Epidermal Growth Factor-like Motifs Research Articles

Host-mediated RNAi of a Notch-like receptor gene in Meloidogyne incognita induces nematode resistance.

GLP-1 (abnormal germline proliferation) is a Notch-like receptor protein that plays an essential role in pharyngeal development. In this study, an orthologue of Caenorhabditis elegans glp-1 was identified in Meloidogyne incognita. A computational analysis revealed that the orthologue contained almost all the domains present in the C. elegans gene: specifically, the LIN-12/Notch repeat, the ankyrin repeat, a transmembrane domain and different ligand-binding motifs were present in orthologue, but the epidermal growth factor-like motif was not observed. An expression analysis showed differential expression of glp-1 throughout the life cycle of M. incognita, with relatively higher expression in the egg stage. To evaluate the silencing efficacy of Mi-glp-1, transgenic Arabidopsis plants carrying double-stranded RNA constructs of glp-1 were generated, and infection of these plants with M. incognita resulted in a 47-50% reduction in the numbers of galls, females and egg masses. Females obtained from the transgenic RNAi lines exhibited 40-60% reductions in the transcript levels of the targeted glp-1 gene compared with females isolated from the control plants. Second-generation juveniles (J2s), which were descendants of the infected females from the transgenic lines, showed aberrant phenotypes. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and this effect was accompanied by disruption around the metacorporeal bulb of the pharynx. The present study suggests a role for this gene in organ (pharynx) development during embryogenesis in M. incognita and its potential use as a target in the management of nematode infestations in plants.

LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation.

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor β (TGF-β), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-β, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9–14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.

Thrombomodulin Links Coagulation to Inflammation and Immunity

Thrombomodulin (TM) is a type 1 membrane bound glycoprotein that has a C-type lectin domain at its Nterminus, 6 copies of the epidermal growth factor-like (EGF) motif and serine/threonine rich domain carrying a glycosoaminoglycan external to the membrane. TM binds thrombin changing thrombin's substrate specificity from procoagulant and pro-inflammatory to anti-coagulant and anti-inflammatory because of the activation of protein C (PC) and thrombin-activatable fibrinolysis inhibitor (TAFI). Thrombin's anion binding site 1 binds to TM's EGF domains 5 and 6. EGF4 is required for PC activation and EGF3 and 4 for TAFI activation in addition to EGF56. The X-ray structure of thrombin bound to TM has been solved and shows few major alterations in the active site of thrombin. The lectin domain can bind high mobility group box protein 1 (HMGB1) and a sugar, Lewis Y. TM's lectin domain behaves as an antagonist to HMGB1 endowing it with intrinsic anti-inflammatory activity. Treatment of dendritic cells with TM converts them from immunogenic to tolerogenic. TM is necessary for maintenance of pregnancy as well as prevention of coagulation throughout life. Soluble TM has been developed as an anticoagulant possessing favorable pharmacokinetics that has been approved for treatment of disseminated intravascular coagulation in Japan.

