Potential utilization of milk oligosaccharides of cows and other domestic farm animals Tadasu...

Potential utilization of milk oligosaccharides of cowsand other domestic farm animals

Tadasu Urashima1, Takashi Terabayashi2, Minoru Morita3

1Obihiro University of Agriculture and Veterinary Medicine, 2Kitasato University, and 3Toko Pharmaceutical Industries Co.,Ltd.

What are lactose and milk oligosaccharides?

Human milk1. Lactose 6%2. Lipids 3.5%3. Milk oligosaccharides 1.2∼1.3%4. Proteins 1.1%5. Ash 0.2%

Human milk oligosaccharides12∼13 g/L in mature milk22∼24 g/L in colostrummore than 200 varietiesmore than 100 characterized structures

Lactose and milk oligosaccharides

β1,4α2,3

Sialyllactose

β1,4 β1,3 β1,4

Lacto-N-neotetraose

Lactose

β1,4

β1,4

β1,6

β1,3

β1,4

Lacto-N-hexaose

Fucosyllactose

α1,2 β1,4

: Galactose

: Glucose

: Fucose

: Sialic acid

: N-acethylglucosamine

β1,4

Bio functions of human milk oligosaccharides

1. Anti infection as receptor analogues

Campylobacter jejuni, Entamoeba histolytica,Streptococcus pneumoniae, enteropathogenic E. coli,Uropathogenic E. coli, enterotoxigenic E. coli, etc

2. Prebiotics

3. Immuno modulating factor etc

Gal(1-4)Glc

Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc


Gal(1-4)GlcNAc(1-6)


Gal(1-4)GlcNAc(1-6)


Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc


Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-6)


Gal(1-4)GlcNAc(1-3)


Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-6)


Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc

LactoseLacto-N-tetraoseLacto-N-neotetraoseLacto-N-hexaose

Lacto-N-neohexaose

para-Lacto-N-hexaose

para-Lacto-N-neohexaose

Lacto-N-octaose

Lacto-N-neooctaose

iso-Lacto-N-octaose

para-Lacto-N-octaose

Gal(1-4)GlcGal(1-4)GlcNAc(1-6)

Gal(1-3)GlcNAc(1-3)

Gal(1-4)GlcNAc(1-6)

Gal(1-3)GlcNAc(1-3) Lacto-N-decaose

Gal(1-4)GlcNAc(1-6)


Gal(1-4)GlcNAc(1-6)

Gal(1-4)GlcNAc(1-3) Lacto-N-neodecaose

The 13 core structures of human milk oligosaccharides

Structures Name

Bovine milk oligosaccharides (BMOs)

More than 1 g/L exists in colostrum immediately post partum

The concentration rapidly decreases 48 hr after partum to very low level

Around 40 varieties of structures have been characterized

Flow of this lecture

1. Historical aspects of BMO study

2. BMO chemical structures

3. Quantitative aspect of BMO

4. Our project of bovine colostrum (April, 2005 ～ March, 2010)

5. Potential utilization of bovine milk oligosaccharides

Acidic BMO Kuhn and Gauhe (1965)

Neu5Ac(α2-3)Gal Neu5Ac(α2-6)Gal(β1-4)Glc (6’-SL) Neu5Gc(α2-3)Gal(β1-4)Glc Neu5Ac(α2-6)Gal(β1-4)GlcNAc (6’-SLN) Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(β1-4)Glc (DSL)

Cumar et al. (1965) Gal(β1-4)Glc-3’-PO4

Schneier and Rafelson (1966) Neu5Ac(α2-3)Gal(β1-4)Glc (3’-SL)

Veh et al. (1981) Neu5Gc(α2-6)Gal(β1-4)Glc Neu5Gc(α2-6)Gal(β1-4)GlcNAc

Parkkinen and Finne (1985, 1987)

Neu5Ac(α2-6)Gal(β1-4)GlcNAc-1-PO4

Neu5Ac(α2-6)Gal(β1-4)GlcNAc-6-PO4

Neutral BMO

Saito et al. (1985)

GalNAc(β1-4)Glc

Gal(β1-4)GlcNAc (N-acetyllactosamine)

Saito et al. (1987)

Gal(β1-3)Gal(β1-4)Glc (β3’-GL)

Gal(β1-6)Gal(β1-4)Glc (β6’-GL)

Gal(β1-4)[Fuc(α1-3)]GlcNAc

Urashima et al. (1991)

Gal(α1-3)Gal(β1-4)Glc (isoglobotriose)

GalNAc(α1-3)Gal(β1-4)Glc

Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose 1)

These oligosaccharides have never been found in human milk.Isoglobotriose is a major neutral BMO.

