Localized unfolding of collagen explains collagenase cleavage near imino-poor sites

Collagenases cleave all three chains of type III collagen at specific sites characterized by a Gly-Leu or a Gly-Ile bond that is upstream from an imino acid-poor region. Molecular dynamics trajectories were used to calculate the free energy of unfolding for collagen-like model peptides. The free energy profiles suggest that such imino-poor regions can adopt a low-energy, partially unfolded state where one of the peptide chains forms a solvent-exposed loop. The results are consistent with a model for collagenase cleavage where partial unfolding of imino-poor regions enables collagenases to gain access to their cleavage sites.     

Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system

Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.     

Differential susceptibility of type X collagen to cleavage by two mammalian interstitial collagenases and 72-kDa type IV collagenase

We have studied the degradation of type X collagen by human skin fibroblast and rat uterus interstitial collagenases and human 72-kDa type IV collagenase. The interstitial collagenases attacked the native type X helix at two loci, cleaving residues Gly92-Leu93 and Gly420-Ile421, both scissions involving Gly-X bonds of Gly-X-Y-Z-A sequences. However, the human and rat interstitial enzymes displayed an opposite and substantial selectivity for each of these potential sites, with the uterine enzyme catalyzing the Gly420-Ile421 cleavage almost 20-fold faster than the Gly92-Leu93 locus. Values for enzyme-substrate affinity were approximately 1 microM indistinguishable from the corresponding Km values against type I collagen. Interestingly, in attacking type X collagen, both enzymes manifested kinetic properties intermediate between those characterizing the degradation of native and denatured collagen substrates. Thus, energy dependence of reaction velocity revealed a value of EA of 45 kcal, typical of native interstitial collagen substrates. However, the substitution of D2O for H2O in solvent buffer failed to slow type X collagenolysis significantly (kH/kD = 1.1), in contrast to the 50-70% slowing (kH/kD = 2-3) observed with native interstitial collagens. Since this lack of deuterium isotope effect is characteristic of interstitial collagenase cleavage of denatured collagens, we investigated the capacity of another metalloproteinase with substantial gelatinolytic activity, 72-kDa type IV collagenase, to degrade type X collagen. The 72-kDa type IV collagenase cleaved type X collagen at both 25 and 37 degrees C, and at loci in close proximity to those attacked by the interstitial enzymes. No further cleavages were observed at either temperature with type IV collagenase, and although values for kcat were not determined (due to associated tissue inhibitor of metalloproteinases-2), catalytic rates appeared to be substantial in comparison to the interstitial enzymes. In contrast, type X collagen was completely resistant to proteolysis by stromelysin. Type X collagen thus appears to be highly unusual in its susceptibility to degradation by both interstitial collagenase and another member of the metalloproteinase gene family.     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

Spectrophotometric assay for vertebrate collagenase

Collagenase from normal human skin fibroblasts was found to catalyze the hydrolysis of esters and thio esters. This observation led to the development of a rapid, sensitive, continuous spectrophotometric assay for vertebrate collagenase using the thio peptolide Ac-ProLeuGly-S-LeuLeuGly-OC2H5 as substrate in the presence of 4,4'-dithiodipyridine or Ellman's Reagent. A Km of 0.004 M and a kcat of 370,000 h-1 were determined for the thio peptolide-enzyme reaction. The method is able to detect collagenase at concentrations as low as 2 ng/ml.     

Type I collagen contains at least 14 cryptic fibronectin binding sites of similar affinity

There is uncertainty in the literature regarding the number and location of fibronectin binding sites on denatured collagen. Although most attention has focused on a single site near the collagenase-sensitive region of each alpha chain, there is evidence for additional sites in other regions. We treated bovine type I collagen with cyanogen bromide, labeled the resulting mixture with fluorescein, and separated the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent bands were excised from the gel and dialyzed exhaustively to remove detergent. Titration of eight distinct fluorescent-labeled fragments with the 42-kDa gelatin-binding fragment of fibronectin caused increases in anisotropy that were fully reversible with unlabeled gelatin. By fitting the dose responses it was possible to calculate apparent K(d)'s whose values ranged between 1 and 4 microM. The largest fragment, alpha(2)-CB3,5, composing about 2/3 of the alpha(2) chain, when further digested with endoproteinase Lys-C, yielded at least three additional subfragments that also bound with similar affinities. Thus, there appear to be at least 14 distinct fibronectin binding sites of similar affinity in bovine type I collagen, five on each of the alpha(1) chains and four on the alpha(2) chain. Experiments with several synthetic peptides failed to reveal the exact nature of the binding site.     

