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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(1-3)Gal(1-4)Glc
Gal(1-4)GlcNAc(1-6)
Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc
Gal(1-4)GlcNAc(1-6)
Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc
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(1-3)Gal(1-4)Glc
Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-6)
Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc
Gal(1-4)GlcNAc(1-3)
Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-6)Gal(1-4)Glc
Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-6)
Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc
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(1-3)GlcNAc(1-3)Gal(1-4)Glc
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)
20. Neu5Ac(α2-6)Gal(β1-4)GlcNAc(1-6)
GlcNAc(β1-3)Gal(β1-4)Glc
21. Neu5Ac(α2-6)Gal(β1-4)GlcNAc(1-6)
Gal(β1-3) Gal(β1-4)Glc
22. Gal(β1-4)GlcNAc(1-6)
Neu5Ac(α2-3){ Gal(β1-4)GlcGal(β1-4)GlcNAc(1-3)
23. Gal(β1-4)GlcNAc(1-6)
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
Time post partum (h)
6'S
L c
on
ce
ntr
ati
on
(g
/L)
0
0.1
0.2
0 24 48 72 96 120 144 168
Time post partum (h)
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