Nereis cuticle collagen. Proteolysis by marine vibrial and clostridial collagenases

Proteolysis of Nereis cuticle collagen by two bacterial collagenases was investigated using viscosimetry, enzyme kinetics, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ion exchange chromatography of collagenolytic peptides. Collagenase of the marine Vibrio B-30 completely degrades native cuticle collagen at 7 degress C with a turnover number 50 times greater than that of the clostridial collagenase. Although turnover numbers for the two enzymes are comparable when using denatured cuticle collagen as substrate, the vibrial collagenase appears to cleave twice as many peptide bonds per mg of cuticle collagen as does the clostridial enzyme. Sodium dodecyl sulfate gel electrophoresis of collagenase-digested native cuticle collagen reflects the resistance of the collagen to clostridial collagenase; however, the vibrial enzyme completely degrades the cuticle collagen with the formation of one transient intermediate (Mr 400,000). Peptide analysis of fully digested denatured cuticle collagen reveals that the two enzymes have a number of qualitative and quantitative similarities. Despite these, however, only the vibrial collagenase seems capable of extensively degrading native cuticle collagen.     

Some newly characterized collagenases from procaryotes and lower eucaryotes

Summary Chemical and enzymatic properties of four collagenases newly isolated from anaerobic Clostridium histolyticum, aerobic Achromobacter iophagus, and from two lower eucaryotes, the fungus Entomophthora coronata and the insect Hypoderma lineatum are reviewed.The problems of their biosynthesis and precursors, namely the effect of induction of collagenase and neutral proteinase in Achromobacter by their macromolecular substrates are discussed.The two bacterial collagenases are Zn-metallo-enzymes; the highly purified Clostridium collagenase contains cyst(e)ine, serine phosphate and tryptophan additionally to amino acids reported previously. Achromobacter collagenase has the highest specific activity of all collagenases; it yields by autolysis enzymatically active degraded forms. The active dimer is composed of two identical subunits of molecular weight 35,000. Similarities between Achromobacter collagenase, thermolysin and Bacillus subtilis neutral proteinase in molecular weight, amino acid composition, and amino acids important for the active sites are discussed.The two collagenases from low eucaryotes are serine proteinases; Hypoderma collagenase is homologous to the trypsin family in the amino terminal sequence.The initial cleavage of native collagen by highly purified bacterial collagenases occurs in the central helical part of the a chains and not progressively from the amino terminal end. One of the two initial cleavages produced by Achromobacter collagenase is situated in the region cleaved specifically by vertebrate collagenases, but with different bond specificity. The same is true for the insect collagenase. Entomophthora collagenase is a proteinase of broad specificity which also cleaves collagen in its helical parts. All four collagenases also degrade other proteins according to their bond specificity.     

Characterization of extracellular alkaline proteases and collagenase induction in Vibrio alginolyticus

The number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28 000, 22 500 and 19 500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15 500 and 14 500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 degrees C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45 000, 38 500, 33 500 and 31 000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.     

Induction of collagenase production in Vibrio B-30.

The inducible nature of an extracellular collagenase produced by a marine Vibrio (Vibrio B-30, ATCC 21250) was demonstrated by observing the increase in extracellular collagenase activity after the addition of collagen to cell cultures in the latter part of the exponential growth phase. When collagenase-hydrolyzed collagen was added, the lag time required before collagenase production was detected decreased significantly compared with cultures receiving collagen. Cells preinduced to synthesize collagenase did not produce the enzyme when collagen was removed from the culture medium. Incorporation of penicillin G had no effect on final collagenase activity levels in suspensions of Vibrio B-30 in complete medium supplemented with collagen. However, chloramphenicol and tetracycline inhibited collagenase production, indicating that de novo protein synthesis was necessary for the appearance of activity. Attempts to isolate the inducing substance(s) involved filtering hydrolyzed collagen through a series of ultrafiltration membranes. The lowest-molecular-weight fraction of collagen hydrolysate with inducing ability was between 1,000 and 10,000. Gel filtration of this fraction on Sephadex G-50 resulted in the appearance of three protein peaks, two of which were capable of inducing collagenase production. Results from amino acid composition and N-terminal amino acid analysis suggest that the inducing substance originates from the polar helical portion of the collagen molecule.     

