Prolysyl Oxidase Research Articles

Molecular events that contribute to lysyl oxidase enzyme activity and insoluble collagen accumulation in osteosarcoma cell clones.

Maximum collagen synthesis and maximum accumulation of insoluble collagen occur at different phenotypic stages in developing osteoblastic cell cultures. Insoluble collagen accumulation depends in part on the activity of extracellular enzymes including procollagen N-proteinases, procollagen C-proteinase (derived from the BMP1 gene), and lysyl oxidase. In addition to its action on procollagen, procollagen C-proteinase processes prolysyl oxidase to mature 32-kDa lysyl oxidase. The regulation of extracellular activities that control insoluble collagen accumulation has not been studied extensively. The present study compares molecular events that control production of a collagenous mineralized extracellular matrix in vitro among five different murine osteosarcoma cell clones derived from the same tumor, but which differ in their ability to produce an insoluble mineralized matrix. Levels of insoluble type I collagen, insoluble calcium, bone morphogenetic protein 1 (BMP-1), and lysyl oxidase expression, lysyl oxidase biosynthesis, lysyl oxidase activity, and prolysyl oxidase processing activity were determined. Results surprisingly indicate that lysyl oxidase activity is not related closely to lysyl oxidase messenger RNA (mRNA) levels among the different cell clones. However, it appears that BMP-1-dependent prolysyl oxidase processing could contribute to the observed lysyl oxidase activity. Highest collagen and BMP-1 mRNA levels, prolysyl oxidase processing activity, and lysyl oxidase activity occurred in a cell clone (K8) that showed the highest levels of insoluble collagen accumulation. Culture media from a cell clone (K37) that accumulates little insoluble collagen or calcium but expresses high levels of lysyl oxidase mRNA contained low molecular weight fragments of lysyl oxidase protein and showed low lysyl oxidase activity. By contrast the K14 cell line exhibits relatively high lysyl oxidase activity and collagen accumulation, but low levels of mature lysyl oxidase protein. Together, these studies indicate that catabolic as well as anabolic activities are important in regulating insoluble collagen accumulation in osteoblastic cells. In addition, results suggest that products of genes homologous to lysyl oxidase may contribute to observed lysyl oxidase activity.

Procollagen C-proteinase and its enhancer protein as regulators of collagen fibril formation and matrix deposition

Procollagen C-proteinase (PCP) and its enhancer protein (PCPE) are key to collagen fibril-assembly and extracellular matrix formation. PCP cleaves the carboxyl-propeptides of procollagens types I, II, and III and this initiates the self-assembly of collagen fibrils. PCP can also process pro-lysyl oxidase and laminin 5, and it may cleave the type V procollagen N-propeptides. Procollagen processing by PCP is stimulated by PCPE, a glycoprotein that binds to the C-propeptide of type I procollagen through its N-terminal CUB domains. PCPE is also required for normal cell growth and morphology. Two distinct forms of PCP were isolated from mouse and chick sources. These were recently identified as alternatively spliced products of the gene coding for bone morphogenetic protein-1 (BMP-1), a member of a family of Zn-dependent astacin-like metalloendopeptidases implicated in tissue patterning and development. Typically, these are multidomain proteases that, in addition to their catalytic domain, contain a number of EGF-like and CUB protein-protein interaction domains. Recent evidence suggests that PCP/BMP-1 related proteases can activate TGF-β-like growth factors. There is also evidence for additional alternatively spliced PCP variants. Thus, PCPs may have important biological functions in addition to their role in collagen fibril assembly.

Bone Morphogenetic Protein-1 Processes the NH2-terminal Propeptide, and a Furin-like Proprotein Convertase Processes the COOH-terminal Propeptide of pro-α1(V) Collagen

Bone morphogenetic protein-1 (BMP-1) plays key roles in regulating the deposition of vertebrate extracellular matrix; it is the procollagen C-proteinase that processes the major fibrillar collagen types I-III, and it may process prolysyl oxidase to the mature enzyme necessary to the formation of covalent cross-links in collagen and elastic fibers. Type V collagen is a fibrillar collagen of low abundance that is incorporated into and helps regulate the shape and diameter of type I collagen fibrils. Here we show that, in contrast to its action on procollagens I-III, BMP-1 does not cleave the C-propeptide of pro-alpha1(V) homotrimers. Instead, the single BMP-1-specific cleavage site within pro-alpha1(V) chains, lies within the large globular N-propeptide. This cleavage site is immediately upstream of a glutamine, thus redefining the specificity of cleavage for BMP-1-like enzymes. It also produces an NH2 terminus that corresponds to an equivalent NH2 terminus on the processed matrix form of the similar alpha1(XI) chain, thus suggesting physiological significance. Cleavage of the C-propeptide occurs efficiently in recombinant pro-alpha1(V) homotrimers produced in 293-EBNA human embryonic kidney cells, and this cleavage is shown to occur immediately downstream of the sequence RTRR. This is similar to sites cleaved by subtilisin-like proprotein/prohormone convertases and is shown to be specifically cleaved by the recombinant subtilisin-like proprotein/prohormone convertase furin.

Incorporation of copper into lysyl oxidase.

Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation.

Metalloproteinase activity processing prolysyl oxidase to the mature enzyme. potential role of procollagen C-proteinase

Metalloproteinase activity processing prolysyl oxidase to the mature enzyme. potential role of procollagen C-proteinase

Metalloproteinase Activity Secreted by Fibrogenic Cells in the Processing of Prolysyl Oxidase: POTENTIAL ROLE OF PROCOLLAGEN C-PROTEINASE

Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor.

The synthesis and post-translational processing of a lysyl oxidase precursor protein predicted by the cDNA sequence of rat aorta lysyl oxidase were investigated. In vitro transcription of the cloned lysyl oxidase cDNA and cell-free translation of its mRNA transcript yielded a 47-kDa protein in the absence and a 50-kDa glycosylated protein in the presence of pancreatic membranes, each of which were immunoprecipitated with antibody against the 32-kDa bovine enzyme. Similarly, an N-glycosylated, 50-kDa protein band was synthesized by and immunoprecipitated from cultured neonatal rat aorta smooth muscle cells labeled with [35S]methionine. Pulse-chase studies of proteins newly synthesized by these cells demonstrated that the glycosylated 50-kDa precursor is secreted and that it is processed to the 32-kDa molecular form of lysyl oxidase principally in the medium. The presence of an extracellular processing enzyme activity which converts the 50-kDa precursor to the 32-kDa species was demonstrated by incubating conditioned medium of neonatal rat aorta smooth muscle cell cultures as a source of processing activity with conditioned medium containing 35S-labeled precursor synthesized but not processed by a tumorigenic cell line transfected with a lysyl oxidase expression vector. In contrast to the 50-kDa species, the 32-kDa protein does not appear to be N-glycosylated consistent with the loss of N-linked oligosaccharide units during the processing to the smaller species. The modes of biosynthesis and secretion of lysyl oxidase are discussed in terms of other nonproteolytic activities required for activation of prolysyl oxidase.