J. Frederick Woessner

TIMP-3 Binds to Sulfated Glycosaminoglycans of the Extracellular Matrix*

Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B β-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases.

Heparan Sulfate Proteoglycans as Extracellular Docking Molecules for Matrilysin (Matrix Metalloproteinase 7)

Many matrix metalloproteinases (MMPs) are tightly bound to tissues; matrilysin (MMP-7), although the smallest of the MMPs, is one of the most tightly bound. The most likely docking molecules for MMP-7 are heparan sulfate proteoglycans on or around epithelial cells and in the underlying basement membrane. This is established by extraction experiments and confocal microscopy. The enzyme is extracted from homogenates of postpartum rat uterus by heparin/heparan sulfate and by heparinase III treatment. The enzyme is colocalized with heparan sulfate in the apical region of uterine glandular epithelial cells and can be released by heparinase digestion. Heparan sulfate and MMP-7 are expressed at similar stages of the rat estrous cycle. The strength of heparin binding by recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay; theK D is 5–10 nm. Zymographic measurement of MMP-7 activity is greatly enhanced by heparin. Two putative heparin-binding peptides have been identified near the C- and N-terminal regions of proMMP-7; however, molecular modeling suggests a more extensive binding track or cradle crossing multiple peptide strands. Evidence is also found for the binding of MMP-2, -9, and -13. Binding of MMP-7 and other MMPs to heparan sulfate in the extracellular space could prevent loss of secreted enzyme, provide a reservoir of latent enzyme, and facilitate cellular sensing and regulation of enzyme levels. Binding to the cell surface could position the enzyme for directed proteolytic attack, for activation of or by other MMPs and for regulation of other cell surface proteins. Dislodging MMPs by treatment with compounds such as heparin might be beneficial in attenuating excessive tissue breakdown such as occurs in cancer metastasis, arthritis, and angiogenesis.

Elevated tissue levels of collagenase during dilation of uterine cervix in human parturition

Seven biopsy specimens from the cervix and 17 from the lower uterine segment were obtained in 24 women at term (37 to 42 weeks). Collagenase was extracted and assayed on telopeptide-free [3H]collagen; typical collagen cleavage products were found on sodium dodecyl sulfate-gel electrophoresis. There was no significant difference between collagenase levels in the cervix and in the lower uterine segment in women not in labor and with the cervix closed. Levels of active and latent collagenase in 11 such specimens were 0.14 +/- 0.03 and 0.64 +/- 0.90 U/gm wet weight, respectively (mean +/- SEM; 1 U = 1 micrograms collagen digested per minute at 30 degrees C). Thirteen women at term in active labor with cervical dilation of 4 to 8 cm exhibited a thirteenfold increase in mean collagenase activity in the lower uterine segment. Active and latent collagenase increased to 2.06 +/- 0.92 and 8.64 +/- 2.87 U/gm, respectively. This is the first direct evidence that interstitial collagenase increases markedly during cervical dilation in human parturition.

Matrix metalloproteinase inhibition. From the Jurassic to the third millennium.

ABSTRACT: A brief historical introduction to the matrix metalloproteinase (MMP) field, which began in 1962, is followed by an overview of the inhibition of these proteases by natural inhibitors such as α2 macroglobulin and the TIMPs (tissue inhibitors of metalloproteinases) and by synthetic inhibitors, which are largely chelating agents. The latter include thiol, alkylcarbonyl, phosponamidate and hydroxamate compounds, as well as the tetracyclines. A review of the most recent progress concludes with prognostications as to where the field may be going next.

MMPs and TIMPs--an historical perspective.

There are currently 25 known vertebrate matrix metalloproteinases (MMPs) and 4 tissue inhibitors of metalloproteinases (TIMPs). This article reviews these proteases from an historical perspective in terms of who discovered each protein, when the sequence was established, when action on protein substrates was demonstrated, and what names have been used. A similar approach is taken for the TIMPS, and their multiple functions in addition to protease inhibition are emphasized. MMPs from invertebrates, plants, and bacteria are also discussed. This review is an outgrowth and update of a chapter by the same name originally published in Matrix Metalloproteinase Protocols, pp. 1–23, edited by I. M. Clark and published by Humana Press in 2001.

Mammalian collagenase: direct demonstration in homogenates of involuting rat uterus.

Summary Homogenates of involuting rat uterus contain an enzyme capable of digesting native collagen fibers at pH 7.5 and 37°. Collagenase activity and the uterine collagen are both found in the pellet when the homogenates are centrifuged at 6,000 g . Incubation of this pellet at 37° in the presence of calcium leads to the release of soluble degradation products of collagen. It is suggested that the collagenase is firmly bound to its substrate collagen fibers, since it cannot be readily extracted from the pellet.

Neutral Proteases and Cathepsin D in Human Articular Cartilage

Proteolytic enzymes have been studied in extracts of human articular cartilage by the use of micromethods. The digestion of hemoglobin at pH 3.2 and of cartilage proteoglycan at pH 5 was shown to be due chiefly to cathepsin D. Cathepsin D was purified 900-fold from human patellar cartilage. Its identity was established by its specific cleavage of the B chain of insulin. At least six multiple forms of cathepsin D are present in cartilage; these corresponded to bovine forms 4-9. Cathepsin D had no action on proteins at pH 7.4. However, cartilage extracts digested proteoglycan, casein, and histone at this pH. The proteolytic activities against these three substrates were purified about 170-, 160-, and 70-fold, respectively. Each activity appeared in multiple forms on DEAE-Sephadex chromatography. The three activities appear to be different since cysteine inhibited casein digestion, aurothiomalate inhibited histone digestion, and neither inhibited proteoglycan digestion. Tests with a wide range of inhibitors and activators suggest that these three activities differ from other neutral proteases described in the literature.

Extraction of collagenase from the involuting rat uterus.

Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90-95% recovery of the total active and latent collagenase activity in a 6000 X g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60 degrees C for 4 min in 0.04 M Tris - HCl buffer, pH 7.5, containing 0.1 M CaCl2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65-70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.

The pathogenesis of osteoarthritis

This article reviews the etiology and pathogenesis of osteoarthritis, particularly one of several current concepts concerning the possible central mechanisms regulating degradation of cartilage. According to this theory, degradation involves diffuse or focal exposure of the extracellular matrix to active neutral metalloproteinases, which then results in injury as well as initiation of repair processes. Diffuse matrix exposure is probably not a physiologic aberrancy but rather a pathologic result of either physical injury to local chondrocytes or inflammatory mediators.

A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryhänen et al. (L. Ryhänen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.