Collagenase enzymes from Clostridium: characterization of individual enzymes.

Four collagenases have been purified to apparent homogeneity from extracts of Clostridium histolyticum and partially characterized. The four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen. The enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase I = 5.50, collagenase II = 5.65, and collagenases IIIa and IIIb = 5.90-6.00. Collagenases IIIa and IIIb can be subsequently separated on diethylaminoethylcellulose. The four purified enzymes show single bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Calibration of the molecular weights on the basis of migration distance shows a marked dependence on gel porosity. At high acrylamide concentration, collagenases I, II, and IIIa appear to converge to a limiting molecular weight congruent to 81 000, while collagenase IIIb has a distinctly lower value congruent to 72 000. The similarity between these molecular weight values and those derived from the sedimentation and diffusion coefficients of the native enzyme indicates that each collagenase is a single polypeptide chain. All of the collagenases have comparable catalytic activities against a series of natural and synthetic substrates and are immunologically cross-reactive. Although all four enzymes are evident upon initial electrofocusing of the crude extract, it is possible that the multiplicity of forms is, at least in part, a consequence of lysis following initial secretion from the cell.     

Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase.

The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403. Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH. A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C. histolyticum. While the former degraded both native and denatured collagen, the latter degraded only denatured collagen. Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other. Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence. These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving off the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers. The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C. histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.     

Differences in the degradation of native collagen by two microbial collagenases.

The early stages of degradation of native collagen by two bacterial collagenases were studied by electron microscopy and by automatic Edman degradation. The purified collagenase from Clostridium histolyticum was shown to cleave native collagen at several sites, but not progressively from the N-terminus, as had been previously suggested. The homogeneous collagenase from Achromobacter iophagus cleaves native collagen preferentially at two sites corresponding to the interbands 33-34 and 41-42. The latter lies within the region cleaved by the eukaryotic collagenases.     

Purification and characteriazation of collagenase from guinea pig skin.

Guinea pig skin col-agenase, isolated from culture medium of whole skin, was separated into two enzymatically active fractions. These two fractions have been purified extensively. Peak II fraction has been purified to homogeneity as examined by polyacrylamide gel electrophoresis. Their molecular weights are approximately 130 000 (peak I) and 40 000 (peak II). Both guinea pig skin collagenase fractions are capable of degrading the native collagen fibrils and are inhibited by serum, cysteine and EDTA. They appear to be glycoproteins. Guinea pig skin (peak II) and human skin collagenase were compared. They are both glycoproteins and have similar molecular size (Mr = 40 000). Immunodiffusion assay showed that no cross-reactivity was seen between the enzymes, indicating species specificity among collagenases.     

Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene.

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.     

Limited proteolysis of type I collagen at hyperreactive sites by class I and II Clostridium histolyticum collagenases: complementary digestion patterns

The initial proteolytic events in the hydrolysis of rat tendon type I collagen by the class I and II collagenases from Clostridium histolyticum have been investigated at 15 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been used to detect the initial cleavage fragments of both the alpha 1(I) and alpha 2 chains, which migrate at different rates in the buffer system employed. Experiments with the class I collagenases indicate that the first cleavage occurs across all three chains of the triple helix close to the C-terminus to produce fragments whose alpha chains have molecular weights of approximately 88,000. The second cleavage occurs near the N-terminus to reduce the molecular weight of the alpha chains to 80,000. Initial proteolysis by the class II collagenases occurs across all three chains at a site in the interior of the collagen triple helix to give N- and C-terminal fragments with alpha-chain molecular weights of 35,000 and 62,000, respectively. The C-terminal fragment is subsequently cleaved to give fragments with alpha-chain molecular weights of 59,000. These results indicate that type I collagen is degraded at several hyperreactive sites by these enzymes. Thus, initial proteolysis by these bacterial collagenases occurs at specific sites, much like the mammalian collagenases. These results with the individual clostridial collagenases provide an explanation for earlier data which indicated that collagen is degraded sequentially from the ends by a crude clostridial collagenase preparation.     