FokI requires two specific DNA sites for cleavage

FokI is a bipartite restriction endonuclease that recognizes a non-palindromic DNA sequence, and then makes double-stranded cuts outside of that sequence to leave a 5' overhang. Earlier kinetic and crystallographic studies suggested that FokI might function as a dimer. Here, we show, using dynamic light-scattering, gel-filtration and analytical ultracentrifugation, that FokI dimerizes only in the presence of divalent metal ions. Furthermore, analysis of the DNA-bound complex reveals that two copies of the recognition sequence are incorporated into the dimeric complex and that formation of this complex is essential for full activation of cleavage. These results have broad implications for the mechanism by which monomeric type II endonucleases achieve high fidelity.     

New Achromobacter collagenase and its immunological relationship with a vertebrate collagenase

Evidence is presented that Achromobacter iophagus produces two distinct collagenases. Achromobacter collagenases A and B were separated by high-performance liquid chromatography from partially purified enzyme. The main collagenase, A (EC 3.4.24.8), which has been already described, was eluted in the region of molecular mass 110-90 kDa. A minor collagenase B eluted in the region of 320 kDa, although in SDS-gel electrophoresis the apparent molecular masses of its main active forms were estimated as 55 and 110 kDa. The specificities of collagenases A and B are different. Collagenase A splits in its synthetic substrate Pz-Pro-Leu-Gly-Pro-DArg the bond Leu-Gly, collagenase B does not split this substrate. Both collagenases split bonds Gln-Gly and Leu-Gly in synthetic peptides DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-DArg-OH and DNP-Pro-Leu-Gly-Ile-Ala-Gly-DArg-NH2, respectively. Collagenase B is twice as active as A on the native collagen type I. Both enzymes are inhibited by EDTA. The antibodies raised against the human tooth collagenase specifically inhibited the collagenase B, but did not influence the activity of collagenase A. These results indicate, to our knowledge for the first time, an immunological relationship between a bacterial and a vertebrate collagenase.     

Microtubule-associated protein 8 contains two microtubule binding sites

Microtubule-associated proteins (MAPs) are critical regulators of microtubule dynamics and functions, and have long been proposed to be essential for many cellular events including neuronal morphogenesis and functional maintenance. In this study, we report the characterization of a new microtubule-associated protein, we named MAP8. The protein of MAP8 is mainly restricted to the nervous system postnatally in mouse. Its expression could first be detected as early as at embryonic day 10, levels plateau during late embryonic and neonatal periods, and subsequently decrease moderately to remain constant into adulthood. In addition to its carboxyl terminal binding site, the MAP8 polyprotein also contains a functional microtubule-binding domain at its N-terminal segment. The association of the carboxyl terminal of the light chain with actin microfilaments could also be detected. Our findings define MAP8 as a novel microtubule associated protein containing two microtubule binding domains.     

Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.     

Structural properties of a collagenous heterotrimer that mimics the collagenase cleavage site of collagen type I

Collagens contain sequence- and conformation-dependent epitopes responsible for their digestion by collagenases at specific loci. A synthetic heterotrimer construct containing the collagenase cleavage site of collagen type I was found to mimic perfectly native collagen in terms of selectivity and mode of enzymatic degradation. The NMR conformational analysis of this molecule clearly revealed the presence of two structural domains, i.e. a triple helix spanning the Gly-Pro-Hyp repeats and a less ordered portion corresponding to the collagenase cleavage site where the three chains are aligned in extended conformation with loose interchain contacts. These structural properties allow for additional insights into the very particular mechanism of collagen digestion by collagenases.     

An improved viscosimetric assay for vertebrate collagenase activity

An improved viscosimetric assay for vertebrate collagenase acitivity is described. The assay is carried out at 35 degrees C in the presence of 1 M glucose to prevent fibril formation. The decrease in viscosity is linear with ime and proportional to enzyme concentration.     

PvuII endonuclease contains two calcium ions in active sites

Restriction endonucleases differ in their use of metal cofactors despite having remarkably similar folds for their catalytic regions. To explore this, we have characterized the interaction of endonuclease PvuII with the catalytically incompetent cation Ca(2+). The structure of a glutaraldehyde-crosslinked crystal of the endonuclease PvuII-DNA complex, determined in the presence of Ca(2+) at a pH of approximately 6.5, supports a two-metal mechanism of DNA cleavage by PvuII. The first Ca(2+) position matches that found in all structurally examined endonucleases, while the second position is similar to that of EcoRV but is distinct from that of BamHI and BglI. The location of the second metal in PvuII, unlike that in BamHI/BglI, permits no direct interaction between the second metal and the O3' oxygen leaving group. However, the interactions between the DNA scissile phosphate and the metals, the first metal and the attacking water, and the attacking water and DNA are the same in PvuII as they are in the two-metal models of BamHI and BglI, but are distinct from the proposed three-metal or the two-metal models of EcoRV.     

