Mammalian tolloid-like 1 binds procollagen C-proteinase enhancer protein 1 and differs from bone morphogenetic protein 1 in the functional roles of homologous protein domains.
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Bone morphogenetic protein 1 (BMP1) is the prototype of a subgroup of metalloproteinases with manifold roles in morphogenesis. Four mammalian subgroup members exist, including BMP1 and mammalian Tolloid-like 1 (mTLL1). Subgroup members have a conserved protein domain structure: an NH2-terminal astacin-like protease domain, followed by a fixed order of CUB and epidermal growth factor-like protein-protein interaction motifs. Previous structure/function studies have documented those BMP1 protein domains necessary for secretion, and activity against various substrates. Here we demonstrate that, in contradiction to previous reports, the most NH2-terminal CUB domain (CUB1) is not required for BMP1 secretion nor is the next CUB domain (CUB2) required for enzymatic activity. The same is true for mTLL1. In fact, secreted protease domains of BMP1 and mTLL1, devoid of CUB or epidermal growth factor-like domains, have procollagen C-proteinase (pCP) activity and activity for biosynthetic processing of biglycan, the latter with kinetics superior to those of the full-length proteins. Structure-function analyses herein also suggest differences in the functional roles played by some of the homologous domains in BMP1 and mTLL1. Surprisingly, although BMP1 has long been known to be Ca2+-dependent, a property previously assumed to apply to all members of the subgroup, mTLL1 is demonstrated to be independent of Ca2 levels in its ability to cleave some, but not all, substrates. We also show that pCP activities of only versions of BMP1 and mTLL1 with intact COOH termini are enhanced by the procollagen C-proteinase enhancer 1 (PCOLCE1) and that mTLL1 binds PCOLCE1, thus suggesting reappraisal of the accepted paradigm for how PCOLCE1 enhances pCP activities. (+info)
Temporal and spatial action of tolloid (mini fin) and chordin to pattern tail tissues.
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In vertebrates, a bone morphogenetic protein (BMP) signaling pathway patterns all ventral cell fates along the embryonic axis. BMP activity is positively regulated by Tolloid, a metalloprotease, that can eliminate the activity of the BMP antagonist Chordin. A tolloid mutant in zebrafish, mini fin (mfn), exhibits a specific loss of ventral tail tissues. Here, we investigate the spatial and temporal requirements for Tolloid (Mfn) in dorsoventral patterning of the tail. Through chimeric analyses, we found that Tolloid (Mfn) functions cell non-autonomously in the ventral-most vegetal cells of the gastrula or their derivatives. We generated a tolloid transgene under the control of the inducible hsp70 promoter and demonstrate that tolloid (mfn) is first required at the completion of gastrulation. Although tolloid is expressed during gastrulation and dorsally and ventrally within the tail bud, our results indicate that Tolloid (Mfn) acts specifically in the ventral tail bud during a approximately 4 h period extending from the completion of gastrulation to early somitogenesis stages to regulate BMP signaling. Examination of the temporal requirements of Chordin activity by overexpression of the hsp70-tolloid transgene indicates that Chordin is required both during and after gastrulation for proper patterning of the tail, contrasting Tld's requirement only during post-gastrula stages. We hypothesize that the gastrula role of Chordin in tail patterning is to generate the proper size domains of cells to enter the ventral and dorsal tail bud, whereas post-gastrula Chordin activity patterns the derivatives of the tail bud. Thus, fine modulation of BMP signaling levels through the negative and positive actions of Chordin and Tolloid, respectively, patterns tail tissues. (+info)
Mammalian tolloid alters subcellular localization, internalization, and signaling of alpha(1a)-adrenergic receptors.
