A collagen-binding protein in the endoplasmic reticulum of myoblasts exhibits relationship with serine protease inhibitors (original) (raw)
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Human 92 kDa type IV collagenase: Functional analysis of fibronectin and carboxyl-end domains
Kidney International, 1993
Human 92 kDa type IV coIL.. i.tional analysis of fibronectin and carboxyl-end domains. Two closely related secreted metalloproteases 72 and 92 kDa type IV collagenases (72-and 92T4C1) consist of several structural domains, the functions of which are poorly understood. Both metalloproteases can bind to gelatin as well as form complexes with specific inhibitors in the proenzyme form. The biologic role of the proenzyme-inhibitor complex formation remained unclear. Here we summarize results demonstrating that the fibronectin-like domain of 92T4C1 mediates gelatin binding of the proenzyme, while the hemopexin like carboxy-terminal domain is essential for the complex formation of the proenzyme with TIMP. The formation of a 92T4C1 proenzyme complex with TIMP prevents dimerization, formation of the novel complex with Cli proenzyme, and activation of the 92T4C1 by stromelysin. Conversely, formation of the covalent 92T4C1 homodimer excludes the formation of a proenzyme-TIMP complex, thus allowing this form of enzyme to enter into the proteolytic cascade of activation. Both components of the 92T4C1-ClI complex can be activated in a fashion similar to that of free enzymes, yielding a complex active against both gelatin and fibrillar collagen. The extracellular matrix (ECM) metalloproteases secreted by eucaryotic cells can initiate degradation of ECM macromolecules such as collagens and proteoglycans and thus play an important role in tissue remodeling [reviewed in 1, 2]. A clear understanding of the structure-function relationship within the structural domains of these enzymes and their role in mechanisms governing regulation of enzymatic activity in extracellular space has been a focus of our efforts. All known ECM metalloproteases contain the amino terminal, zinc binding, and hemopexin-like carboxyl terminal (with the exception of PUMP-l) domains. Type IV collagenases are distinguished from the other members of the family by the presence of three contiguous copies of the fibronectin (FN) type II homology unit (T2HU). The 92T4C1 protease contains yet an additional, collagen-like domain [3]. The aminoterminal domain of these enzymes is responsible for maintenance of the proenzyme state [4, 5]. The presence of the fibronectin-like domain in both 72 and 92 kDa type IV collagenases [3, 6] coincides with their ability to bind gelatin in a proenzyme form. The zinc binding domain comprises an active center by analogy with the similar structure of the well characterized bacterial metalloendopeptidase thermolysin [7, 8]. The functions of the collagen
The human 72-kDa (CLG4A) and 92-kDa (CLG4B) type IV collagenases contain a domain consisting of three contiguous copies of the fibronectin (FN)-derived type I1 homology unit (TSHU), T2HU-1, T2HU-2, and T2HU-3. To investigate the functional role of this domain, we have constructed plasmids expressing @-galactosidase fusion proteins with one or more of the CLG4B-derived T2HU. The gelatin binding assays demonstrate that a single copy of T2HU-2 renders 8galactosidase capable of binding gelatin. The three repeats, however, differ dramatically in their capacity to bind gelatin, with T2HU-1 and T2HU-3 having significantly less binding activity than T2HU-2. Using alanine scanning mutagenesis we have defined the amino acid residues (A&", AspSog, AsnS1', TyrS2', AspSa3) that are critical for gelatin binding of T2HU-2. The low gelatin binding of T2HU-1 compared to T2HU-2 was traced to the non-conserved residues Ala228-Ala and L e~~~~-P r o .
