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A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesi

s of infectious colitisKristina K. Hansen, Philip M. Sherman, Laurie Cellars, Patricia Andrade-Gordon, Zhengying Pan, Amos Baruch,John L. Walla

ce, Morley D. Hollenberg, and Nathalie Vergnolle

PNAS June 7, 2005 vol. 102 no. 23 8363–8368

生物科技學系四年級邱英哲

91390330

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Escherichia coli Pathogen Citrobacter rodentium

( C. rod. )

Human Host Murine Release extracellular serine proteinase Mechanism ？

ResultPAR2 activation ？

Abstract

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Abstract1. Proteinase-activated receptor ( PAR ) Family

PAR1~4

2. PAR2’s activation induces acute inflammation

proteinase

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Proteinase

Cleavage the N-terminal

PAR2 Activation

G protein Activation

Ion channel Activation

Ion mobilization

Ca2+ mobilization

Inhibitor

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Experimental Procedures

1.mice : six-week-old C57BL/6 wild-type mice、 PAR2-deficient ( - / - )C57BL/6 background2.C. rodentium 107 CUF in 0.1ml or equal volume of broth (Control)3.soybean trypsin inhibitor ( STI ) treatment : orally gavaged(100μl) with

40mg/day STI4.Colon tissue : flushed with 0.5 ml PBS pH 7.4

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1. Reference : a. C. rodentium release the trypsin-like proteinaseb. Using the substrate, Boc-Gln-Ala-Arg-AMC for testing the

proteinase activity 2. Hydrolysis rate over 20 min at RT

luminal fluid 5μlsubstrate solution 200 μl

3. Determined by using a Fluoroskan Ascent microplate fluorometer (螢冷光儀 )

Trypsin-like activity

7FIG. 1.

p<0.05, n=8 in each group

(Control)

Sham Sham Sham Inhibitor

Substrate hydrolysis

C. rod.- 較 sham- 有 trypsin-like activity

Inhibitor 明顯降低 proteinase activity

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Proteinase

Cleavage the N-terminal

PAR2 Activation

G protein Activation

Ion channel Activation

Ion mobilization

Ca2+ mobilization

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1. Kirsten virus-transformed rat kidney (KNRK) cell vector-transfected with rat PAR2 (KNRK-PAR2) 10≒ 6 cell/ml

2. Homogenates : 30-40 mg of colonic tissue in 1.5 ml 3. Using calcium ionophore A23187 (2μl) as a standard ( ac

tivated PAR2 )4. Desensitization of PAR2 ： by using SLIGRL-NH2

( activated PAR2 )

Calcium mobilization

Sample(s)

calcium ionophore A23187= %

10FIG. 2.

n=12 n=26 n=3 n=4n=4

29±6 ％

12±7 ％

p<0.05 代表 protease 會引起 Calcium mobilization

11FIG. 2.

2nd1st

5 min

E530: Calcium-mediated fluorescence

代表 SLUGRL-NH2 和 C. rod.- 作用在同一處 PAR2

12FIG. 2.

Direct correlation between the trypsin-like activity and degree of calcium signaling in PAR2-transfected KNRK cell

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Identified the activating proteinase(s)

1. Using fluorescent in-gel assays

2. To identify the molecular masses and substrate cleavage by proteinase with trypsin-like

14FIG. 3.

60kDa

Control

24kDa

SDS PAGE有可能的 proteinases 大約是 60kDa 和 24kDa

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Purify of C. rodentium-infected luminal fluid : By affinity chromatography and

STI-agarose column Mass spectral analysis of eluent fraction granzyme A kallikrein B (serine protease) trypsinogen 16 Tryptic digestion、mass fingerprinting、

sequencing of tryptic peptides 、 databaseseaching and protein identification：trypsin 3、 trypase 4 trypsinogen 9、 trypsinogen11trypsinogen 16

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FIG. 3.

28 kDa

ABP labling

• SDS / PAGE gel and Western blotting techniques

• Activity-based probe (ABPs) identify serine proteinases

unstable

C. rod.- sample 中帶有 serine protease 但 size 大小不完全相同

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Western blotting techniques to detect the size of proteinases

FIG. 3.

expression

expression代表 C. rod.- 有 Trypsin 和 Granzyme A

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The activity of Granzyme A• Using P20 peptide, corresponds to cleavage/activation site

of the wild type – rat PAR2

30GPNSKGR↓SLIGRLDT45P-YGGC

(↓ trypsin cleavage site )

• Treatment with P20

Reactant ％ n Time

Sham-infected 0 n=4 2 hr

Granzyme A 22±2 n=3 20 hr

Luminal fluid 94±4 n=4 2 hr

Positive control(trypsin) 94±2 n=3 20 min

Granzyme A activates PAR2

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Inflammatory indicesA

B

p<0.05, n=8 in each group

FIG. 4.

20FIG. 4.

C Inflammatory indices p<0.05, n=8 in each group

D

Myeloperoxydase (MPO)

Stand for the granulocyte filtration

After 10 days infection, bacterial translocation

Inflammatory 和 PAR2 有關

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Conclusion

Major findings of this study

1. Enteric bacterial infection can liberate PAR2-

activating proteinases in Vivo

2. The presence of PAR2 and proteolytic activity in

the colon can play a major role in host

inflammatory response to enteric bacterial

infection