Bacterial collagenases - A review.

Bacterial collagenases are metalloproteinases involved in the degradation of the extracellular matrices of animal cells, due to their ability to digest native collagen. These enzymes are important virulence factors in a variety of pathogenic bacteria. Nonetheless, there is a lack of scientific consensus for a proper and well-defined classification of these enzymes and a vast controversy regarding the correct identification of collagenases. Clostridial collagenases were the first ones to be identified and characterized and are the reference enzymes for comparison of newly discovered collagenolytic enzymes. In this review we present the most recent data regarding bacterial collagenases and overview the functional and structural diversity of bacterial collagenases. An overall picture of the molecular diversity and distribution of these proteins in nature will also be given. Particular aspects of the different proteolytic activities will be contextualized within relevant areas of application, mainly biotechnological processes and therapeutic uses. At last, we will present a new classification guide for bacterial collagenases that will allow the correct and straightforward classification of these enzymes.

No ancient DNA damage in Actinobacteria from the Neanderthal bone.

BACKGROUND: The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized. PRINCIPAL FINDINGS: Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences. CONCLUSIONS/SIGNIFICANCE: Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

Tadpole collagenase, the single parent of such a large family.

This editorial review comments the development of the field of the matrix metalloproteinases that was initiated by the demonstration of the tadpole collagenase in 1962.

Collagenases in cancer.

Three mammalian collagenases (MMP-1, MMP-8, and MMP-13) belong to family of matrix metalloproteinases and are the principal secreted endopeptidases capable of cleaving collagenous extracellular matrix. In addition to fibrillar collagens, collagenases can cleave several other matrix and non-matrix proteins including growth factors, and this way regulate cell growth and survival. Collagenases are important proteolytic tools for extracellular matrix remodeling during organ development and tissue regeneration, but they also apparently play important roles in many pathological situations and tumor progression and metastasis. Because of their potentially destructive characteristics the expression and activity of collagenases are strictly controlled. Synthesis of collagenases is regulated by extracellular signals via cellular signal transduction pathways at transcriptional and post-transcriptional level. Collagenases are synthesized as inactive pro-forms, and once activated, their activity is inhibited by specific tissue inhibitors of metalloproteinases, TIMPs, as well as by non-specific proteinase inhibitors. In this review we discuss the current view on the role of collagenases in tumor growth, invasion, and metastasis, as a basis for their feasibility in diagnosis and prognostication, as well as therapeutic targets in cancer patients.

Secretion of different types of gelatinases from cultured human keratinocytes.

We investigated gelatinolytic enzymes derived from cultured human keratinocytes. Using a zymograph, we detected 92 kDa and 72 kDa gelatinases as major components and 83 kDa as a minor component from a conditioned cultured medium (BSL-K110, Kyokutoseiyaku Co.) of keratinocytes. After several passages, the 72 kDa band disappeared, and the 83 kDa band became dominant in another conditioned culture medium (K-GM, Kurashikibouseki Co.); these keratinocytes had a relatively differentiated appearance. These results suggest that some mechanism may regulate the secretion of selected types of gelatinase from keratinocytes under different conditions.

Identification and characterization of a novel collagenase in Xenopus laevis: possible roles during frog development.

Matrix metalloproteinases (MMPs) participate in extracellular matrix remodeling and degradation and have been implicated in playing important roles during organ development and pathological processes. Although it has been hypothesized for > 30 years that collagenase activities are responsible for collagen degradation during tadpole tail resorption, none of the previously cloned amphibian MMPs have been biochemically demonstrated to be collagenases. Here, we report a novel matrix metalloproteinase gene from metamorphosing Xenopus laevis tadpoles. In vitro biochemical studies demonstrate that this Xenopus enzyme is an interstitial collagenase and has an essentially identical enzymatic activity toward a collagen substrate as the human interstitial collagenase. Sequence comparison of this enzyme to other known MMPs suggests that the Xenopus collagenase is not a homologue of any known collagenases but instead represents a novel collagenase, Xenopus collagenase-4 (xCol4, MMP-18). Interestingly, during development, xCol4 is highly expressed only transiently in whole animals, at approximately the time when tadpole feeding begins, suggesting a role during the maturation of the digestive tract. More importantly, during metamorphosis, xCol4 is regulated in a tissue-dependent manner. High levels of its mRNA are present as the tadpole tail resorbs. Similarly, its expression is elevated during hindlimb morphogenesis and intestinal remodeling. In addition, when premetamorphic tadpoles are treated with thyroid hormone, the causative agent of metamorphosis, xCol4 expression is induced in the tail. These results suggest that xCol4 may facilitate larval tissue degeneration and adult organogenesis during amphibian metamorphosis.

