Drosophila Tolloid Research Articles

Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity.

In Xenopus embryos, a dorsal-ventral patterning gradient is generated by diffusing Chordin/bone morphogenetic protein (BMP) complexes cleaved by BMP1/Tolloid metalloproteinases in the ventral side. We developed a new BMP1/Tolloid assay using a fluorogenic Chordin peptide substrate and identified an unexpected negative feedback loop for BMP4, in which BMP4 inhibits Tolloid enzyme activity noncompetitively. BMP4 binds directly to the CUB (Complement 1r/s, Uegf [a sea urchin embryonic protein] and BMP1) domains of BMP1 and Drosophila Tolloid with high affinity. Binding to CUB domains inhibits BMP4 signaling. These findings provide a molecular explanation for a long-standing genetical puzzle in which antimorphic Drosophila tolloid mutant alleles displayed anti-BMP effects. The extensive Drosophila genetics available supports the relevance of the interaction described here at endogenous physiological levels. Many extracellular proteins contain CUB domains; the binding of CUB domains to BMP4 suggests a possible general function in binding transforming growth factor-beta (TGF-beta) superfamily members. Mathematical modeling indicates that feedback inhibition by BMP ligands acts on the ventral side, while on the dorsal side the main regulator of BMP1/Tolloid enzymatic activity is the binding to its substrate, Chordin.

A Complete Domain Structure of Drosophila Tolloid Is Required for Cleavage of Short Gastrulation

Drosophila tolloid (TLD) is a member of a family of proteinases that play important roles in development and includes mammalian tolloid (mTLD) and bone morphogenetic protein (BMP)-1. TLD accentuates the activity of decapentaplegic (DPP), a transforming growth factor beta superfamily growth factor, by cleaving its antagonist Short gastrulation (Sog). Similarly, the activity of BMP-2/4 (vertebrate homologues of DPP) is augmented by cleavage of chordin. However, whereas TLD is an effective Sogase, mTLD is a poor chordinase and is functionally replaced by its smaller splice variant BMP-1, which lacks the most C-terminal epidermal growth factor (EGF)-like and CUB domains of mTLD. Moreover, the minimal chordinase activity resides in the N-terminal half of BMP-1. This study showed that the proteolytic activity of TLD is considerably enhanced by Ca2+ and tested the hypothesis that the Sogase activity of TLD resides in the N-terminal half of the proteinase. Unexpectedly, it was found that TLD lacking the CUB4 and CUB5 domains and/or the EGF-like domains was unable to cleave Sog. Loss of function mutations have been reported in the tld gene that result in amino acid substitutions at E835K (in CUB4), S915L (in CUB5), and N760I (in EGF2) in TLD. The CUB mutants were found to be ineffective Sogases, but the activity of the EGF2 mutant was unchanged. The results show that substrate recognition and cleavage by Drosophila tolloid and mTLD are different despite their identical domain structure and homologous functions in patterning. The result that the N760I mutant has full Sogase activity suggests that novel substrates for TLD exist.

Matching catalytic activity to developmental function: Tolloid-related processes Sog in order to help specify the posterior crossvein in theDrosophilawing

