Matrix Metalloproteinases

Henning Birkedal‐Hansen1, Susan Yamada1, Jack Windsor2, Anne Havernose Pollard3, Guy Lyons4, William Stetler‐Stevenson5, Bente Birkedal‐Hansen5

1 National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, 2 University of Indiana School of Dentistry, Indianapolis, Indiana, 3 University of Copenhagen School of Dentistry, Copenhagen, 4 Kanematsu Laboratories, Royal Prince Alfred Hospital, Sydney, 5 Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.8
DOI:  10.1002/0471143030.cb1008s40
Online Posting Date:  September, 2008
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Abstract

Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods—cell‐mediated dissolution of type‐1 collagen fibrils, direct and reverse zymography, enzyme capture based on α2‐macroglobulin and TIMP‐1 and ‐2, and demonstration of cryptic thiol groups in metalloproteinase precursors—that are used to characterize the functions of matrix metalloproteinases and their inhibitors. Curr. Protoc. Cell Biol. 40:10.8.1‐10.8.23. © 2008 by John Wiley & Sons, Inc.

Keywords: matrix metalloproteinases; type‐1 collagen; zymography; α2‐macroglobulin; TIMP‐1 and ‐2

     
 
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Table of Contents

  • Basic Protocol 1: Dissolution and Degradation of Collagen Fibrils by Live Cells
  • Support Protocol 1: Preparing Rat Tail Tendon Collagen Type I
  • Support Protocol 2: Labeling of Collagen
  • Basic Protocol 2: Gelatin/Casein Zymography
  • Basic Protocol 3: Reverse Zymography
  • Basic Protocol 4: α2‐Macroglobulin (α2M) Capture
  • Alternate Protocol 1: TIMP Capture
  • Basic Protocol 5: Fluorescent Labeling of Cryptic Cys‐Residue in MMPs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Dissolution and Degradation of Collagen Fibrils by Live Cells

  Materials
  • 3 mg/ml rat tail tendon type I collagen in 13 mM HCl (see protocol 2)
  • 13 mM HCl, 4°C
  • Neutralizing buffer (see recipe), 4°C
  • Phosphate‐buffered saline (PBS) without Ca2+ and Mg2+ (CMF‐PBS; appendix 2A) supplemented with 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate
  • Cells of interest (e.g., fibroblasts, keratinocytes, or tumor cells)
  • DMEM ( appendix 2A) supplemented with 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate with and without 10% (v/v) FBS (or other medium appropriate for cell type)
  • Growth factors/cytokines: e.g., IL‐1β, TNF‐α, TGF‐α, or TPA; or phorbol ester (12‐O‐tetradecanoylphorbol‐13‐acetate, TPA, or phorbol myristate acetate, PMA)
  • 1% (v/v) Triton X‐100
  • 0.05% (w/v) trypsin/0.53 mM EDTA (Invitrogen)
  • Coomassie blue stain (see recipe)
  • 6‐well cell culture plates
  • Additional reagents and equipment for trypsinizing and counting cells (unit 1.1)

Support Protocol 1: Preparing Rat Tail Tendon Collagen Type I

  Materials
  • Tails of ∼400 g rats (freshly removed or stored frozen at −80°C)
  • 0.5 M NaCl in 50 mM Tris⋅Cl, pH 7.4 (see appendix 2A for Tris⋅Cl)
  • 5 mM, 50 mM, and 0.5 M acetic acid
  • NaCl (solid)
  • 0.02 M Na 2HPO 4
  • 13 mM HCl
  • Neutralizing buffer (0.2 M NaP i)
  • Glass wool or cheesecloth
  • 500‐ml centrifuge bottles
  • High‐speed centrifuge (Sorvall with SS‐34 and GSA rotors, or equivalent centrifuge and rotors)
  • 10,000 to 14,000 MWCO dialysis membrane
  • One large (25‐liter) or several smaller (4‐liter) dialysis tanks
  • Sterile scissors
  • 125‐ml glass Wheaton bottles
  • Additional reagents and equipment for dialysis ( appendix 3C)

