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WO2005061700A1 - Moteur moleculaire a collagenase interstitielle - Google Patents

Moteur moleculaire a collagenase interstitielle Download PDF

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Publication number
WO2005061700A1
WO2005061700A1 PCT/US2004/038236 US2004038236W WO2005061700A1 WO 2005061700 A1 WO2005061700 A1 WO 2005061700A1 US 2004038236 W US2004038236 W US 2004038236W WO 2005061700 A1 WO2005061700 A1 WO 2005061700A1
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WO
WIPO (PCT)
Prior art keywords
collagen
mmp
enzyme
fibril
interstitial collagenase
Prior art date
Application number
PCT/US2004/038236
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English (en)
Inventor
Gregory Goldberg
Saveez Saffarian
Ivan E. Collier
Barry L. Marmer
Elliot L. Elson
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Washington University
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Publication of WO2005061700A1 publication Critical patent/WO2005061700A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)

Definitions

  • the present invention relates to the field of interstitial collagenase
  • the extracellular matrix (ECM) of vertebrates is a three- dimensional scaffold consisting of highly organized macromolecular assemblies that vary in structure and composition to define and maintain the shapes and mechanical properties of tissues.
  • ECM extracellular matrix
  • Many physiological and pathophysiological processes from morphogenesis to wound healing, tumor progression and metastatic invasion are characterized by intensified tissue remodeling that begins with degradation of the existing ECM (refs.1 , 2, below).
  • Collagen is the most abundant component of the ECM.
  • Monomers of fibrillar collagens have a unique triple-helical structure that self assembles to produce tightly packed periodic fibrils (ref. 3) up to 500 nm in diameter that are highly resistant to proteolytic degradation.
  • Resident cells of tissues can secrete a specialized group of enzymes, matrix metalloproteases (MMPs) that degrade ECM macromolecules including collagens (ref. 4).
  • MMPs matrix metalloproteases
  • interstitial collagenase (MMP-1), (ref. 5) is principally responsible for fibrillar collagen turnover.
  • the enzyme cleaves all three a chains of the collagen monomer at a single site located approximately three-fourths of the way from its NH 2 terminus (ref. 6).
  • the assembled collagen fibril contains multiple equidistantly distributed cleavage sites 300 nm apart.
  • a “Brownian Ratchet” can be powered by coupling to a non- equilibrium chemical reaction driving the particle between two states (refs.14-16). Recently a “Burnt Bridge” model of a Brownian ratchet has been described (ref.17). In this model the diffusion bias is created because a moving particle can destroy weak places on a track in a way that inhibits its ability to diffuse back.
  • the present invention relates to a novel interstitial collagenase molecular motor and method of use operating extracellularly. More particular, the invention comprises an interstitial collagenase (MMP-1) acting as an ATP-independent molecular motor driven by the proteolysis of substrate collagen.
  • MMP-1 interstitial collagenase
  • MMP-1 is the state-of-the-art enzyme nomenclature for interstitial collagenase. MMP-1 is known to degrade fibrillar collagen (types I, II, and III) but not collagen types IV and V. The complete cDNA and primary structure of interstitial collagenase from human skin fibroblasts is described in U.S. Patent 4,772,557 (see also ref. 5). [0008] It is demonstrated in accordance with the invention that the digestion of a collagen fibril occurs when the bound MMP-1 undergoes biased diffusion along the fibril encountering cleavage sites without noticeable dissociation. The MMP-1 transport mechanism is akin to a Brownian ratchet with biased diffusion independent of ATP hydrolysis but coupled to collagen proteolysis instead.
  • MMP-1 as thus described herein is believed to be the first example of an ATP-independent extracellular molecular motor.
  • the disclosure herein of MMP-1 acting as a molecular ratchet tethered to the cell surface of a collagen fibril supports its use as a research tool in the tissue remodeling and cell-matrix interaction.