A mass-spectrometric approach to primary screening of collagenolytic enzymes

A comparative analysis of MALDI TOF mass spectra of low-molecular products resulting from the hydrolysis of native collagen I by collagenases of various classes (bacterial metallocollagenase from Clostridium histolyticum, serine collagenase from the Morikrasa commercial preparation, cysteine collagenase from Serratia proteomaculans, and cysteine collagenases from larvae of beetles Dermestesfrischi and D. maculates) was carried out. The spectra contain a number of ion peaks common for all collagenases; nevertheless, the mass spectra of each hydrolysate contains a unique set of peaks ("fingerprint") characteristic of each enzyme. This is especially true for the peaks of major products with relative intensities of more than 50%. At the same time, the enzymes of one class (cysteine collagenases) exhibit in their mass spectra peaks of identical major products. The results show a potential opportunity for MALDI TOF application in the primary screening of collagenases according to the fingerprints of collagen hydrolysis products.     

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.     

Characterization of the individual collagenases from Clostridium histolyticum

The six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) from Clostridium histolyticum isolated in the preceding paper [Bond, M. D., & Van Wart, H. E. (1984) Biochemistry (first paper of three in this issue)] have been characterized in detail. The molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis range from 68 000 to 125 000. Isoelectric focusing experiments demonstrate that the isoelectric points of the collagenases are in the 5.35-6.20 range. These experiments also reveal that the subspecies of alpha- and gamma-collagenases (alpha1 vs. alpha 2 and gamma 1 vs. gamma 2) have different isoelectric points but the same molecular weights. Microheterogeneity is also observed for the beta- and epsilon-collagenases. The amino acid compositions of all six collagenases have been determined, and analysis for neutral sugars and hexosamines shows that none of the enzymes have a significant carbohydrate content. Zinc and calcium are the only metals that copurify with the collagenases. The purified enzymes contain approximately 1 mol of zinc/mol of protein and a calcium content that varies from about 2 mol/mol for alpha-collagenase to about 7 mol/mol for beta-collagenase. All of the collagenases are 5-10 times more active against gelatin than collagen. The alpha-, beta-, and gamma-collagenases are significantly less active toward the synthetic peptide substrates examined than the delta-, epsilon, and zeta-collagenases. This property, taken together with data on the stabilities and amino acid compositions of these enzymes, strongly supports their assignment to two distinct classes. This establishes clearly that C. histolyticum does, indeed, produce more than one different type of collagenase.     

Purification and characterization of a streptomycete collagenase

A soil streptomycete designated as Streptomyces sp. A8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-a rginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration. The purified enzyme had an apparent molecular weight of about 75,000 by SDS-polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as alpha-alpha-dipyridyl and 8-hydroxyquinoline. Ethylene diamine tetraacetate completely inhibited the enzyme activity. Among the cations tested only Ca2+ and Mg2+ enhanced the collagenase activity. Heavy metal ions like Pb2+, Ag+, Cu2+ and Zn2+ strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca2+. Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.     

Effect of divalent metal ions on collagenase from Clostridium histolyticum.

The collagenase from Clostridium histolyticum (EC 3.4.24.3) degrades type IV collagen with Km 32 nM, indicating a high affinity for this substrate. Ferrous and ferric ions can inhibit Clostridium collagenase. Inhibition by Fe++ was of the mixed, non-competitive type, with Ki 90 microM. The inhibitory effect of Fe++ may be due to Zn++ displacement from the intrinsic functional center of this metalloprotease, since in the presence of excess amounts of Zn++ enzyme activity is retained. This inhibitory effect of Fe++ may be common for all types of collagenases, since this ion can also inhibit type IV collagenase purified from Walker 256 carcinoma, with IC50 80 microM. Cu++ can only partially inhibit Clostridium collagenase, while other divalent metal ions such as Cd++, Co++, Hg++, Mg++, Ni++ or Zn++ are devoid of any inhibitory effect on the enzyme.     