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In the present study, we identified the CUB5 domain of mammalian Tolloid (mTLD) as a novel protein binding to alpha(1A)-adrenergic receptor (AR) using the yeast two-hybrid system. Whereas CUB5 did not couple to either alpha(1B)-AR or alpha(1D)-AR. It was determined that amino acids 322 to 359 of alpha(1A)-AR were the major binding region for CUB5. The direct interaction between alpha(1A)-AR cytoplasmic tail and CUB5 was discovered by glutathione S-transferase pull-down assay. We confirmed the interaction of mTLD with alpha(1A)-AR in human embryonic kidney (HEK) 293 cells by immunoprecipitation, immunofluorescence, and fluorescence resonance energy transfer. Although mTLD did not affect the density and affinity of receptors in crudely prepared membranes from HEK293 cells stably expressing alpha(1A)-AR, it significantly altered the subcellular localization of the receptors. Moreover, mTLD reduced the level of cell surface alpha(1A)-ARs, delayed the initial rate of agonist-induced receptor internalization, and facilitated agonist-induced calcium transient. We have demonstrated that mTLD interacts with alpha(1A)-AR directly, alters the subcellular localization of receptor, and influences agonist-induced alpha(1A)-AR internalization and calcium signaling. (+info)
bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis.
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Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis. (+info)
At the next stop sign turn right: the metalloprotease Tolloid-related 1 controls defasciculation of motor axons in Drosophila.
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Navigation of motoneuronal growth cones toward the somatic musculature in Drosophila serves as a model system to unravel the molecular mechanisms of axon guidance and target selection. In a large-scale mutagenesis screen, we identified piranha, a motor axon guidance mutant that shows strong defects in the neuromuscular connectivity pattern. In piranha mutant embryos, permanent defasciculation errors occur at specific choice points in all motor pathways. Positional cloning of piranha revealed point mutations in tolloid-related 1 (tlr1), an evolutionarily conserved gene encoding a secreted metalloprotease. Ectopic expression of Tlr1 in several tissues of piranha mutants, including hemocytes, completely restores the wild-type innervation pattern, indicating that Tlr1 functions cell non-autonomously. We further show that loss-of-function mutants of related metalloproteases do not have motor axon guidance defects and that the respective proteins cannot functionally replace Tlr1. tlr1, however, interacts with sidestep, a muscle-derived attractant. Double mutant larvae of tlr1 and sidestep show an additive phenotype and lack almost all neuromuscular junctions on ventral muscles, suggesting that Tlr1 functions together with Sidestep in the defasciculation process. (+info)
Cleavage and oligomerization of gliomedin, a transmembrane collagen required for node of ranvier formation.
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Gliomedin, which has been implicated as a major player in genesis of the nodes of Ranvier, contains two collagenous domains and an olfactomedin-like domain and belongs to the group of type II transmembrane collagens that includes collagens XIII and XVII and ectodysplasin A. One characteristic of this protein family is that constituent proteins can exist in both transmembrane and soluble forms. Recently, gliomedin expressed at the tips of Schwann cell microvilli was found to bind axonal adhesion molecules neurofascin and NrCAM in interactions essential for Na(+)-channel clustering at the nodes of Ranvier in myelinating peripheral nerves. Interestingly, exogenously added olfactomedin domain was found to have the same effect as intact gliomedin. Here we analyze the tissue form of gliomedin and demonstrate that the molecule not only exists as full-length gliomedin but also as a soluble form shed from the cell surface in a furin-dependent manner. In addition, gliomedin can be further proteolytically processed by bone morphogenetic protein 1/Tolloid-like enzymes, resulting in release of the olfactomedin domain from the collagen domains. Interestingly, the later cleavage induces formation of higher order, insoluble molecular aggregates that may play important roles in Na(+)-channel clustering. (+info)
Characterization of a novel reptilian tolloid-like gene in the pond turtle, Pseudemys scripta elegans.
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The Tolloid metalloproteases are pleiotropic enzymes that are important for many developmental processes. This study describes the isolation and characterization of a novel Tolloid family member from the pond turtle Pseudemys scripta elegans. The turtle Tolloid, designated tTll, is found in two forms. The first, tTlls, contains a signal sequence which may provide a mechanism for secretion. The second, tTllc, does not contain a signal sequence and is likely cytoplasmic. Sequence analysis of tTll revealed that it is most closely related to chicken Tll-2 although tTll domain structure is different. We examined the expression of tTll mRNA by real-time RT-PCR and found the highest expression in the cerebellum with lower levels in the brain stem and cortex. This expression pattern is similar to the expression of the Tolloid mouse orthologues Tll-1 and Tll-2 with highest levels of expression in the cerebellum and lower levels in the brain stem and cortex. (+info)
Genetic analysis of the role of proteolysis in the activation of latent myostatin.
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