The Drosophila tolloid (tld) and tolloid related (tlr) gene products belong to a family of developmentally important proteases that includes Bone Morphogenetic Protein 1 (Bmp1). Tld is required early in Drosophila development for proper patterning of dorsal embryonic structures, whereas Tlr is required later during larval and pupal stages of development. The major function of Tld is to augment the activity of Decapentaplegic (Dpp) and Screw (Scw), two members of the Bmp subgroup of the Tgf beta superfamily, by cleaving the Bmp inhibitor Short gastrulation (Sog). In this study, we provide evidence that Tlr also contributes to Sog processing. Tlr cleaves Sog in vitro in a Bmp-dependent manner at the same three major sites as does Tld. However, Tlr shows different site selection preferences and cleaves Sog with slower kinetics. To test whether these differences are important in vivo, we investigated the role of Tlr and Tld during development of the posterior crossvein (PCV) in the pupal wing. We show that tlr mutants lack the PCV as a result of too little Bmp signaling. This is probably caused by excess Sog activity, as the phenotype can be suppressed by lowering Sog levels. However, Tld cannot substitute for Tlr in the PCV; in fact, misexpressed Tld can cause loss of the PCV. Reducing levels of Sog can also cause loss of the PCV, indicating that Sog has not only an inhibitory but also a positive effect on signaling in the PCV. We propose that the specific catalytic properties of Tlr and Tld have evolved to achieve the proper balance between the inhibitory and positive activities of Sog in the PCV and early embryo, respectively. We further suggest that, as in the embryo, the positive effect of Sog upon Bmp signaling probably stems from its role in a ligand transport process.

Identification of an astacin-like metallo-proteinase transcript from the infective larvae of Strongyloides stercoralis

Strongyloides stercoralis, an important nematode pathogen of humans, is transmitted by contact with soil contaminated with the microscopic larvae of the parasite. We determined the cDNA sequence and deduced amino acid structure of a metallo-proteinase that is abundantly transcribed expressed by infective stage larvae of S. stercoralis. This deduced structure of the enzyme revealed a multi-domain protein that included an NH 2-terminal peptidase. This peptidase consisted of a signal peptide, a pro-enzyme region, and a mature peptidase domain that included the metal ion co-ordinating motifs, HETSHALGVIH and SIMHY (“Met-turn”), characteristic of the catalytic active site of members of the metzincin superfamily of zinc metallo-endopeptidases. It was phylogenetically and structurally similar to astacin from the digestive gland of the crayfish Astacus astacus, to the HCH-1 peptidase of Caenorhabditis elegans required for hatching and migration of a post-embryonic neuroblast, and to the morphogenetically important peptidases, bone morphogenetic protein-1 (BMP-1) and Drosophila tolloid. In addition, the Strongyloides enzyme, designated strongylastacin, includes a central epidermal growth factor (EGF) domain followed by a carboxyl CUB (complement sub component C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein) domain. Inspection of the dbEST database revealed the presence of at least 9 transcript clusters that are related to greater or lesser extent to strongylastacin; based on these expressed sequence tags, strongylastacin was expressed only in the infective third stage larvae, whereas other transcript clusters were expressed both in filariform and rhabditiform stages or only in the rhabditiform stage. Based on the deduced sequence, structure, and expression profile, strongylastacin is the probable candidate for the zinc-dependent metalloprotease, Ss40, known to be deployed by larvae of S. stercoralis to penetrate human skin to initiate infection.

Domain deletions outside of the catalytic domain of drosophila tolloid result in loss of protease activity