92-kDa type IV collagenase and TIMP-3, but not 72-kDa type IV collagenase or TIMP-1 or TIMP-2, are highly expressed during mouse embryo implantation.

Expression of 72 kDa and 92 kDa type IV collagenases and the metalloproteinase inhibitors TIMPs 1, 2, and 3 was studied by in situ hybridization in implanting mouse embryos of days 5.5 to 7.5. The 92 kDa type IV collagenase was strongly expressed in invading trophoblasts, signals above background not being observed in the embryonic proper or placental tissue. In contrast, signals above background were not seen for the 72 kDa enzyme in any cells of the implantation region, including trophoblasts and stromal cells of the decidual tissue. Only cells in the mucosal stroma outside the decidual region displayed some expression. TIMP-3 was intensily expressed in maternal cells in the area surrounding the invading embryonic tissue. No expression was observed for TIMP-1 or TIMP-2 in the embryo proper, trophoblasts, or the area of the uterine decidual reaction. Weak signals appeared for TIMP-1 only in the circular layer of myometrial smooth muscle and in some uterine stroma cells distant from the site of embryo implantation. The results suggest a central role for 92 kDa type IV collagenase and TIMP-3 in the extracellular proteolysis associated with implantation of the early embryo.

Multiple forms of gelatinases/type IV collagenases in saliva and gingival crevicular fluid of periodontitis patients.

The aim of the present study was to characterize the eventual presence and molecular forms of gelatinase/type IV collagenase activities in gingival crevicular fluid (GCF) and saliva in different forms of periodontitis; patients with clinically healthy periodontium served as controls. Enzyme activities were monitored electrophoretically by zymography using gelatin and type IV collagen as substrates and analyzed visually and/or densitometrically. Both saliva and GCF collected from adult periodontitis, localized juvenile periodontitis and type II diabetes mellitus periodontitis patients contained species moving identically with gelatinase isolated from human neutrophils or MMP-9 (mean 98 kD), and species with mobility similar to gelatinase in fibroblast cell culture supernatants or MMP-2 (mean 76 kD). Hitherto, undescribed high molecular weight forms (mean 128 kD), were found, possibly representing polymerized or complexed enzyme active/activated in situ in the gel matrix. Small molecular forms of gelatinases (mean 51 kD and 46 kD), unable to cleave type IV collagen, were also found, most likely representing in vivo proteolytically activated, truncated enzymes. Although multiple forms of gelatinases/type IV collagenases in saliva and GCF may take part in the tissue destruction in periodontitis, their profile judged according to molecular weights does not differentiate between different forms of periodontitis.

Sequence homology between a bacterial metalloproteinase and eukaryotic matrix metalloproteinases.

The origin and evolution of matrix metalloproteinases represent an exciting subject of study. Recently, various reports have searched for a relationship between bacterial and eukaryotic metalloproteinases. In this report, we constructed a phylogenetic tree using the amino acid sequence of one bacterial metalloproteinase and eight eukaryotic matrix metalloproteinases and performed multiple alignments with some of these sequences. We concluded that there is a familial relationship between members of the four major branches represented in the tree.

Urinary type IV collagenase: elevated levels are associated with bladder transitional cell carcinoma.

