Gal-Gal-(GlcN)5 Gal-Gal-Gal-(GlcN)

Measured Mass (Da) 2204.8849 2161.8854 2119.8750 2077.8624 2000.8215 1917.7775 1874.7822 1714.6989 1672.6779 1471.5760 1470.5855 1469.5893 1310.5067 1309.5378 1308.5296 1148.4479 1146.4698 1145.4932 986.3933 985.4063 984.4209 1148.4457 1147.4640 987.3800

Error (Da) 0.0172 0.0123 0.0125 0.0104 0.0172 0.0104 0.0095 0.0111 0.0007 0.0101 0.0036 -0.0086 0.0096 0.0005 0.0005 0.0036 -0.0064 0.0009 0.0018 -0.0011 0.0000 0.0015 0.0037 0.0046

Gal-Gal-(GlcN)4

986.3921

0.0007

Gal-(GlcN)5

985.4026

-0.0048

Gal-Gal-(GlcN)3

825.3326

0.0001

Compounds1, 2)

3

Gal-COS F4

Gal-(GlcN)4

824.3348 -0.0038 (GlcN)5 823.3563 0.0017 Gal-Gal-(GlcN)2 664.2489 -0.0049 (GlcN)4 662.2852 -0.0006 (GlcN)3 501.2179 0.0009 1) The position of N-acetyl-glucosamine groups does not correspond to its position in different oligosaccharides; however, as a result of enzymatic catalysis, galactose units are at the non-reducing end. 2) Nomenclature: Gal, Galactose; GlcN, N-glusosamine; GlcNAc, N-acetyl-glucosamine 26 ACS Paragon Plus Environment

Page 27 of 37




Page 28 of 37

Table 4. List of compounds identified in acetylated Gal-COS elution fractions and their mass accuracy Oligosaccharides fractions

Acetylated Gal-COS F2



Compounds

Measured mass (Da)

Error (Da)

Compounds

Measured mass (Da)

Error

Gal-(GlcN)(GlcNAc)10 Gal-Gal-Gal-(GlcN)(GlcNAc)8 Gal-(GlcNAc)10 Gal-Gal-(GlcNAc)9 Gal-(GlcN)(GlcNAc)9 Gal-Gal-Gal-(GlcNAc)8 Gal-Gal-(GlcN)(GlcNAc)8

2371.9303 2289.8933 2210.8655 2169.8457 2168.8497 2128.8214 2127.8300

0.0044 0.0204 0.0084 0.0151 0.0031 0.0173 0.0010

Gal-Gal-Gal-(GlcN)-(GlcNAc)7 Gal-(GlcNAc)9 Gal-(GlcN)(GlcNAc)8 Gal-Gal-Gal-(GlcN)(GlcNAc)6 Gal-(GlcN)(GlcNAc)7 Gal-Gal-(GlcN)(GlcNAc)6

2086.8016 2007.7821 1965.7672 1883.7253 1762.6841 1721.6646

0.0081 0.0044 0.0000 0.0113 -0.0037 0.0033

Gal-Gal-(GlcN)(GlcNAc)8 Gal-Gal-GlcN-(GlcNAc)7 Gal-Gal-Gal-(GlcN)(GlcNAc)4 (GlcNAc)7 Gal-(GlcNAc)6 Gal-Gal-(GlcNAc)5 Gal-Gal-(GlcN)(GlcNAc)4 Gal-Gal-(GlcN)(GlcNAc)4 Gal-Gal-(GlcN)(GlcNAc)3 Gal-Gal-Gal-(GlcN)(GlcNAc)2 Gal-(GlcN)(GlcNAc)3

2127.8303 1924.7427 1477.5656 1439.5653 1398.5427 1357.5210 1315.5033

0.0103 0.0020 0.0103 -0.0009 0.0030 -0.0017 0.0008

Gal-Gal-Gal-(GlcN)(GlcNAc)3 (GlcNAc)6 Gal-(GlcNAc)5 Gal-(GlcN)(GlcNAc)4 Gal-Gal-(GlcN)2(GlcNAc)2 Gal-Gal-(GlcN)(GlcNAc)3

1274.4845 1236.4851 1195.4652 1153.4464 1070.4117 1112.4238

0.0085 -0.0017 0.0050 -0.0033 -0.0009 0.0007

1315.5069 1112.4237 1071.4047 950.3716

0.0044 0.0006 0.0081 0.0012

Gal-Gal-(GlcN)(GlcNAc)2 Gal-Gal-Gal-(GlcN)(GlcNAc) (GlcNAc)4 Gal-Gal-(GlcN)(GlcNAc)

909.3452 868.3246 830.3283 706.2670

0.0015 0.0074 0.0002 0.0026

1) The position of N-acetyl-glucosamine groups does not correspond to its position in different oligosaccharides; however, as a result of enzymatic catalysis, galactose units are at the non-reducing end. 2) Nomenclature: Gal, Galactose; GlcN, N-glusosamine; GlcNAc, N-acetyl-glucosamine


Page 29 of 37


Table 5. Inhibition of erythrocyte agglutination by ETEC K88 strain with acetylated GalCOS elution fractions at various concentrations. Hemagglutination was determined with E. coli K88 ECL13795 (O149, virotype STb:LT:EAST1:F4), and results were based on three independent assays. Different letters indicate statistical differences (P 10


5.83 ± 0.96a


6.11 ± 0.96a


> 10


> 10

Sample



Page 30 of 37

Figure legends. Figure 1. HPAEC-PAD results of transgalactosylation reaction with lactose and COS and purification of the active oligosaccharide fractions. Panel A, Chromatographic trace of unpurified enzymatic reactions; chromatograms of lactose and COS are shown for comparison. Panel B. Chromatographic traces of charged oligosaccharides purified by cation exchange chromatography. The corresponding fraction obtained with separation of COS is shown for comparison. Chromatograms are representative of three independent reactions. Galactose, glucose and lactose elute at 11.2, 11.6 15.9 min, respectively. Figure 2. Size exclusion results for different fractions of Gal-COS elution. Fraction 2 is the largest followed by fraction 3 and followed by fraction 4 and fraction 5 was the smallest fraction collected. LMW dextran (450,000-650,000), inulin, stachyose (DP 4), raffinose (DP3), and lactose (DP 2), were used as molecular weight standards. Chromatography was based on three independent reactions. Figure 3. Size exclusion results for different fractions of acetylated Gal-COS elution. Fraction 2 is the largest followed by fraction 3 and followed by fraction 4 and fraction 5 was the smallest fraction collected. LMW dextran (450,000-650,000), inulin, stachyose (DP 4), raffinose (DP3), and lactose (DP 2), were used as molecular weight standards. Chromatography was based on three independent reactions. Figure 4. Quantification of E. coli K88 ECL13795 binding to porcine erythrocytes with ELISA targeting K88 antibodies. ETEC were incubated with erythrocytes without addition of glycans (no glycan control), or with addition of 10 g/L of the carbohydrates as indicated on the x-axis. Results are reported as means ± standard deviation of three independent assays. The values of columns that do not share a common superscript differ significantly (P

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