GalNAc(α1-3)Gal(β1-4)Glc

Urashima et al. Biochim. Biophys. Acta 1073, 225 (1991)

13C-NMR of GalNAc(α1-3)Gal(β1-4)Glc, separated from bovine colostrum

Recent study of BMO structures

Method for milk oligosaccharide profiling by 2-aminobenzamideLabeling and hydrophilic interaction chromatography

K. Marino, J.A. Lane, J.L. Abraham, W.B. Struwe, D.J. Harvey,M. Marotta, R.M. Hickey, P.M. Rudd

Glycobiology 21, 1317, 2011

Neutral Bovine Milk Oligosaccharides Characterized by Marino et al

1. GalNAc(1-4)GlcNAc

2. GalNAc(1-4)Glc

3. Gal (1-4)GlcNAc (N-acetyllactosamine)

4. Fuc(α1-2)Gal(β1-4)Glc (2’-FL) 5. GalNAc(α1-3)Gal(β1-4)Glc

6. GlcNAc(β1-3)Gal(β1-4)Glc

7. Gal(1-6)Gal(β1-4)Glc (β6’-GL)

8. Gal(α1-3)Gal(β1-4)Glc (isoglobotriose)

9. Gal(β1-3)Gal(β1-4)Glc (β3’-GL)

10. Gal(β1-4)Gal(β1-4)Glc (β4’-GL)

12. Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc (lacto-N-neotetraose)

11. Fuc(α1-2)Gal(β1-4)Glc

GalNAc(α1-3)

13. Gal(1-4)GlcNAc(1-6)Gal(1-4)Glc

GlcNAc(β1-3)

14. Gal(1-4)GlcNAc(1-6)Gal(1-4)Glc (lacto-N-novopentaose 1) Gal(β1-3)

15. Gal(1-4)GlcNAc(1-6)Gal(1-4)Glc (lacto-N-neohexaose) Gal(1-4)GlcNAc(1-3)

Acidic Bovine Milk Oligosaccharides Characterized by Marino et al.

16. Neu5Ac(α2-3)Gal(β1-4)GlcNAc(1-3)Gal(β1-4)Glc

17. 　 Neu5Ac(α2-6)Gal(β1-4)GlcNAc(1-3)Gal(β1-4)Glc

24. 　 Neu5Ac(α2-6)GalNAc(1-4) GlcNAc

25. 　 Neu5Ac(α2-3)Gal(1-4) Glc (3’-SL)

18. 　 Neu5Ac(α2-3)Gal(β1-4)GlcNAc(1-6)

GlcNAc(β1-3)Gal(β1-4)Glc

19. Gal(β1-4)GlcNAc(1-6)

　　 Neu5Ac(α2-3)｛ Gal(β1-4)GlcGal(β1-3)


GlcNAc(β1-3)Gal(β1-4)Glc


Gal(β1-3) Gal(β1-4)Glc


　　 Neu5Ac(α2-3)｛ Gal(β1-4)GlcGal(β1-4)GlcNAc(1-3)


　　 Neu5Ac(α2-6) Gal(β1-4)GlcGal(β1-4)GlcNAc(1-3)｛

Acidic Bovine Milk Oligosaccharides Characterized by Marino et al.

