Collagen Zymography as a Sensitive and SpecificTechnique for the Determination of SubpicogramLevels of Interstitial Collagenase

Shin ae Lee 13.04.20

※The use of casting native collagen type in SDS–polyacrylamide gel Ⅰ(collagen zymography) for the determination of interstitial collagenase

Contents

1. Introduction

2. Enzymes and methods

3. Result and discussion

4. Conclusion

IntroductionIntroduction

Native interstitial type I III collagensthe main fibrillar macromolecules of most connective tissueshighly resistant to degradation by nonspecific neutral proteinases

Collagenasecleaves interstitial collagen into two (3 /4 and 1/4 length) fragments which are susceptible to further proteolysis

a key role in the remodeling of interstitial collagens

ex)72kDa type Ⅳ collagenase(MMP2), 92kDa type Ⅳ collagenase(MMP9)can degrade, alone or in synergy, all the matrix macromolecules

Methods used to quantify collagenaselack sensitivity or specificity

ex) ELISA : cannot discriminate between active and inactive forms of enzyme

Substrate-containg polyacrylamide gel electrophoresis

Reliable technique for the resolution of 72- and 92-kDa type IV collagenases

could be also adapted to a quantitative assay

문제점the incorporation of native collagen fibrils in polyacrylamide gels

해결책First step, A detergent such as SDS could disrupt most of the fibrillar

organization of the polymer

Second Step, detergent exchange Triton X-100 vs SDS, during incubation could restore part of its original triple-helical conformation, allowing its degration by collagenase

IntroductionIntroduction

Enzymes and methodsEnzymes and methods

Materials•Human fibroblast collagenase, 72-kDa gelatinase, stromelysin•rabbit polyclonal antibody to collagenase•Zymogens were activated using 2 mM aminophenylmercuric acetate (progelatinase and procollagenase) for 3 h at 37°C and 8 h at 37°C for prostromelysin•Calf skin collagen type I (C 4530) and porcine skin gelatin (G 2500) •Stained rainbow molecular weight markers•interleukin-1b (IL-1) and 4-(2-aminoethyl)benzenesulfonyl fluoride (Pefabloc) ,Batimastat (a matrixin inhibitor )

Fibroblast culturesHuman gingival fibroblasts

Immunoassaysdot blot on nitrocellu- lose membrane and Western blot on polyvinyl difluor- ide membrane U =S (pixels) X gray level.

Zymography•Running gel : 10 % polyacrylamide gels contained 1 mg/ml of gelatin or 0.2–1 mg/ml collagen •Stacking gel : 4% polyacrylamid gel•Run under Laemmli conditions (24 mM Tris, 192mM glycine, 3.47 mM SDS; 40 mA, 1 h)•washed twice in 200 ml of 2.5% Triton X-100•stirring and incubated in 100 mMTris–HCl, 5 mM CaCl2 , 0.005% Brij-35, 0.001% NaN3 , pH 8.0 for 6–48 h at 37°C.•Staining and destaing•the gels were incubated for 1 h in 5% methanol, 7.5% acetic acid and kept under cellophane.

Quantification•CFR 126 video camera•Complete digestion of gelatin or collagen substrates corresponded to 255 gray levels and absence of digestion (of hydrolysis) corresponded to the background gray level with a 0 gray level. •U = Surface (pixels) 1 gray levels of the lysis band�

Enzymes and methodsEnzymes and methods

Results and DiscussionResults and Discussion

Influence of Substrate in the Migration of Proteins in Polyacrylamide Gels

FIG.1 Migration of standard protein markers▲control (10% polyacrylamide gel, m)

Lysozyme, trypsin inhibitor, carbonic anhydrase, ovalbumin, phosphorylase ,myosin

■gelatin (1 mg/ml in 10% polyacrylamide gel, j)●collagen (0.5 mg/ml, l). 0.1% SDS–10% PAGE.

500μg/ml collagen cocentration within gels induced a 9.6% reduction og Mr of the standard protein

Sensitivity of Collagen Zymography for Collagenase Quantification

FIG. 2. Collagen zymography of 50 pg of interstitial procollagenase.

6h : detected

Up to 36h : increased linearly with time

Results and DiscussionResults and Discussion

Results and DiscussionResults and Discussion

FIG. 3. Comparison of collagen zymograms obtained using a range of 10–50 pg procollagenase (A) Interstital procollagenase (57 kDa□ 52 kDa■) (B)APMA activated procollagenase

FIG. 4. Quantification of procollagenase using an dot-blot immunoassay

•Zymography : 0.1pg of collagenase discriminate pro and active form•Dot blot : 100pg of collagenase

Specificity of Collagen Zymography for Collagenase

FIG. 5. Specifity of the collagen zymography technique

(A) Lane A 300 pg of procollagenase depositedLane b, contained 10 mM EDTA;lane c, contained 2 mM 1,10 phenanthroline; lane d, contained 3 nM Batimastat;lane e, contained 2 mM Pefabloclane f, APMA-activated procollagenase; lane g, APMA-activated pro 72-kDa gelatinaselane h, APMA-activated prostromelysin; lane i: 72 kDa gelatinase.

(B) Lane j, APMA-activated procollagenase lane k, APMA-activated pro 72-kDa gelatinase; lane l, 72-kDa gelatinase.

Results and DiscussionResults and Discussion

Results and DiscussionResults and Discussion

Use of Collagen Zymography for Evaluating Influence of Interleukin 1 on Collagenase Expression by Human Gingival Fibroblast

FIG. 6. Influence of interleukin 1 on interstitial collagenase expression by cultured human gingival fibroblasts.

▷ Interleukin 1 :enhance collagenase expression from several cell types in culture

A similar extent of collagenase variation could be obtained using collagen zymography instead of immunoblot assay

•collagen zymography 21 ng/ml for control, 47 ng/ml for IL-1-treated cells•immunoblot assay19 ng/ml for control, 50 ng/ml for IL-1-treated cells

ConclusionConclusion

Collagen Zymography

: collagenase identification and quantification

Level as 0.1pg of active enzyme ,10pg of proenzyme were detected

The specificity of method for collagenase was demonstrated and its simplicity could justify broader laboratory and clinical use