Support Protocol 2: Labeling of Collagen

  Materials
  • 3 mg/ml rat tail tendon type I collagen originally dissolved in or dialyzed into 13 mM hydrochloric acid (see protocol 2)
  • Neutralizing buffer (see recipe)
  • Borate buffer: 0.05 M NaB 4O 7⋅10H 2O, pH 9.3, containing 0.04 M NaCl, filter sterilized
  • 20 to 30 mg tetramethylrhodamine‐5‐(and 6)‐isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) stock solutions, dissolved in DMSO
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 20 mM and 1 M hydrochloric acid, sterile
  • 125‐ml glass Wheaton bottle, autoclaved
  • Platform shaker

Basic Protocol 2: Gelatin/Casein Zymography

  Materials
  • Gelatin (bovine skin, Sigma‐Aldrich type B6‐6269) or casein (Sigma‐Aldrich, technical, C‐0376)
  • 2.0 M Tris⋅Cl, pH 8.8 ( appendix 2A)
  • 30/0.8 acrylamide/bisacrylamide (unit 6.1)
  • Glycerol
  • 10% (w/v) SDS ( appendix 2A)
  • TEMED
  • 10% (w/v) ammonium persulfate
  • MMP preparation of interest (for standards, use 1 to 5 ng purified MMP)
  • 5× electrophoretic sample buffer (see recipe)
  • Electrophoretic running buffer (see recipe)
  • Gel washing buffers 1 to 4 (see recipe)
  • Coomassie blue stain (see recipe)
  • Gel destaining solution (see recipe)
  • 50‐ml centrifuge tubes
  • 57°C water bath
  • Whatman no. 1 filter paper or 0.5‐µm syringe filter
  • Gel washing tray of appropriate size
  • Additional reagents and equipment for preparing SDS‐PAGE gels according to Laemmli (unit 6.1)
NOTE: The following procedure is based on a standard 10% SDS‐PAGE according to Laemmli (Laemmli, ; unit 6.1) using a 4% stacking gel and a pH 8.3 running buffer. It is important to avoid heating and/or reduction during sample preparation and running of the gel.

Basic Protocol 3: Reverse Zymography

  Materials
  • 8.7 mg/ml gelatin solution (see recipe)
  • MMP‐2 (Gelatinase A)
  • 5× electrophoretic sample buffer (see recipe)
  • 2.5% (w/v) Triton X‐100
  • Incubation solution (see recipe)
  • Additional reagents and equipment for “forward” zymography (see protocol 4)

Basic Protocol 4: α2‐Macroglobulin (α2M) Capture

  Materials
  • MMP solution to be tested
  • 2 to 3 mg/ml purified α2M in 50 mM Tris⋅Cl standard buffer (see recipe for buffer)
  • 100 µg/ml TPCK‐treated trypsin (e.g., Sigma) in 50 mM Tris⋅Cl standard buffer (see recipe), pH 7.4
  • 1.0 mg/ml soybean trypsin inhibitor in 50 mM Tris⋅Cl standard buffer (see recipe), pH 7.4
  • 5× electrophoretic sample buffer (see recipe)
  • Antibodies to MMPs of interest
  • Nitrocellulose paper
  • Additional reagents and equipment for SDS‐PAGE according to Laemmli (unit 6.1) and for immunoblotting (unit 6.2)

Alternate Protocol 1: TIMP Capture

  Materials
  • 0.1 to 1.0 mg/ml TIMP‐1 (Oncogene Research Products, Chemicon International; also see Bodden et al., ) in 50 mM Tris⋅Cl standard buffer (see recipe), pH 7.4
  • 10.0 mM NH 2PheHgAc (APMA; Sigma) in Tris⋅Cl standard buffer (see recipe), pH 7.4
  • 5× electrophoretic sample buffer (see recipe, but use only 0.5% w/v SDS)
  • Antibodies to MMPs and TIMP‐1 of interest (Calbiochem, Chemicon International)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)

Basic Protocol 5: Fluorescent Labeling of Cryptic Cys‐Residue in MMPs

  Materials
  • MMP‐containing samples
  • 20 µM fluorescent maleimide N‐(7‐(di‐methylamino‐4‐methyl‐3‐coumarinyl) maleimide (DACM) in Tris⋅Cl standard buffer (see recipe for buffer; prepare from 1 mM DACM stock in DMSO or ethanol)
  • 2‐mercaptoethanol stock in electrophoretic sample buffer (see recipe for buffer): concentration appropriate to obtain 5% final concentration in reaction mixture
  • Fluorescent lamp
  • Photographic equipment
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1)
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Figures

Videos

Literature Cited

Literature Cited
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