The developmentally regulated type X collagen gene contains a long open reading frame without introns

Type X collagen is a recently discovered product of hypertrophic chondrocytes that is localized to presumptive mineralization zones of hyaline cartilage. Thus, in the epiphyseal growth plate of long bones it is present only in the zone of hypertrophic chondrocytes and absent in the resting and rapidly growing cartilage and in bone. Type X collagen represents, therefore, a transient and developmentally regulated collagen which is synthesized by a subpopulation of chondrocytes. We report here the isolation and characterization of cDNA and genomic clones specific for the chicken protein. The results demonstrate that the polypeptide chains of this collagen contain three distinct domains: a short non-collagenous, amino-terminal region, a collagenous domain of 460 amino acid residues, and a non-collagenous, carboxyl-terminal domain of 170 amino acid residues. The nucleotide sequence of the gene shows that these domains are encoded by a long open reading frame that is not interrupted by introns. Examination of the amino acid sequence derived from this nucleotide sequence reveals the presence of a hydrophobic segment localized 10 amino acid residues upstream from the translational stop codon. The length and sequence characteristics of this segment raise the intriguing possibility that Type X collagen polypeptides may contain a transmembrane segment.     

Type X collagen, a product of hypertrophic chondrocytes.

The synthesis of collagen types IX and X by explants of chick-embryo cartilages was investigated. When sternal cartilage labelled for 24h with [3H]proline was extracted with 4M-guanidinium chloride, up to 20% of the 3H-labelled collagen laid down in the tissue could be accounted for by the low-Mr collagenous polypeptides (H and J chains) of type IX collagen; but no type X collagen could be detected. Explants of tibiotarsal and femoral cartilages were found to synthesize type IX collagen mainly in zones 1 and 2 of chondrocyte proliferation and elongation, whereas type X collagen was shown to be a product of the hypertrophic chondrocytes in zone 3. Pulse-chase experiments with tibiotarsal (zone-3) explants demonstrated a time-dependent conversion of type X procollagen into a smaller species whose polypeptides were of Mr 49 000. The processed chains [alpha 1(X) chains] were shown by peptide mapping techniques to share a common identity with the pro alpha 1(X) chains of Mr 59 000. No evidence for processing of type IX collagen was obtained in analogous pulse-chase experiments with sternal tissue. When chondrocytes from tibiotarsal cartilage (zone 3) were cultured on plastic under standard conditions for 4-10 weeks they released large amounts of type X procollagen into the medium. However, 2M-MgCl2 extracts of the cell layer were found to contain mainly the processed collagen comprising alpha 1(X) chains. The native type X procollagen purified from culture medium was shown by rotary shadowing to occur as a short rod-like molecule 148 nm in length with a terminal globular extension, whereas the processed species comprising alpha 1(X) chains of Mr 49 000 was detected by electron microscopy as the linear 148 nm segment.     

The identification of two subcellular sites for cleavage of gamma-glutamyltranspeptidase propeptide

In order to determine the subcellular site(s) of rat renal gamma-glutamyltranspeptidase propeptide cleavage labeled immunoprecipitates were obtained from preparations of either intracellular membranes or brush border membrane vesicles. Heterodimer accounts for 25% of the label associated with transpeptidase in intracellular membranes from 5 to 40 min postinjection of [35S]methionine, consistent with a cotranslational cleavage of propeptide in the endoplasmic reticulum. Labeled propeptide and heterodimer appear in the brush border membrane fraction between 20-30 min postinjection and accumulate for 1 h and 4h, respectively. Subsequently, the propeptide disappears with a half-life of 1 h while the heterodimer is relatively stable. These results confirm our previous proposal for two distinct subcellular sites for transpeptidase propeptide cleavage (Capraro, M.A. and Hughey, R.P. (1983) FEBS Lett. 157, 139-143).     

A collagen film microassay for tissue collagenase.

A quantitative microassay for the detection of bacterial and tissue collagenase is presented. Collagen in acetic acid solution forms a thin, tenacious film upon contact with a dried agar surface. The digestion of the film by collagenase is detected by subsequent staining with Coomassie blue. Undigested film is stained dark blue while areas of collagenase digestion remain clear. The technique can be employed in two ways: with collagen-coated glass coverslips as a rapid screening method or with collagen-lined capillary tubes for precise quantitation. The assay has a sensitivity comparable to those assays using radioactively labeled collagen substrates and requires only 5 to 60 min of incubation.