Introduction The tolloid (TLD)‐related zinc metalloproteinases serve multiple functions in development, being involved in both extracellular matrix assembly and TGF‐β signalling. Signalling by bone morphogenetic proteins BMPs, which form a subgroup of the TGF‐β superfamily, is a highly regulated process which involves cleavage of extracellular BMP antagonists by TLD‐like (TLL) proteases. In vertebrates, BMP‐1 and mTLL‐1 cleave the BMP antagonist chordin to release free BMPs, while in invertebrates, TLD cleaves the BMP antagonist short gastrulation (SOG) to release decapentaplegic (DPP). Mammalian TLD (mTLD), TLL‐1 and TLL‐2 share the same domain structure as drosophila TLD, while BMP‐1 is a shorter splice variant of mTLD which lacks the EGF‐2, CUB‐4 and CUB‐5 domains.Material and methods Site‐directed mutagenesis was used to produce TLD constructs lacking various domains. These were subcloned into the vector pAc5.1V5His, along with wild‐type TLD and full‐length SOG. Transient transfections in drosophila S2 cells were used to produce recombinant proteins, and aliquots of medium containing the appropriate recombinant proteins were dialysed or desalted as required. In vitro digests of SOG by TLD and the TLD mutants were performed overnight at 25°C in the presence of recombinant DPP (R & D Systems). Digests were analysed by SDS Page and Western blotting, and full‐length SOG and SOG fragments were detected using an anti‐V5 antibody (Invitrogen).Results Wild‐type TLD was able to cleave SOG at three distinct sites as previously reported (Marques et al. 1997). A ‘BMP1‐like’ TLD construct, truncated after the CUB3 domain, was efficiently secreted by S2 cells but was unable to cleave SOG. Wild‐type TLD was also found to have enhanced activity in the presence of calcium ions, indicating that it is a calcium‐dependent protease. Removal of the putative calcium‐binding domains from TLD (EGF1 and EGF2) resulted in a decreased amount of enzyme present in the medium of the transfected S2 cells and less (ΔEGF1) or abolished (ΔEGF2 and double deletion) protease activity.Discussion These preliminary findings indicate that the EGF‐2, CUB‐4 and CUB‐5 domains are required for cleavage of SOG by TLD. This is in direct contrast to the cleavage of chordin which is actually cleaved most efficiently by the short‐splice variant BMP‐1 (Scott et al. 1999). In vitro cleavage of SOG also requires the presence of DPP, whereas chordin is cleaved in the absence of exogenous BMPs. It is possible that these domains could be involved in interactions with the substrate or alternatively with DPP itself. The putative calcium‐binding domains, EGF1 and EGF2, appear to be required both for secretion and stability of the protein as well as for protease activity. The mechanism whereby calcium augments the protease activity of TLD is unknown, but it could aid the binding of enzyme to substrate or create specific conformational changes in the enzyme to increase activity.

Isolation of connective-tissue-specific genes involved in Xenopus intestinal remodeling: thyroid hormone up-regulates Tolloid/BMP-1 expression.

To clarify connective-tissue-specific genes involved in adult epithelial development during amphibian intestinal remodeling, we have isolated 16 cDNA clones derived from the anterior part of Xenopus laevis intestine cultured in vitro by using subtractive suppression hybridization. Among four genes identified, the expression of Xtld, a Xenopus homolog of Drosophila Tolloid closely related to bone morphogenic protein-1 (BMP-1), was most remarkably up-regulated during metamorphosis. To further explore the roles of Xtld in intestinal remodeling, we examined its developmental expression in the X. laevis intestine by in situ hybridization and northern blot analysis. Xtld mRNA first became detectable in the connective tissue just before the appearance of adult epithelial primordia. Subsequently, the level of Xtld mRNA reached a high in the connective tissue, concomitantly with adult epithelial development along the anteroposterior axis of the intestine. Thereafter, towards the completion of metamorphosis, the expression of Xtld mRNA was down-regulated. Thus, the expression profile of Xtld mRNA spatiotemporally correlates well with adult epithelial development in vivo. Furthermore, the present culture study has shown that thyroid hormone (TH) up-regulates the expression of Xtld mRNA organ-autonomously in the anterior part of the intestine, but not in its posterior part, and that TH up-regulation of Xtld expression is not mediated by the epithelium. These results suggest that TH directly up-regulates Xtld expression in the connective tissue along the anteroposterior axis, which in turn plays important roles in adult epithelial development during amphibian intestinal remodeling.

Mammalian BMP-1/Tolloid-Related Metalloproteinases, Including Novel Family Member Mammalian Tolloid-Like 2, Have Differential Enzymatic Activities and Distributions of Expression Relevant to Patterning and Skeletogenesis

Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal–ventral patterning by releasing TGFβ-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis.

The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart.

Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c. embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1-/- embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum.

Failure of ventral body wall closure in mouse embryos lacking a procollagen C-proteinase encoded by Bmp1, a mammalian gene related to Drosophila tolloid.