A mass-spectrometric approach to primary screening of collagenolytic enzymes

A comparative analysis of MALDI TOF mass spectra of low-molecular products resulting from the hydrolysis of native collagen I by collagenases of various classes (bacterial metallocollagenase from Clostridium histolyticum, serine collagenase from the Morikrasa commercial preparation, cysteine collagenase from Serratia proteomaculans, and cysteine collagenases from larvae of beetles Dermestes frischi and D. maculatus) was carried out. The spectra contain a number of ion peaks common for all collagenases; nevertheless, the mass spectra of each hydrolysate contains a unique set of peaks (“fingerprint”) characteristic of each enzyme. This is especially true for the peaks of major products with relative intensities of more than 50%. At the same time, the enzymes of one class (cysteine collagenases) exhibit in their mass spectra peaks of identical major products. The results show a potential opportunity for MALDI TOF application in the primary screening of collagenases according to the fingerprints of collagen hydrolysis products.     

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.     

Metal content and biochemical analyses of a recombinant collagenase PrtV from Vibrio parahaemolyticus

PrtV is an extracellular metalloprotease of Vibrio parahaemolyticus and regarded as a collagenase. Inductively coupled plasma-optical emission spectrometry analysis indicated that the recombinant PrtV contains 1 mol of zinc per mol of the native enzyme. On the basis of a kinetic study using 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA, the specific substrate for bacterial collagenase) as a substrate, it was suggested that metal ions may play a significant role in the binding and catalytic steps of the substrate. PrtV hydrolyzed type I, II, III, and IV collagens; however, it did not hydrolyze type V. In addition, the hydrolysis of native proteins and synthetic substrates revealed that PrtV possesses higher activity toward collagen and collagen-like sequences. The result of the thermal stability study indicated that PrtV was thermostable up to 40 C; at 50 C, stability gradually decreased. In addition, PrtV showed higher storage stability at -20 and 4 C, respectively, than at 25 C. Compared with collagenases from Clostridium histolyticum and Vibrio alginolyticus, PrtV was immunologically different and had no significant effect on the growth of CHO, HeLa, and Vero cells. Taken together, the results of the studies described in this paper advance our knowledge concerning the metal content and biochemical properties of PrtV.     

Collagenase production by Achromobacter iophagus.

Achromobacter iophagus produced collagenase (EC 3.4.24.3) when cultured aerobically in buffer containing 5% peptone. The bacterium is non-pathogenic and tests on rabbits indicated that the culture medium was atoxic. The collagenase, which hydrolyzed insoluble and soluble native collagen, was purified by (NH4)2 SO4 precipitation, starch block electrophoresis, and gel filtration. It was shown to be serologically distinct from Clostridium histolyticum collagenase and to have molecular weight and sedimentation coefficient values of approx. 112 000 and 5.3 S, respectively.     

A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1

A collagenolytic protease was purified to homogeneity from thermophilic Bacillus sp. strain MO-1. The protease from strain MO-1 showed high activity toward type I and IV collagens and gelatin. However, peptide substrates (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and 2-furylacryloyl-Leu-Gly-Pro-Ala) for collagenases were inert as substrates. The collagenolytic protease cleaved oxidized insulin B-chain at 11 sites and degraded type I and IV collagens into anonymous small pieces, suggesting that the protease digests collagens at multiple sites. The collagenolytic protease was far more thermostable than a mesophilic Clostridium histolyticum collagenase. The collagenolytic protease possesses two salient features: (1) it has a very large molecular mass, 210 kDa, and consists of two, identical 105-kDa subunits; (2) it belongs to a serine protease group. The high molecular mass is unique among serine proteases but common for collagenases. The features of the enzyme from strain MO-1 suggest that it is a new collagenolytic protease which is distinct from previously reported collagenases and serine proteases.