  • MMP-1 has been known for many years to be useful in the treatment of hypertrophic scars, keloids and intervertebral disc disease. It is implicated in conditions in which degeneration of connective tissue is an important part of the pathology of normal repair and damage. MMP-1 is also recognized as a drug target for the treatment of inflammation, would healing and cancer. Accordingly, the invention as described and claimed herein has significant utility in the screening and development of inhibitors of MMP-1 and as a research tool for evaluating the activity and effect of drug candidates.
  • the interstitial collagenase molecular motor as disclosed herein also is useful in the field of nanotechnology whereby the molecular motor can manipulate molecules one at a time. This is illustrated herein by Fluorescence Correlation Spectroscopy (FCS) in which an individual MMP-1 decorated collagen fibril represents single molecules of the enzyme passing through the laser beam.
  • FCS Fluorescence Correlation Spectroscopy
  • the interstitial collagenase can thus act as an ATP-independent motor for cargo delivery in nanotechnology.
  • interstitial collagenase acting as an ATP-independent extracellular molecular motor as disclosed herein is further useful in the field of drug delivery whereby attached drug molecules can be transported by the molecular motor.
  • FIGS. 1A, 1B, and 1C Mobility of MMP-1 Bound to a Collagen Fibril, shown in 3 parts, FIGS. 1A, 1B, and 1C.
  • the nano-positioning stage was instructed to center a selected individual collagen fibril under the laser beam.
  • a constant average fluorescence indicating a steady state was achieved after the initial exposure of the fibril spot to a 5 mw laser beam for 120 sec.
  • a primary fluorescent signal was collected for 300 sec at 400 /sec intervals immediately following the initial exposure
  • FIG. 1A Primary fluorescent intensity record of a MMP-1 decorated (Red) and an untreated collagen fibril (Black) recorded at 400 ⁇ sec intervals. Both records are dominated by shot noise.
  • FIG. 1B Primary fluorescent intensity record shown in FIG. 1A with the time interval increased to 80 msec to reduce the shot noise. Spikes of fluorescence intensity are observed in the experiments performed on MMP-1 decorated fibrils. Background fluorescence from untreated collagen fibrils shows no spikes in the record.
  • FIG. 1 C The data at 80 msec time resolution in FIG.1 B were used for finding the position of the spikes in the fluorescence record using a threshold method with the threshold set to 5 times the standard deviation calculated from the average signal in each of the 2 sec windows. Isolated from the background, the spikes of fluorescence intensity represent single molecules of MMP-1 passing through the laser beam.
  • the single molecule fluorescence was measured in FCS experiments with MMP-1 in solution.
  • the average value of 870 Hz (Black line) and a maximum of 2.4 KHz (Gray line) were calculated by either averaging the fluorescence over the entire observation volume or assuming the molecule traveled through the center of the beam respectively.
  • the intensity of spikes is within the expected range for a single molecule intensity.
  • Activated MMP-1 Is a ATP-independent motor enzyme driven by proteolysis of its substrate, collagen.
  • w is the beam waist
  • D is the diffusion coefficient
  • V is the flow velocity
  • the shown fit of the WT MMP-1 data has a local diffusion coefficient of 8+1.5 x10 "9 cm 2 sec "1 and a transport velocity of 4.5 ⁇ 0.36 ⁇ m sec "1 .
  • the correlation function of the inactive mutant exhibits a long tail characteristic of an unbiased 1-D diffusion , with a local diffusion coefficient of
  • FIG. 3 The unequal flux of MMP-1 molecules around a "no transport" block on the collagen fibril.
  • Photo-bleaching of MMP-1 decorated collagen fibrils with a laser beam intensity below 50 mw results in the recovery of fluorescence after termination of exposure. Raising the laser intensity to 80 -90 mw prevents the recovery after photo-bleaching indicating a blockage of the enzyme transport across the bleached area due to a damage of a fibril.
  • FIG. 4A The rules of the road.
  • the enzyme molecules perform a random walk in one-dimension along a collagen fibril. Once they reach a cleavage recognition site, a successful cleavage occurs with a set probability Pj and the enzyme molecule responsible for the cleavage will always end up on one side of the cleaved peptide bond.