Journal of Biological Chemistry
The complete structure of the human gene for 92-kDa type IV collagenase was determined. Two overlapping genomic clones spanning 26 kilobases (kb) of genomic DNA were shown to contain the entire 7.7-kb structural gene together with 15 and 3.5 kb of 5'-end and 3'-end flanking regions, respectively. The 92-kDa type IV collagenase gene contains 13 exons as does the 72-kDa type IV collagenase gene. All intron locations of the 92-kDa enzyme gene coincided with intron locations in the 72-kDa enzyme gene. Exons 5,6, and 7 which were 174, 174, and 177 base pairs long, respectively, each encoded one complete internal repeat which resembles the collagen-binding domains of fibronectin. The sequence coding for a unique 48-residue segment in the 92-kDa type IV collagenase that has no counterpart in other metalloproteinases was not present in a separate exon, but was contained in exon 9 which also codes for sequences with homology to the other metalloproteinases. The initiation site for transcription was determined by primer extension analysis. Sequencing analysis of 599 base pairs of the 5'end flanking region showed that the promoter does not have a TATA motif, but a TTAAA sequence at position -29 to -25. A CAAT motif was not observed but there was one GC box. Two putative 12-0-tetradecanoylphorbol-13-acetate (TPA) response elements, that might serve as binding sites for the transcription factor AP-1 and a consensus sequence of a transforming growth factor j 31 (TGF-j31) inhibitory element were found in the promoter region. Gelatinase assay of enzyme secreted by cultured human fibrosarcoma cells (HT-1080) revealed only low levels of 92-kDa type IV collagenase activity, whereas considerable activity of the 72-kDa enzyme was present. Northern hybridization analysis confirmed these findings. Treatment of the HT-1080 cells with TPA resulted in induction of the secretion of 92-kDa type IV collagenase activity. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Complete structure of the human gene for 92-kDa type IV collagenase
The complete structure of the human gene for 92-kDa type IV collagenase was determined. Two overlapping genomic clones spanning 26 kilobases (kb) of genomic DNA were shown to contain the entire 7.7-kb structural gene together with 15 and 3.5 kb of 5'-end and 3'-end flanking regions, respectively. The 92-kDa type IV collagenase gene contains 13 exons as does the 72-kDa type IV collagenase gene. All intron locations of the 92-kDa enzyme gene coincided with intron locations in the 72-kDa enzyme gene. Exons 5,6, and 7 which were 174, 174, and 177 base pairs long, respectively, each encoded one complete internal repeat which resembles the collagen-binding domains of fibronectin. The sequence coding for a unique 48-residue segment in the 92-kDa type IV collagenase that has no counterpart in other metalloproteinases was not present in a separate exon, but was contained in exon 9 which also codes for sequences with homology to the other metalloproteinases. The initiation site for transcription was determined by primer extension analysis. Sequencing analysis of 599 base pairs of the 5'end flanking region showed that the promoter does not have a TATA motif, but a TTAAA sequence at position -29 to -25. A CAAT motif was not observed but there was one GC box. Two putative 12-0-tetradecanoylphorbol-13-acetate (TPA) response elements, that might serve as binding sites for the transcription factor AP-1 and a consensus sequence of a transforming growth factor j 31 (TGF-j31) inhibitory element were found in the promoter region. Gelatinase assay of enzyme secreted by cultured human fibrosarcoma cells (HT-1080) revealed only low levels of 92-kDa type IV collagenase activity, whereas considerable activity of the 72-kDa enzyme was present. Northern hybridization analysis confirmed these findings. Treatment of the HT-1080 cells with TPA resulted in induction of the secretion of 92-kDa type IV collagenase activity. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The Journal of Cell Biology
A major collagen-binding heat shock protein of molecular mass 47,000 D was found to bind to collagen by a pH-dependent interaction; binding was abolished at pH 6.3. Native 47-kD protein could therefore be purified from chick embryo homogenates in milligram quantities by gelatin-affinity chromatography and gentle acidic elution. Rat monoclonal and rabbit polyclonal antibodies were generated against the purified 47-kD protein. Immunofluorescence microscopy of cultured chick embryo fibroblasts with these antibodies revealed bright, granular perinuclear staining as well as a weaker reticular network structure towards the cell periphery, suggesting that this protein was located in the endoplasmic reticulum. No immunofluorescence staining was detected on the cell surface. Doublestaining experiments with these antibodies and fluorescently labeled wheat-germ agglutinin suggested that the 47-kD protein was absent from the Golgi apparatus. Localization of the 47-kD protein in the endoplasmic reticulum but not in the Golgi complex was confirmed by immunoelectron microscopy. In vivo localization studies using immunohistochemistry of cryostat sections of chick liver revealed that the 47-kD protein was present in fibrocytes, Kupffer cells, and smooth muscle cells. It was absent from hepatocytes and the epithelia of bile ducts or sinusoidal endothelium. This major transformation-and heat shockregulated glycoprotein is thus localized intracellularly, is expressed in only certain cells, and functions in a pH-regulated manner. These findings suggest that this glycoprotein is not likely to be a general cell-surface collagen receptor, but may instead play roles in intracellular protein processing or translocation.
European Journal of Biochemistry, 1979
An increase of about 4-9-fold was found in prolyl and lysyl hydroxylase activities per cell when 3T6 fibroblasts were grown from the early-log to the stationary phase, whereas essentially no changes were found in hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase activities. A control experiment using a different assay procedure indicated that the increase in prolyl hydroxylase activity was not due to any methodological artefact. The results demonstrate for the first time that these four intracellular enzymes of collagen biosynthesis are not regulated in an identical manner within one cell type.