The mouse bone morphogenetic protein1 (Bmp1) gene encodes a secreted astacin metalloprotease that cleaves the COOH-propeptide of procollagen I, II and III. BMP-1 is also related to the product of the Drosophila patterning gene, tolloid (tld), which enhances the activity of the TGFbeta-related growth factor Decapentaplegic and promotes development of the dorsalmost amnioserosa. We have disrupted the mouse Bmp1 gene by deleting DNA sequences encoding the active site of the astacin-like protease domain common to all splice variants. Homozygous mutant embryos appear to have a normal skeleton, apart from reduced ossification of certain skull bones. However, they have a persistent herniation of the gut in the umbilical region and do not survive beyond birth. Analysis of the amnion of homozygous mutant embryos reveals the absence of the fold that normally tightly encloses the physiological hernia of the gut. At the electron microscopic level, the extracellular matrix of the amnion contains collagen fibrils with an abnormal morphology, consistent with the incorporation of partially processed procollagen molecules. Metabolical labelling and immunofluorescence studies also reveal abnormal processing and deposition of procollagen by homozygous mutant fibroblasts in culture.

Bone Morphogenetic Protein-1: The Type I Procollagen C-Proteinase

Bone morphogenetic proteins (BMPs) are bone-derived factors capable of inducing ectopic bone formation. Unlike other BMPs, BMP-1 is not like transforming growth factor-beta (TGF-beta), but it is the prototype of a family of putative proteases implicated in pattern formation during development in diverse organisms. Although some members of this group, such as Drosophila tolloid (TLD), are postulated to activate TGF-beta-like proteins, actual substrates are unknown. Procollagen C-proteinase (PCP) cleaves the COOH-propeptides of procollagens I, II, and III to yield the major fibrous components of vertebrate extracellular matrix. Here it is shown that BMP-1 and PCP are identical. This demonstration of enzymatic activity for a BMP-1/TLD-like protein links an enzyme involved in matrix deposition to genes involved in pattern formation.

The tolkin gene is a tolloid/BMP-1 homologue that is essential for Drosophila development.

The Drosophila decapentaplegic (dpp) gene, a member of the transforming growth factor beta superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interactions between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp.

Characterization of tolloid-related-1: A BMP-1-like Product That Is Required during Larval and Pupal Stages of Drosophila Development

The Drosophila tolloid (tld) gene product belongs to a family of developmentally important proteins that includes bone morphogenetic protein-1 (BMP-1). In Drosophila, tld is required at the blastoderm stage to establish pattern within the dorsal half of the embryo. Genetic analysis suggests that the major function of tld is to augment the activity of the decapentaplegic gene product, a close relative of the TGF-β superfamily members, BMP-2 and BMP-4. In this report, we describe a new gene called tolloid-related-1 (tlr-1) that maps immediately proximal to tld . Sequence analysis indicates that tlr-1 has a large N-terminal extension relative to tld, but otherwise shows the same general organization of sequence motifs found in tld and other BMP-1 family members. These include a region of similarity to astacin, a crayfish metalloprotease, five copies of a repeat first found in complement proteins C1r and C1s, and two copies of an epidermal growth factor-like sequence. In situ hybridization experiments show that tlr-1 expression partially overlaps tld expression in early embryos, but shows unique transcriptional patterns in late stage embryos that are not seen with tld . In larval stages, both genes are expressed in identical patterns in imaginal discs and in the optic lobes of the brain, but tlr-1 is more abundant than tld. Deletions that eliminate tlr-1 expression cause lethality during larval and pupal stages of development. A small proportion of homozygous mutant flies eclose and show wing veination defects. Transgenic animals in which a tlr-1 cDNA is driven by the tld promoter fail to rescue tld mutations, and extra copies of tld fail to rescue tlr-1 mutations, implying that these genes have evolved functionally distinct features. We propose that tld and tlr-1 arose by gene duplication and that each has evolved independently to acquire distinct tissue specific roles in Drosophila development.