26. 　 Neu5Gc(α2-3)Gal(1-4)Glc

27. 　 Neu5Ac(α2-6)Gal(1-4)GlcNAc (6’-SLN)

28. Neu5Gc(α2-6)Gal(1-4)GlcNAc

29. Neu5Ac(α2-6)Gal(1-4)Glc (6’-SL)

30. Neu5Gc(α2-6)Gal(1-4)Glc

32. Neu5Ac(α2-3)Gal(1-4)Gal(β1-4)Glc

34. Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(β1-4)Glc (DSL)

35. Neu5Gc(α2-8)Neu5Ac(α2-3)Gal(β1-4)Glc

36. Neu5Ac(α2-8)Neu5Gc(α2-3)Gal(β1-4)Glc

37. Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(β1-4)GlcNAc

31. Neu5Ac(α2-6)Gal(1-4)Glc

GlcNAc(1-3)

33. Neu5Ac(α2-6)Gal(1-4)Glc

Gal(1-3)

Differences between the milk oligosaccharides of human and cows

HMOs22 ～ 24 g/L in colostrum

12 ～ 13 g/L in mature milk

more than 200 varietiesGal(β1-3)GlcNAc containing HMO (type ) >Ⅰ Gal(β1-4)GlcNAc containing HMO (type )Ⅱ

neutral HMO > acidic HMO

The ratio of fucosyl oligosaccharides to total HMOs is high

×Gal(α1-3)Gal(β1-4)Glc

×Neu5Gc containing HMO

×GalNAc(α1-3)Gal(β1-4)Glc×Sialyl galactosyllactose

BMOsmore than 1 g/L in colostrumsmall concentration in mature milkaround 40 varieties

○ type BMOs, × type BMOsⅡ Ⅰ

acidic BMO > neutral BMO,70% ; Neu5Ac(α2-3)Gal(β1-4)GlcThe concentration of fucosyl oligosaccharides is very low

○ Gal(α1-3)Gal(β1-4)Glc, GalNAc(α1-3)Gal(β1-4)Glc

○Neu5Gc containing BMO

○Sialyl galactosyllactose

Minor fucosyl BMOs characterizaed in recent study

Annotation and structural elucidation of bovine milk oligosaccharides and determinationof novel fucosylated structures

D.L.Aldredge, M.R.Geronimo, S.Hua, C.C.Nwosu, C.B.Lebrilla, D.Barile

Glycobiology 23,664,2013

H2N1F1

H4N1F1

H3N2F1

Fuc

HexNAc GlcGal

HexNAcGal Gal Gal

Fuc

Glc

HexNAc HexNAc

Fuc

Gal

Gal

Glc

R. Mehra, D. Barille, M. Marotta, C.B. Lebrilla, C. Chu, J.B. German PLOS one, 9, e96049, 2014

Novel high-molecular weight fucosylated milk oligosaccharides identified in dairy streams

H3N4F1, H4N4F1, H3N5F1, H5N4F1, H4N5F1, H3N6F1

Quantitative aspect of BMOs

Time post partum (h)

0

0.2

0.4

0.6

0.8

1.0

1.2

0 24 48 72 96 120 144 168

3’S

L c

on

ce

ntr

ati

on

(g

/L)

0

0.1

0.2

0 24 48 72 96 120 144 168


6'S

L c

on

ce

ntr

ati

on

(g

/L)

0

0.1

0.2

0 24 48 72 96 120 144 168


6'S

LN

co

nc

en

tra

tio

n (

g/L

)

Changes in the concentrations of individual sialyloligosaccharides of bovinecolostrum during early lactation. 3’SL, Neu5Ac(α2-3)Gal(β1-4)Glc; 6’SL, Neu5Ac(α2-6)Gal(β1-4)Glc; 6’SLN, Neu5Ac(α2-6)Gal(β1-4)GlcNAc. Values areindicated as means±SD (n=4).

T. Nakamura et al., J. Dairy Sci., 86, 1315, 2003

Bovine milk oligosaccharide analysis by HPLC-HRSRM: typical total ion current trace of oligosaccharideStandards (a) followed by their extracted ion chromatograms (c-f) and total ion current trace of bovineMilk oligosaccharide (b) followed by the extracted ion chromatograms (g-j).