  • the molecules are not allowed to cross the cleaved triple helix from either side but are allowed to jump to a neighboring triple helix track with a small probability Pj
  • This mechanism akin to a "Brownian ratchet" produces a net transport with velocity V which depends on a diffusion coefficient, the probabilities defined above and a spatial distribution of the cleavage sites.
  • FIG. 4B Monte Carlo simulations of the interaction of MMP-1 with a collagen fibril.
  • 100 enzyme molecules on a 30 ⁇ m long fibril with a cross- section of 100 triple helixes of collagen and cleavage sites 0.3 ⁇ m apart were monitored.
  • a value of Pj was set at 1/200 sec.
  • the size of random walk steps has a Gaussian distribution as shown. Walkers encounter a reflective boundary condition at each of the "burnt bridges”.
  • Figure 5 Monte Carlo simulations of the experimental correlation functions and concentration profile of MMP-1 on collagen fibril, shown in 2 parts, FIGS. 5A and 5B.
  • FIG. 5B The simulated concentration profiles of WT (Red) and inactive mutant (Black) enzymes on a collagen fibril.
  • the simulation was composed by monitoring 100 molecules on a 30 ⁇ m long fibril with a cross-section of 100 triple helices and proteolytic sites spaced at 0.3 ⁇ m producing an average occupation number of 1 for each of the 0.3 ⁇ m segments.
  • the concentration profile was summed over 24 sec of the simulation.
  • FIG. 6 Monte Carlo simulation of the flux of single MMP-1 molecules around a "no transport" block on a collagen fibril.
  • Monte Carlo simulations were performed as in the Fig. 5B with a reflective boundary condition set in the middle of the fibril representing the "no transport" block. The number of molecules on each side of the block was recorded to calculate the asymmetry ratios as in the Fig. 4. The simulations demonstrate that the asymmetry ratio is highly dependent on the value of Pc.
  • the asymmetry ratios obtained experimentally with active MMP-1 , its inactive mutant and MMP-1 inhibited by the protease inhibitor, Galadrin or heavy water are indicated.
  • MMP-1 Human recombinant Interstitial Collagenase
  • the enzyme was labeled with Alexa 488 fluorescent dye using the Alexa Fluor Protein labeling kit (Molecular Probes, A-10235).
  • the active center mutant "E219Q” was constructed using PCR site directed mutagenesis and MMP-1 cDNA as a template (ref. 5). The resulting mutant was sub-cloned into expression vector p ⁇ RHyg and transfected into p2AHT2a cells for expression (ref. 33). The mutant "E219Q” has been previously characterized (ref. 34) as having a normal binding activity but completely inactive in collagen proteolysis.
  • the enzyme mixture was incubated for 1 hr at 37° C and the plasmin activity was inhibited by addition of a 5 fold molar excess of aprotinin.
  • the activation of MMP-1 was visualized on SDS-PAGE as conversion of pro-enzyme to 42 kDa MW enzyme.
  • FCS Fluorescence Correlation Spectroscopy
  • the FCS setup consisted of a titanium sapphire laser coupled to an Olympus IX-70 inverted microscope equipped with a p527.3CL piezzo electric stage and e-710.4CL controller (Physik Instrumente, Germany) was described previously (ref.35), The 5 mwatts intensity laser beam was tuned to 810 nm for all FCS experiments.
  • the scanning or move commands were sent to the stage controller by the home written Labview program.
  • the collagen gel chambers were mounted on top of the piezzo electric stage and a fluorescent image was produced by scanning an area of the gel across the laser beam. A selected individual fibril was scanned with a higher zoom of 100 nm pixels.
  • a selected experimental spot was centered under the beam and its position was verified by observation of a high fluorescence at the time of initial contact.
  • the spot was exposed for 120 sec to achieve a constant average fluorescence indicating a steady state.
  • the fluorescence intensities were recorded for 300 sec at400 ⁇ sec intervals starting immediately after the initial exposure.