Embryonic Expression of Mouse Bone Morphogenetic Protein-1 (BMP-1), Which Is Related to the Drosophila Dorsoventral Gene tolloid and Encodes a Putative Astacin Metalloendopeptidase

cDNAs encoding a mouse gene closely related to human bone morphogenetic protein-1 (BMP-1) have been isolated from an 8.5-day p.c. embryo cDNA library. Analysis of the predicted amino acid sequence shows that the protein contains a putative zinc-binding astacin metalloendopeptidase domain, five Clr/s domains, and two potential Ca2+-binding EGF-like repeats. In overall domain organization it more closely resembles the Drosophila tolloid gene product than human BMP-1. By RNase protection, transcripts can be detected in the embryo at 6.5 days p.c. and persist at least until 16.5 days p.c. By in situ hybridization, transcripts are seen at high levels in the maternal deciduum and in the late-gastrulation stage embryo at low levels throughout the mesoderm. This low level of mesodermal expression continues through development but somewhat higher levels are seen in developing membranous and endochondral bone, in the submucosa of the intestine, the dermis of the skin, and the mesenchyme of the spleen and lung. High levels of BMP-1 transcripts are also seen from 9.5 days p.c. in the floorplate region of the developing neural tube. Potential therefore exists for the interaction of BMP-1 protein with products of TGF-β-related genes expressed during embryogenesis.

Mutational analysis of the Drosophila tolloid gene, a human BMP-1 homolog

Seven zygotically active genes have been identified in Drosophila that determine the fate of dorsal cells in the developing embryo. decapentaplegic (dpp), a member of the transforming growth factor-beta (TGF-beta) family, appears to play the central role in dorsal ectoderm formation, as mutations in this gene confer the most severe mutant phenotype of this group of genes. dpp's activity is modulated by tolloid, which also has a role in the determination of dorsal cell fate. tolloid encodes a protein that contains a metalloprotease domain and regulatory domains consisting of two EGF motifs and five C1r/s repeats. We have generated several mutant tolloid alleles and have examined their interaction with a graded set of dpp point alleles. Some tolloid alleles act as dominant enhancers of dpp in a trans heterozygote, and are therefore antimorphic alleles. However, a tolloid deficiency shows no such genetic interaction. To characterize the nature of the tolloid mutations, we have sequenced eighteen tolloid alleles. We find that five of the seven alleles that act as dominant enhancers of dpp are missense mutations in the protease domain. We also find that most tolloid alleles that do not interact with dpp are missense mutations in the C-terminal EGF and C1r/s repeats, or encode truncated proteins that delete these repeats. Based on these data, we propose a model in which the tolloid protein functions by forming a complex containing DPP via protein-interacting EGF and C1r/s domains, and that the protease activity of TOLLOID is necessary, either directly or indirectly, for the activation of the DPP complex.(ABSTRACT TRUNCATED AT 250 WORDS)

The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1

Mutations in the Drosophila tolloid (tld) gene lead to a partial transformation of dorsal ectoderm into ventral ectoderm. The null phenotype of tld is similar to, but less severe than decapentaplegic (dpp), a TGF-β family member required for the formation of all dorsal structures. We have cloned the tld locus by P element tagging. At the blastoderm stage, tld RNA is expressed dorsally, similar to that described for dpp. Analysis of a tld cDNA reveals three sequence motifs: an N terminal region of similarity to a metalloprotease, two EGF-like repeats, and five copies of a repeat found in human complement proteins C1r and C1s. tld sequence is 41% identical to human bone morphogenetic protein 1 (BMP-1); the closest members to dpp within the TGF-β superfamily are BMP-2 and BMP-4, two other bone morphogenetic proteins. These findings suggest that these genes are members of a signal generating pathway that has been conserved between insects and mammals.