B. Fong, K. Ma & P. McJarrow: J. Agric. Food. Chem., 59, 9788, 2011

3’SL ( mg/L) 6’SL (mg/L) 6’SLN ( mg/L) DSL ( mg/L) GNL ( mg/L)

Skim milk 1 51 ± 4 (30,9%) 6.3 ±0.4 (8,6%) 0.13 ± 0.02 (2,14%)a 1.5 ± 0.1 (2,6%) 2.6 ± 0.3 (2,10%)a

Skim milk 2 55 ± 4 (2, 6%) 9 ±0.2 (2, 2%) 0.10 ±0.02 (2, 17%)a 2.1 ± 0.3 (2, 14%) 3.4 ± 0.4 (2, 13%)a

Homogenized milk 48 ± 4 (6, 8%) 9.6 ± 0.8 (3, 8%) 0.1 ±0.03 (3, 14%)a 3.1 ± 0.2 (3, 6%) 2.4 ± 0.1 (3, 2%)a

Unpasteurized milk 47 ± 4 (6, 9%) 3.6 ±0.3 (2, 9%) <LOD (2) 0.54 ± 0.01 (2,14%)a <LOD (2%)

Mature milk

(Martin-Sosa et al.) 94-119 67-88 145-176 41-77

Mature milk

(?g/mL, McJarrow and van

Amelsfort-Schoonbeek)

35-50 14-25 9-12 2-7 3-4

Mature milk

(7 d post partum, Nakamura et al.) 30 25 12 ND

Colostrum (second milking) 1245 ± 82 (7,

7%)

85 ± 6 (7, 7%) 119 ± 7 (7, 6%) 126 ± 8 (2, 8%) 1 ± 0.1 (2, 12%)a

Colostrum (fourth milking) 739 ± 53 (5, 7%) 73 ± 2 (5, 2%) 117 ± 10 (5, 8%) 80 ±7 (2, 9%) 1 ± 0.1 (2, 10%)a

Colostrum

(Martin-Sosa et al.) 354 147 210 135

Concentrations of Oligosaccharides in Samples of Bovine Milk, Bovine Colistrum, and Infant Formula Number of samples and coefficient of variation in parentheses

“Development of the innovative utilizations of bovine colostrum components”

Financially supported by the Research and Development Program for New Bio-Industry Initiatives, the Bio-oriented Technology Research Advancement Institution (BRAIN), National Agriculture and Food Research Organization (NARO), Japan

April, 2005 ～ March, 2010

1. Obihiro University of Agriculture & Veterinary Medicine, Graduate course of Food Hygiene

2. Obihiro University of Agriculture & Veterinary Medicine, Protozoa Disease Institution

3. Kitasato University

4. Toko Pharmaceutical Industries Co.Ltd

5. National Animal Hygiene Institution

1. Anti influenza activity by the condensation products of sialyl oligosaccharides from bovine colostrum with fatty acids

Dr. Takashi Terabayashi (Kitasato University)Dr. Minoru Morita (Toko Pharmaceutical Industries Co. LtD)

2. Biofunctional proteins from bovine colostrum

Bovine colostrum odrant binding protein

Dr. Kenji Fukuda (Obihiro University of Agriculture & Veterinary Medicine)Poster :

Binding of influenza A virus to the host cell via sialyl-galactosyl glyco-chain on the receptor

Hemagglunitin(HA)

Neuraminidase (NA)

Human influenza A virusAvian influenza A virus

Neu5Aca2-3Gal- Neu5Aca2-6Gal-

Neu5Ac-Gal-

OH

O

HN

O

O

OH

OOH

O

OH

O

COOH

OHAcHN

HO OH

HOOH

OOH

HO

n

Competitive inhibition against binding of the viral HA to the receptor sugar chain

O

OH

OOH

O

OH

O

COOH

OHAcHN

HO OH

HOOH

O

OH

OH

HO

Anti-influenza virus activity

Anti-influenza virus activity

3’-sialyllactose(3’-SL)

Ganglioside GM3

Neu5Aca2-3Galb1-4Glcb1-Cer

Neu5Aca2-3Galb1-4Glc -NH-C12

[Terabayashi T., Carbohydrate Res., 341 (2006) 2246-2253]

Ganglioside analogue ( single tailed amphiphile ) 　

New type !