Accumulating experimental evidence has linked the overproduction of extracellular matrix-degrading metalloproteinases with tumor cell invasion. In the present study one member of the metalloproteinase family, type IV collagenase (M(r) 72,000 gelatinase), is shown to be elevated in the urine of patients with transitional cell carcinoma of the bladder. The form of the enzyme in the urine was studied by three independent methods: enzyme-linked immunosorbent assay, Western immunoblotting; and gelatin zymography. Immunoblotting revealed that the enzyme was present as a series of fragments, each retaining the amino terminus of the mature proenzyme. A prominent M(r) 43,000 fragment was associated with the transitional cell carcinoma cases. Zymography demonstrated that multiple enzyme species with gelatinase activity were present in urine and that high-molecular-weight bands of substrate lysis corresponded to complexes between type IV collagenase and tissue inhibitor of metalloproteinases 2. The total amount of type IV collagenase antigen was significantly elevated in the urine of 37 transitional cell carcinoma patients (range, 0-1081 ng/ml; mean, 318.4 +/- 147.3) compared to 19 normal controls (P < or = 0.004) and 17 inflammatory disease controls (P < or = 0.011). Immunohistochemical staining of bladder tumor biopsies verified that the transitional cell carcinoma cells were producing the M(r) 72,000 enzyme. Thus, M(r) 72,000 type IV collagenase, which is present in the urine in many forms including fragments and complexes with inhibitors, may be a useful marker for bladder cancer diagnosis or prognosis.

Human neutrophil collagenase.

Human neutrophil collagenase (HNC) has been purified from extracts of fresh and outdated buffy coats and from the exudates of phorbol myristate acetate-stimulated neutrophils. The HNC present in the starting material from such preparations can be either latent or active, or have an approximate molecular weight of 75 or 58 kDa, depending upon whether the extraction buffer contains protease inhibitors and/or antioxidants. The purification of these different forms of HNC is described and is made possible by taking appropriate precautions to stabilize the HNC. For example, a purification protocol is described that allows the purification to homogeneity of the active and PCMB-active latent 58 kDa forms of HNC in high yield with specific collagenase activities that greatly exceed that of trypsin-activated human fibroblast collagenase (HFC). The pattern of activation of the latent 58 and 75 kDa species by trypsin, organomercurials and oxidants has been investigated. HNC is shown to preferentially hydrolyze type I over types II and III collagens in solution. The specificity of HNC toward the hydrolysis of 60 octapeptides has been examined and compared with HFC. HNC is shown to be a glycoprotein that contains complex N-linked oligosaccharides.

Salivary gland morphogenesis: possible involvement of collagenase.

When 12-day rudiments of mouse submandibular glands were cultured, they formed an average two clefts within 24 h. The average number of the clefts, however, increased up to five in the presence of bovine tissue inhibitor of metalloproteinases (TIMP). Contrary to this, either bacterial or bovine interstitial collagenase in the medium completely inhibited the cleft formation of the glands. Electron microscopic observations revealed that the amounts of collagen bundles at the cleft points significantly increased with TIMP, but decreased with bacterial collagenase. These results strongly support the idea that endogenous collagenase regulates the cleft formation through the modulation of fibrillar architectures containing collagen in the extracellular matrix. Recently, our immunohistochemical studies clarified that collagen III, known to be rich in embryonic tissue, accumulated preferentially at the cleft points of the epithelium, and may play a crucial role in submandibular gland morphogenesis.

Expression of 92 kDa phagocyte gelatinase by inflammatory and connective tissue cells.

It is now apparent that multiple proteinases cooperate in the degradation of the collagenous matrix. Among these proteinases are two biochemically related proteinases which were originally identified by their ability to degrade denatured collagens. The higher molecular weight proteinase (M(r) = 92,000) is predominantly expressed by inflammatory cells while the lower molecular weight proteinase (M(r) = 72,000) is secreted constitutively by fibroblasts. To explore the expression of the 92 kDa proteinase, the gelatinase from human neutrophils was purified and characterized and specific immunologic probes developed for its study in other cell types. Studies of leukemic cell lines (U937s and HL60s) which can be differentiated to a monocyte/macrophage phenotype revealed that after differentiation with phorbol myristic acetate the 92 kDa proteinase was the major proteinase produced. Additional studies of human alveolar macrophages and monocytes differentiated in vitro suggest that these cells secrete a proteinase immunologically and biochemically similar to human neutrophil gelatinase. Although the major gelatinase secreted by the fibroblasts is the 72 kDa proteinase, they also produce the 92 kDa proteinase in a regulated manner. These studies suggest that the regulation of these similar gelatinolytic proteinases varies dependent on cell type. Further studies of their regulation will be important to define the roles of these proteinases in physiologic and pathological processes.