Structural Analysis and Promoter Characterization of the Human Collagenase3 Gene (MMP13
Genomics, 1997
The matrix metalloproteinases (MMPs) 2 constitute a composed of 10 exons and 9 introns and spans over family of structurally related zinc-dependent endopep-12.5 kb. The overall organization of the collagenasetidases that play a major role in the remodeling of the 3 gene is similar to that of other MMP genes clustered connective tissue occurring in many normal processes at chromosome 11q22, including fibroblast collagenlike embryonic growth and development, wound healase (MMP-1), matrilysin (MMP-7), and macrophage ing, bone growth and resorption, and uterine involution metalloelastase (MMP-12), but is more distantly re- (Woessner, 1991;; Birkedal-Hansen et lated to genes coding for stromelysin-3 (MMP-11), al., 1993). In addition, abnormal expression of these gelatinase-A (MMP-2), and gelatinase-B (MMP-9), proteolytic enzymes may contribute to a variety of which map outside of this gene cluster. Nucleotide pathological processes such as rheumatoid arthritis sequence analysis of about 1 kb of the 5-flanking re-(Murphy and Hembry, 1992), atherosclerosis (Henney gion of the collagenase-3 gene revealed the presence et al., 1991), and tumor invasion and metastasis (Liotta of a TATA box, an AP-1 motif, a PEA-3 consensus se- et al., 1991). Recently, we have reported the cloning of quence, an osteoblast specific element (OSE-2), and a human gene coding for a novel member of this family a TGF-b inhibitory element. Transient transfection of proteolytic enzymes that has been designated collaexperiments in HeLa and COS-1 cells with chloramgenase-3 (MMP13) , since it reprephenicol acetyltransferase (CAT)-containing consents the third member of the collagenase subfamily structs showed that the AP-1 site is functional and of MMPs, the others being fibroblast and neutrophil responsible for the observed inducibility of the recollagenases (Goldberg et al., 1986;.
Journal of Biological Chemistry, 1995
72-kI)a gelatinase/type lv collagenase is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens (gelatin). These proteolytic processes are required for pathologic tissue destruction and physiologic tissue remodeling. To investigate the molecular determinants of substrate specificity of this enzyme, a 21-kDa domain of 72-kDa gelatinase, consisting of three tandem fibronectin type Il-like modtiles, was expressed in Escherichia coil. Similar to fulllength 72-kDa gelatinase and the type II modules in fibronectin, the recombinant (r) fibronectin-like domain of this proteinase bound denatured type I collagen with an apparent Kd in the micromolar range. This domain, designated the collagen-binding domain (rCBD123), possesses at least two collagen-binding sites that can each be simultaneously occupied. rCBD123 also avidly bound elastin and denatured types LV and V collagens, but neither native types lv and V collagens nor fibronectin, all of which are substrates of the enzyme. Although 72-kDa gelatinase is involved in basement membrane degradation, rCBD123 also did not bind reconstituted basement membrane, laminin, or SPARC. Native type I collagen, which is not degraded by 72-kDa gelatinase, competed with gelatin for a shared binding site on rCBD123. rCBD123 also displaced full-length 72-kDa gelatinase bound to native type I collagen, further demonstrating that the collagen binding properties of the recombinant domain closely mimicked those of the full-length enzyme. Since rCBD123 showed reduced binding to pepsin-cleaved type I collagen, either or both of the collagen telopeptide ends contain recognition sites for the 72-k.Da gelatinase fibronectin-like domain. This was confirmed by the avid binding of rCBD 123 to the al(I) collagen cyanogen bromide fragment CB2 from the NH 2-terminal telopeptide. rCBD123 also bound al(I)-CB7, which encompasses the fibronectinbinding site, and to al(I)-CB8, a fragment not bound by fibronectin Thus, type I collagen contains multiple binding sites for rCBD123 which are partially masked by the triple helical conformation of native collagen and fully exposed upon unfolding of the triple helix. The potential of the fibronectin-like collagen binding domain of 72-kDa gelatinase to bind extracellular matrix proteins may fa-cilitate enzyme localization in connective tissue matrices.
Matrix Biology, 1997
Procollagen N-proteinase (EC 3.4.24.14) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers-Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the Nproteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I procollagen. The protein contained zincbinding sequences of the clan MB of metallopeptidases that includes procollagen C-proteinase͞BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor  or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The Journal of biological chemistry, 1991
The complete structure of the human gene for 92-kDa type IV collagenase was determined. Two overlapping genomic clones spanning 26 kilobases (kb) of genomic DNA were shown to contain the entire 7.7-kb structural gene together with 15 and 3.5 kb of 5'-end and 3'-end flanking regions, respectively. The 92-kDa type IV collagenase gene contains 13 exons as does the 72-kDa type IV collagenase gene. All intron locations of the 92-kDa enzyme gene coincided with intron locations in the 72-kDa enzyme gene. Exons 5, 6, and 7 which were 174, 174, and 177 base pairs long, respectively, each encoded one complete internal repeat which resembles the collagen-binding domains of fibronectin. The sequence coding for a unique 48-residue segment in the 92-kDa type IV collagenase that has no counterpart in other metalloproteinases was not present in a separate exon, but was contained in exon 9 which also codes for sequences with homology to the other metalloproteinases. The initiation site for t...