Anti-influenza virus reagent

O

OH

OOH

O

OH

O

COOH

OHAcHN

HO OH

HOOH

O

NH

OH

HOOC

O

OH

OOH

O

OH

O

COOH

OHAcHN

HO OH

HOOH

O

NH2

OH

HO

O

OH

OOH

O

OH

O

COOH

OHAcHN

HO OH

HOOH

O

OH

OH

HO

* DMT-MM; 4-(4,6-dimathoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride

amination

acyl amidation

3’- and/or 6’- sialyllactoses were substituted

by an amino group in saturated NH4HCO3 / H2O,

with starring for four days at room temperature.

removal of NH4HCO3 by rotary evaporation

acyl amidation with long chain fatty acids

( CH3(CH2)nCOOH; n = 12 ~ 18),

using a reagent for amidation, DMT-MM*

N-3’ –sialyllactosyl-mylistinamide (3’-SL-N-C14)

3’ -sialyllactose (3’- SL)

3’ -sialyllactosylamine (3’ –SL-NH2)

Synthesis of N-sialyllactosyl-acylamide

Inhibitor IC50 (mg/ml)

3’-SL-N-C12 286.5

3’-SL-N-C14 56.5 ± 21.9

3’-SL-N-C16 22.5 ± 4.7

3’-SL-N-C18 4.0

6’-SL-N-C14 99.4 ± 51.6

6’-SLN-N-C14 85.3 ± 11.1

Oseltamivir1 0.9 ± 0.13

Zanamivir 2 0.5

Amantadine 3 30.9

IC50 of N-sialyllactosyl-acylamide

1 Tamiful® (Roche); oseltamivir phosphate 2 Relenza® (GlaaxoSmithKline); zanamivir hydrate 3 1-Aminoadamantane (Aldorich)

Anti-influenza virus activity was evaluated by plaque reduction assays, using MDCK (Madin-Darby canine kidney) cells and Influenza virus A/PR/8/34 (H1N1).

The anti-influenza virus 　 A/PR/8/34 　 effect of each sialyllactosyl-acylamide substance (Survival ratio)

100

7

50

Su

rviv

al (

%)

Days after infection

5 60 1 2 3 4 8 9

3'-SL-N-C14

6'-SL-N-C14

6'-SLN-N-C14

3'-SL-N-C18

Oseltamivir

The 1 mg/kg of each substance was administered to mice twice per day.

3’-SL-N-C14

3’-SL-N-C18

6’-SL-N-C14

6’-SLN-N-C14

oseltamivir

PBS

0

5000

10000

15000

The action of sialyllactosyl-acylamide substance to the viral replication in the lung tissue of influenza virus A/PR/8/34 infection mice

(PFU/ml)

Potential utilization of bovine milk oligosaccharides

2. Anti infection agentsAgainst Campylobacter jejuniJ.A. Lane et al., Int. J. Food Microbiol. 157, 182, 2012

Against Escherichia coli P1422J.A. Lane et al., J. Microbiol. Methods 90, 53, 2012

Against rotavirusS.N. Hester et al., Br. J. Nutr. 110, 1233, 2013

1. Separation of oligosaccharides from bovine colostrum or lactose removedcheese whey using membrane technology

R. Mehra et al., PLOS one, 9, e96049, 2014

　　　　 3. Immuno modulating agents

　　　　 Transcritional response of HT-29 intestinal epithelial cells　　　　 J.A. Lane et al., Br. J. Nutr. 110, 2127, 2013

　　　　 Activation for intestinal VD11C ＋ cells

　　　　 E. Kurakevich et al., PNAS, 110, 17444, 2013

　　　　 4. Stimulating agents for maturation of intestinal epithelial cells　　　　 H.D. Holscher et al., J. Nutr. 144, 586, 2014

Thank you

Potential utilization of milk oligosaccharides of cows and other domestic farm animals Tadasu...

Documents

Transcript of Potential utilization of milk oligosaccharides of cows and other domestic farm animals Tadasu...