  • the Spatial Filter Reducing the time resolution of the fluorescent record from 400 ⁇ sec to 80 msec revealed spikes of intensity present in MMP-1 decorated fibrils that were absent in control.
  • the 80 msec fluorescent record was scanned with a 2 sec window to determine a local fluorescence intensity average.
  • a fluorescent signal exceeding the 5 fold the standard deviation of the local background signal was defined as a spike and its location in time and its average local background were saved.
  • the 400 ⁇ sec data were processed so that the intensities around the spikes were put equal to zero, while the points within the window containing a spike were put to the original intensity minus the average local background contribution.
  • the statistics of the background signal were poissonian.
  • the resulting data were fed to a software correlator to produce a correlation function.
  • a Gaussian laser beam 300 nm in waist was considered in the simulations. Molecules traveling through the beam produced fluorescence intensity proportional to the laser intensity at their position. Each molecule participating in the simulation was allowed to contribute a maximum number of photons predetermined by a random number following a Gaussian distribution with an average of 500 and a width of 500 photons. After the total number of photons exceeded the maximum the molecule was removed from the simulation. To maintain the total number of the molecules on the fibril a replacement fresh molecule was placed on the fibril at a random location.
  • the control record was obtained from the undecorated fibrils using collagen luminescence for imaging.
  • the primary fluorescence data from both the experiment and the control were dominated by shot noise.
  • Decreasing the time resolution of the primary record from 400 ⁇ sec to 80 msec revealed large spikes of fluorescence in the record obtained from MMP-1 decorated fibrils that were absent in the control record. Since the single molecules of Alexa 488 labeled MMP-1 are much brighter than the background, the presence of the spikes can be explained by passage of enzyme molecules through the laser beam.
  • the 80 msec time resolution data (Fig. 1B) was filtered using a threshold method (described above under "The Spatial Filter”) with the threshold set to 5 times the standard deviation calculated from the average signal in each of the 2 sec windows (Fig. 1C).
  • Biased diffusion is a characteristic of a molecular motor and requires energy dissipation. In the classical molecular motors the required energy is supplied by hydrolysis of ATP (refs. 20-22). In the absence of ATP it was hypothesized that the proteolysis of collagen monomers catalyzed by the active enzyme is a possible source of energy.
  • Example 2 investigated the properties of MMP-1 transport near a microscopically small observation volume. To verify these results and to determine whether the bias component dominates the transport process on a macroscopic scale, the flux of single MMP-1 molecules around a "no transport" block created on a collagen fibril (Fig. 3) was measured. Photo bleaching of MMP-1 - decorated collagen fibrils with a laser beam intensity below 50 mw is followed by a recovery of fluorescence after termination of the bleach pulse. Raising the laser intensity to 80 - 90 mw prevents the fluorescence recovery, indicating a blockage of the enzyme transport across the bleached area due to damage to the fibril.
  • the model described herein entails a large scale interaction of the enzyme molecules diffusing on a fibril since cleavage of a triple helical track by a passing enzyme acts as a road block for all the following molecules traveling on the same track. With time this translates into the accumulation of enzyme molecules that are restricted to free diffusion between neighboring cleavage sites 300 nm apart, creating a traffic jam. At room temperature the dissociation of the digested monomer from the surface of the fibril is slow so that the traffic jam can be observed experimentally as a large portion of the fluorescent signal bleached within the first 120 seconds of beam exposure prior to establishment of a steady state.
  • the high energy of enzyme activation was associated with the highly polymerized state of collagen fibrils since the activation energy for digestion of the triple helical collagen monomer was 4 times lower (ref. 23).
  • the high energy of activation for collagenolysis is in good agreement, however, with the apparent energy of activation for collagen fibril unfolding (124 kcal mol "1 ) measured more recently (ref.25).
  • the activation energy for the dissociation of a cleaved monomer is expected to be lower than that of an intact collagen fibril, these results are consistent with the idea that at certain temperatures the rate-limiting step of collagen fibril digestion is the dissociation of the cleaved monomers from the surface of the fibril.
