Glycolipid from lipoteichoic acid and composition comprising the same

This technology relates to Lipoteichoic acid(pLTA) isolated from Lactobacillus plantarum K8 (KCTC 10887BP) which is one of the safe GRAS strains. pLTA of this technology, as a major immunostimulating component in the cell wall of Gram positive bacteria, has immune regulation e�ects including anti-in�ammatory, prevention and treatment of sepsis, alleviate atopic dermatitis, anti-atherosclerosis, inhibition e�ects of collagenase by controlling the immune response(di�erentiation) of Th1/Th2 immune cells. Therefore, pLTA can be applied, as an immunomodulator, to foods, cosmetics and pharmaceuticals based on the aforementioned properties.

pLTA

Abstract

Dae Kyun Chung (Kyung Hee University, Professor)B.S. Seoul National University, Food science and technology. 1985M.S. Pennsylvania State University, Molecular biology. 1988Ph.D. Cornell University, Microbial genetics. 1991CEO. RNA Inc. Publications● Kim HG, Lee SY, Kim NR, Lee HY, Ko MY, Jung BJ, Kim CM, Lee JM, Park JH, Han SH, Chung DK. (2011) Lactobacillus plantarum lipoteichoic acid down-regulated Shigella �exneri peptidoglycan-induced in�ammation. Molecular Immunology 48, 382-391.● Jang KS, Baik JE, Han SH, Chung DK, Kim BG HG. (2011) Multi-spectrometric analyses of lipoteichoic acids isolated from Lactobacillus plantarum. Biochem Biophys Res Commun. 407, 823-30. ● Zeng RZ, Kim HG, Kim NR, Lee HY, Jung BJ, Ko MY, Lee SY, Chung DK. (2010) Protein Expression Changes in Human Monocytic THP-1 Cells Treated with Lipoteichoic Acid from Lactobacillus plantarum and Staphylococcus aureus. Mol Cells. 29, 585-594.● Ryu YH, Baik JE, Yang JS, Kang SS, Im J, Yun CH, Kim DW, Lee K, Chung DK, Ju HR, Han SH. (2009) Di�erential immunostimulatory e�ects of Gram-positive bacteria due to their lipoteichoic acids. Int Immunopharmacol. 9, 127-33.

Inventor information

E�cacy of LTA

Th1 Th2Th1

Th2

● allergy● septic shock

● atherosclerotic● gut & skin health

● Healthy Immune regulation1. Induction of Th1 cytokines (IL-12, IL-23)2. Inhibition of Th2 cytokines (IL-4, IL-10, TNF-α , IL-8)

pLTA

Anti-in�ammatory Prevention and treatment of sepsis

Alleviate atopicdermatitis

Anti-atherosclerosis Inhibition of collagenase

1

Patent information

● Kim HG, Lee SY, Kim NR, Ko MY, Lee JM, Yi TH, Chung SK, Chung DK. (2008) Inhibitory E�ects of Lactobacillus plantarum Lipoteichoic Acid (LTA) on Staphylococcus aureus LTA-induced Tumor Necrosis Factor-Alpha Production. J Microbiol Biotechnol. 18, 1191-6.● Kim HG, Kim NR, Gim MG, Lee JM, Lee SY, Ko MY, Kim JY, Han SH, Chung, DK. (2008) Lipoteichoic acid isolated from Lactobacillus plantarum inhibits lipopolysaccharide – induced TNF-alpha production in THP-1 cells and endotoxin shock in mice. J Immunol. 180, 2553-61.● Kim HG, Gim MG, Kim JY, Hwang HJ, Ham MS, Lee JM, Hartung T, Park JW, Han SH, Chung DK. (2007) Lipoteichoic acid from Lactobacillus plantarum elicits both the production of interleukin-23p19 and suppression of pathogen-mediated interleukin-10 in THP-1 cells. FEMS Immunol Med Microbiol. 49,205-14.

Fig. 1 Immune activities of LTA is controlled by the structure of glycolipid, i.e. glucose units or length and number of fatty acid chains thereof, of which di�erences from glycolipids of S. aureus(aLTA) and L. plantarum(pLTA), which have the induction of excessive in�ammatory responses and the inhibition of in�ammatory responses respectively, was con�rmed.

2

PCT patent application Patent titlePriority NumberAssigneeStatus

PCT/KR2010/05755(WO11/025286)Glycolipid from lipoteichoic acid and composition comprising the sameKR2010-008319(2010.08.26)RNA Inc.Entered the PCT national phase in the United States

Key Technology Highlights

Structure analysis of glycolipid of pLTA

Fig. 2 (a) It showed that Expression of TNF-α decreases as the pretreatment concentration in pLTA-pretreated cell increases.(b) Peptideglycan induced TNF-α and inhibition of expression of IL-1β by pretreatment of pLTA are accompanied by the inhibition of expression of NOD(receptor related sepsis).

