zymography

Zymography

Prepared by: Krisztina Kupai2011

Theoretical course: Basic biochemical methods and ischemic heart models

Supported by: HURO/0901/069/2.3.1 HU-RO-DOCS

Definition of Zymography

• Zymography is an electrophoretic technique, based on SDS PAGE for measuring enzyme activity

• The technique is particularly useful for analyzing the proteinase composition of complex biological samples

• Zymography is also widely used to study various aspects of matrix metalloproteinase (MMP) function

Prepared by Krisztina Kupai

Types of zymography

– SUBSTRATE ZYMOGRAPHY:• Gelatin Zymography• Casein Zymography• Collagen Zymography• Heparin-Enhanced Substrate Zymography

– REVERSE ZYMOGRAPHY– In Situ ZYMOGRAPHY


SUBSTRATE ZYMOGRAPHY: when specific substrate is co-polymerized with the

acrylamide • In zymography, the proteins are separated by

electrophoresis under denaturing [sodium dodecyl sulfate (SDS)], nonreducing conditions

• The separation occurs in a polyacrylamide gel containing a specific substrate that is co-polymerized with the acrylamide


Source:http://www.invitrogen.com

SUBSTRATE ZYMOGRAPHY:gelatin or casein in the gel

• Gelatin Zymography– Gelatin zymography is mainly used for the

detection of the gelatinases, MMP-2 and MMP-9

• Casein zymography – suitable for the detection of MMP-1, MMP-7,

MMP-12, and MMP-13


• Collagen zymography – used for the detection of MMP-1 and MMP-13,

but MMP-2 and MMP-9 can also be detected – The incorporation of native collagen fibers in

polyacrylamide gels appears unsuitable for zymography because of their complicated structure, but SDS disrupts most of the fibrillar organization of the collagen, allowing proteins to run into the gel.


SUBSTRATE ZYMOGRAPHY: collagen in the gel

Heparin-Enhanced Substrate Zymography

• It is known that the extraction of MMPs from tissue in the presence of heparin results in an enhancement of MMP activity.

• The addition of heparin to the samples during or prior to electrophoresis also enhances MMP activity.

• Used for MMP-7• The mechanisms by which heparin seem to enhance MMP-7

activity in zymography are:– (i) the induction of a conformational change– (ii) the facilitation of refolding – (iii) the reduction of autolysis– (iv) the increase of anchorage of the MMP

in the gel


Reverse Zymography• TIMPs can be detected by reverse zymography,

which is a modification of zymography for MMPs

• Besides gelatin, an MMP is also incorporated into the gel, usually MMP-2.

• During the activation step after electrophoresis, the MMP-2 only digests the gelatin in areas where TIMPs are absent.

• Thus, after staining, the gel will be colorless, except for the TIMP bands


In Situ ZYMOGRAPHY

• In situ zymography allows the localization of MMPs in tissue sections

• in situ zymography uses a substrate that is deposited on or under a frozen section of an unfixed tissue sample

• During incubation, the substrate will be digested by the activated MMPs in a time- and dose-dependent manner

• The degradation of the substrate is detected by light microscopy or fluorescence microscopy, depending on the type of substrate. Prepared by Krisztina Kupai

In Situ ZYMOGRAPHY


The red and green signal indicates the immunolocalization of MMP-2

Main steps in gelatin substratezymography:


1. Sample homogenization without a reducing agent or boiling in order for the enzyme to retain its native state (and therefore its proteolytic activity).

2. Preparation of a gel with a final concentration of acrylamide of 8%,containing 2 mg/mL gelatin3. Electrophoresis at 90 V constant voltage (no boiling, no use ofreducing agents in order to preserve enzyme activity)

• 4. Gel washing in Triton X-100 for 40 min at room temperature on an orbital shaker

• 5. Gel washing in incubation buffer for 20 min• 6. Incubation of gels for 20 h at 37 °C 7. Staining of the gels with

Coomassie Blue for 30 min at room temperature. Areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme

• 8. Destaining with a methanol/acetic acid solution for 1 h at room• temperature• 9. Densitometry of gelatinolytic activity appearing as clear bands on• the dark background


Main steps in gelatin zymography:

MMP-9proMMP-2MMP-2

Advantages of substrate Zymography

• Expensive materials are not required (e.g., antibodies) • Proteases with different molecular weights showing

activity towards the same substrate can be detected and quantified on a single gel. – For example, MMPs are released from cells in a

proteolytically inactive proform (zymogen) which is approximately 10 kD larger than the activated form

– Because the proform becomes activated during the process of denaturation and renaturation after gel electrophoresis, the active form and the originally inactive forms degrade gelatin, and both forms can therefore be detected on zymograms.


• MMPs in solution are often associated with endogenous tissue inhibitors of metalloproteases(TIMPs).

• During electrophoresis the inhibitors dissociate from the MMP and do not interfere with detection of the enzymatic activity.

• On the other hand, sandwich ELISA can discriminate between MMP/TIMP complexes and free MMPs, resulting in determination of a potential active fraction (Zucker et al. 1992; Ratnikov et al. 2002; Catterall and Cawston 2003).