  • MMP-1 - collagen system is the first example of a new class of ATP-independent molecular motors operating extracellularly.
  • the mechanism of this motor is akin to a Brownian ratchet that is able to rectify Brownian forces into a propulsion mechanism by coupling to an energy source, in this case collagen proteolysis. Further studies can determine the efficiency of energy coupling in this system.
  • the upper limit of the energy density of the MMP-1 motor can be calculated from the energy of peptide bond cleavage to be 1.4x10 •18 Watts/molecule of MMP-1.

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Abstract

La présente invention concerne une collagénase interstitielle se comportant comme un moteur moléculaire mu par la protéolyse du collagène substrat. L'invention concerne également un procédé par lequel on fait réagir la collagénase interstitielle avec une fibrille de collagène pour contrer plusieurs sites de clivage, sensiblement sans dissociation de ladite collagénase interstitielle, ce qui l'amène à se comporter en moteur moléculaire mu par la protéolyse du collagène substrat. L'invention convient donc particulièrement comme outil de recherche pur le remodelage tissulaire et l'interaction cellulo-matricielle. L'invention convient également particulièrement pour la recherche systématique et la mise au point d'inhibiteurs de la collagénase interstitielle, et comme outil de recherche pour évaluer l'activité et l'effet de candidats médicaments.
PCT/US2004/038236 2003-12-11 2004-12-09 Moteur moleculaire a collagenase interstitielle WO2005061700A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4772557A (en) * 1985-11-12 1988-09-20 Washington University DNA clone of human skin fibroblast collagenase enzyme
US5260059A (en) * 1989-04-14 1993-11-09 The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon Health Sciences University Treatment of open-angle glaucoma by modulation matrix metalloproteinases and their inhibitor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4772557A (en) * 1985-11-12 1988-09-20 Washington University DNA clone of human skin fibroblast collagenase enzyme
US5260059A (en) * 1989-04-14 1993-11-09 The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon Health Sciences University Treatment of open-angle glaucoma by modulation matrix metalloproteinases and their inhibitor

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AIT-HADDOU RACHID ET AL: "Brownian ratchet models of molecular motors.", CELL BIOCHEMISTRY AND BIOPHYSICS, vol. 38, no. 2, 2003, pages 191 - 213, XP008045470, ISSN: 1085-9195 *
FIELDS G B ET AL: "SEQUENCE SPECIFICITY OF HUMAN SKIN FIBROBLAST COLLAGENASE EVIDENCE FOR THE ROLE OF COLLAGEN STRUCTURE IN DETERMINING THE COLLAGENASE CLEAVAGE SITE", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 262, no. 13, 1987, pages 6221 - 6226, XP002324216, ISSN: 0021-9258 *
GOLDBERG G I ET AL: "HUMAN FIBROBLAST COLLAGENASE COMPLETE PRIMARY STRUCTURE AND HOMOLOGY TO AN ONCOGENE TRANSFORMATION-INDUCED RAT PROTEIN", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 261, no. 14, 1986, pages 6600 - 6605, XP002324215, ISSN: 0021-9258 *
SAFFARIAN S. ET AL: "Interstitial collagenase is a Brownian ratchet driven by proteolysis of collagen", SCIENCE, vol. 306, no. 5693, 1 October 2004 (2004-10-01), pages 108 - 111, XP002324217 *
SAFFARIAN SAVEEZ ET AL: "Interaction of Collagenase with collagen fibrils: A proteolysis driven, extracellular molecular motor.", BIOPHYSICAL JOURNAL, vol. 86, no. 1, January 2004 (2004-01-01), & 48TH ANNUAL MEETING OF THE BIOPHYSICAL SOCIETY; BALTIMORE, MD, USA; FEBRUARY 14-18, 2004, pages 602a, XP008045249, ISSN: 0006-3495 *
SCHLIWA MANFRED ET AL: "Molecular motors.", NATURE (LONDON), vol. 422, no. 6933, 17 April 2003 (2003-04-17), pages 759 - 765, XP002324546, ISSN: 0028-0836 *

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