Anti-in�ammatory e�ect of pLTA

840

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

860 880 900 920 940 960 980 1000 1020 1040 1060

873.884

887.884

901.894

915.961

929.963

957.962

971.981

986.0431000.018

1014.0631027.959

1041.949

944.000

(1) MALDI-TOF MS spectrum of glycolipid from S. aureus

Saturated fatty acidGlc2-DAG structure;diglucosyl diacylglycerol-saturated fatty acid

m/z

(2) MALDI-TOF MS spectrum of glycolipid from L. plantarum

1080 1100 1120 1140 1160 1180 1200 1220 1240 1260

1103.771

1117.8361115.919

1129.832

1143.888

1157.856

1171.880

1145.799

1199.854

1185.840

1213.8601227.887

1241.874

1139.638

m/z

1.91.81.71.61.51.41.31.21.11.00.90.80.70.60.50.40.30.20.10.0

mono-unsaturated fatty acidsdi-unsaturated fatty acidsGlc3-DAG structure;triglucosyl diacylglycerol-unsaturated fatty acid

O

OH

OHHO

HO

HOHO

HOO

OO Cx

Cy

O

O

OO

OH

OH

OH

HO

HOHO

HOHO

HO

HO CxCy

O

OO

O

O O

OOOO

2000

1500

1000

500

0

TNF-

α pg

/ml

0.0 0.1 1.0 10.0 100.0

pLTAglycolipid

㎍/ml

2nd: 10㎍/ml �exPGN for 4h

pretreatment:

(a) (b)

300

250

200

150

100

50

0

rel.

NO

D2

mRN

A/G

APD

H

C 0 1 10

*

100

pretreatment; pLTA ㎍/ml

2nd �exPGN 10㎍/ml

Fig. 5 The concentration-dependent inhibition of production MMP-1 (collagenase) The was shown in the sample treated with pLTA when radiated with UV of 75mJ, compared to the control group.

Fig. 6 The production of Reactive Oxygen Species (ROS) was inhibited in the sample treated with pLTA due to reduction of �uorescence, compared to the non-treated control group.

3

Alleviation e�ect of atopic dermatitis by treatment of pLTA

Inhibition e�ect of production of collagenase

Fig. 3 In case of the mice pretreated with PBS, it showed that all mice died within 48 hours when sepsis inducer was injected intraperitioneally. On the other hand, as for the mice pretreated with pLTA, it showed that survival rates was approximately 90% up to four days after and the amount of TNF-α was signi�cantly low.

Fig. 4 Atopic dermatitis was observed to be mitigated by injecting pLTA in mice with atopic dermatitis, and epidermal thickness was restored to its original state.

Prevention and treatment e�ects of sepsis

16

14

12

10

8

6

4

2

0

Surv

ival

(EA

)

injection

day

1 2 3 4

PBSLTA

(a) (b)1500

1000

500

0

TNF-

α pg

/ml

PBS

**

LTA (pretreatment)

n=5

2nd aLTA+MDP

Before treatedNegative control pLTA treated After treated

1

UV 0 10 20 30 40 50 75 100 (ml/cm2)

MMP-1

LTA 0 0.1 1 10 100 (㎍/ml)

MMP-1

after UV radiation

UV LTA+UV

4

K-LAC 8 is a commercialized product made of cellular extract of L.plantarum K8 that contains pLTA by RNA process. In advance, clinical trials for functional food registering would be progressed as immune regulator, intestine & skin health ingredients.

1. RNA process: culture → disruption → adsorption/mix → dry2. pLTA content of K-LAC 8 : 0.17 ~ 0.35 mg/g3. E�cacy: e�cacy of K-LAC 8 is higher than live cell (inhibition of pro-in�ammatory cytokine induction)

Commercialization status

Comparison with other strains: Shows that an ability for inhibiting pro-in�ammatory cytokines induction is higher than other lactic acid bacteria, whereas the lowest induction of pro-in�ammatory cytokine induction

* K-LAC 8 ; K-LAC of L.plantarum K8 Active Compound produced by RNA process

Comparative analysis

4500

4000

3500

3000

2500

2000

1500

1000

500

0

hTN

F-α,

pg/

ml

N LPS 10^8

LiveK-LAC 8

+ LPS

(CFU/ml)

pro-in�ammatory cytokine induction(hTNF-α, pg/ml)

Inhibition of pro-in�ammatory cytokine induction (hTNF-α, %)

Inhi

biti

on R

ate

(%) 60.00

50.0040.0030.0020.0010.00

0.00Live K-LAC 8

3000

2000

1000

0K-LAC 8 L.sakei L.fermentum L.acidophilus

24.32

51.35

80.0

60.0

40.0

20.0

0.0K-LAC 8 L.sakei L.fermentum L.acidophilus