• On the basis of molecular weight markers, the molecular weight of the proteolytic band can be determined, and by comparison with recombinant proteins and the use of specific protease inhibitors the type of protease can be established



Information on the localization of the proteolytic activity in cells or tissues cannot be obtained

on the basis of zymography.


Disadvantages of substrate Zymography

MMPs Introduction• MMPs were discovered in

1962 (ECM degradation is requiered for resorption of

the tadpole tail)

• Play important roles:

Physiological processes:

• embriogenesis

• angiogenesis

• activation of cell surface receptors

Pathological processes:

• tumour metastasis

• inflammation

• arthritis

• Cardiovascular diseases

1. atherosclerosis

2. heart failure

3. aneurism

MMP-2

MMP classification

Based on the ECM substrate which they proteolyse

Groups of MMPs include :1. Collageneses (MMP-1,-8)2. Stromelysins (MMP-10,-11)3. Membrane-type MMP (MMP-14,-15,-17)4. Matrilysins (MMP-26)5. Gelatinases (MMP-2,-9)

heart

MMP in the heart: MMP-2/gelatinase A

• In the normal heart MMPs are present as pro-MMP and often expressed complex with their endogenous inhibitors ,the tissue inhibitors of MMPs (TIMP)

• MMP expressed and found: 1. Cardiac myocites2. Endothelium3. Vascular smooth muscle cells4. fibroblasts

• Localized to the sarcomere within the myocyte

Catalytic (Zn 2+ ) Fibronectin-type

Zn 2+ Hinge

S S

Pre Pro Catalytic (Zn 2+ ) Fibronectin-type

Zn 2+ Hinge

S SSH

HEMOPOEXIN

MMP-2 structre

Inactive/zymogen enzyme C-terminal

Active enzyme

activation

72 kDa

64 kDa


Zn 2+ Hinge

S SSH

HEMOPOEXIN

Inactive/zymogen enzyme C-terminal

• The propeptide domain contains a conserved cysteine that chelates the zinc in the active site• fibronectin type II domain: enhance substrate binding • Hinge: confers specificity • hemopexin domain: regulatory subunit

substrate specificityinvolved in activation as well as inhibition of MMPs calcium binding site (Ca required for full activity)

MMP-2 structure

• Proteolytic activation of MMP-2 by MT1-MMP/TIMP or by other proteases occurs by removal of the autoinhibitory propeptide domain (left arrow) resulting in an active truncated MMP-2.

• presence of oxidative stress (ONOO-) and cellular glutathione (GSH) causes the S-gluathiolation of the critical cysteine residue in the propeptide domain, disrupting its binding to the catalytic Zn2+ ion, resulting in an active full-length enzyme

72 kDa

72 kDa64 kDa

Activation of MMPs and inactivation by ONOOˉ

• Peroxynitrite is formed in vivo by the reaction of NO with superoxide. It is a powerful oxidizing agent that can initiate lipid peroxidation, oxidize sulfhydryls, and nitrate the aromatic residues of proteins.

• The cysteine residue of propeptide domain containing the sulphhydryl groups coordinated to the catalytic Zn2+

• Highly sensitive to changes in the redox environment• 1-20 μM ONOOˉ activates MMP-1,-8,-9 • >100 μM ONOOˉ inactivate MMP-2 likely via the nitration of tyrosine

residues in the sequence


Zn 2+ Hinge

S SSH

HEMOPOEXIN/FIBRONECTIN

Modulation of MMP activity by TIMPs

• TIMP: tissue inhibitor of metalloproteinase• TIMPs are comprised of 4 family members:

• TIMP-1: expression is response to signals such as proinflammatory cytokines

• TIMP-2: expressed constitutively in most of the cell types found in the heart

• TIMP-3: found in greatest concentration in the ECM

• TIMP-4: also known as cardiac inhibitor of MMPs; most abundant in the heart

• all four TIMPs have been observed in the heart and in cardiac myocytes

• Each ~23 kDa in size • They inhibit the activity by forming tight

binding complexes in a 1:1 ratio with MMPs• TIMP-4 is localized to the sarcomers and to

the thin myofilaments, where MMP-2 is also found

Modulation of MMP activity by TIMPs

Therapeutic potential of MMP inhibition

• Since various MMP isoenzymes are involved in the human pathologies, close to 60 MMP inhibitor (MMPi) have been pursued as clinical candidates since the first drug discovery program began

• The clinical developments of most of the MMPi have discontinued due to safety reasons

Therapeutic potential of MMP inhibition

• Doxicycline (Periostat ®)(approved for periodontal disease)– Able to protect the isolated heart from I/R– Inhibit MMP-2– Occured in a concentration range– Thought to have antioxidant properties (scavenge ONOO)

Other synthetic MMP-2 inhibitors

• O-phenantroline• Ilomastat (GM-6001, gelardin)

Zn 2+ chelators

Structure of ilomastat,the Zn-binding group is highlighted in

box

zymography

Documents

modification of zymography

casein zymography suitable

detection of mmp

enhancement of mmp activity

specific substrate

krisztina kupaisource

type of substrate

situ zymographyprepared