Marfan's syndrome

1978 www.thelancet.com Vol 366 December 3, 2005 Marfan’s syndrome is a multisystem connective tissue disorder of autosomal dominant inheritance. Approximately one in every 5000 individuals is affected, though this figure is probably an underestimate. The condition shows no predilection for any particular race or geographical background. It exhibits complete penetrance but variable expression and age dependency. About a quarter of affected individuals have no family history of the disease—ie, they are new mutations. Marfan’s syndrome demonstrates pleiotropy with diverse manifestations in different organ systems. Clinical diagnosis depends on a combination of major and minor signs defined in the revised 1996 Ghent criteria. The hallmark features are noted in the cardiovascular, ocular, and skeletal systems (table). The diagnosis of Marfan’s syndrome can be challenging because of phenotypic variability even between affected individuals of the same family, low specificity of many of the clinical signs, and multiple Marfan-like microfibrillar disorders with overlapping features. The gene for Marfan’s syndrome was localised to chromosome 15q21 in 1990 and cloned in 1991. FBN1 is a huge gene, spanning 110 kb of genomic DNA and comprising 65 exons. It encodes the microfibrillar protein fibrillin 1. Mutational analysis is difficult and impractical at times, in part because of the size of the gene, the heterogeneity of mutations discovered, and the lack of mutational hot spots. Moreover, there are currently no molecular techniques that offer a highly sensitive mutation detection rate of FBN1. Fibrillin 1 is a major component of extracellular microfibrils. It consists of cysteine-rich repeats interspersed by latent transforming growth factor binding protein-like and epidermal growth factor-like motifs. The microfibrils are thought to confer important biomechanical properties in connecting, anchoring, and maintaining tissues and organs. Many mutations associated with fibrillin 1 appear to confer a dominant negative mechanism of pathogenesis, but some associated with fibrillin-1 haploinsufficiency have also been described. These mutations can lead to defects in the synthesis, secretion, modification, folding, stabilisation, incorporation, and assembly of the FBN1 protein. Severe neonatal Marfan’s syndrome is associated with a cluster of mutations in the middle portion of FBN1, but there are no other established correlations between genotype and phenotype. Currently, our understanding of fibrillin biochemistry and microfibrillar assembly is limited. Experiments in mice could provide insight into fibrillin pathogenesis and microfibrillar organisation, leading to a better understanding of the pleiotropic manifestations of Marfan’s syndrome and perhaps development of a cure. Lancet 2005; 366: 1978–81

Vasorin, a transforming growth factor beta-binding protein expressed in vascular smooth muscle cells, modulates the arterial response to injury in vivo.

Growth factors, cell-surface receptors, adhesion molecules, and extracellular matrix proteins play critical roles in vascular pathophysiology by affecting growth, migration, differentiation, and survival of vascular cells. In a search for secreted and cell-surface molecules expressed in the cardiovascular system, by using a retrovirus-mediated signal sequence trap method, we isolated a cell-surface protein named vasorin. Vasorin is a typical type I membrane protein, containing tandem arrays of a characteristic leucine-rich repeat motif, an epidermal growth factor-like motif, and a fibronectin type III-like motif at the extracellular domain. Expression analyses demonstrated that vasorin is predominantly expressed in vascular smooth muscle cells, and that its expression is developmentally regulated. To clarify biological functions of vasorin, we searched for its binding partners and found that vasorin directly binds to transforming growth factor (TGF)-beta and attenuates TGF-beta signaling in vitro. Vasorin expression was down-regulated during vessel repair after arterial injury, and reversal of vasorin down-regulation, by using adenovirus-mediated in vivo gene transfer, significantly diminished injury-induced vascular lesion formation, at least in part, by inhibiting TGF-beta signaling in vivo. These results suggest that down-regulation of vasorin expression contributes to neointimal formation after vascular injury and that vasorin modulates cellular responses to pathological stimuli in the vessel wall. Thus, vasorin is a potential therapeutic target for vascular fibroproliferative disorders.

Epidermal growth factor-like motifs 1 and 2 of Plasmodium vivax merozoite surface protein 1 are critical domains in erythrocyte invasion

Plasmodium vivax merozoite surface protein 1 (PvMSP1) is believed to be important in erythrocyte invasion. However, the detailed mechanism of PvMSP1-mediated invasion has been unclear. We demonstrate that the C-terminal 19 kDa domain (PvMSP1 19) of PvMSP1, the 42-kDa fragment of PvMSP1 is further cleaved to a 33 kDa N-terminal polypeptide and a 19 kDa C-terminal fragment in a secondary processing step, is a critical domain in the binding between parasite ligand and erythrocyte receptor. Also, its cytoadherence was successfully blocked by naturally acquired immunity, was partially sensitive to neuraminidase and trypsin. When expressed separately epidermal growth factor (EGF)-like motifs 1 and 2, subunits of the PvMSP1 19, mediated 64% and 66% of the erythrocyte-binding activity, respectively, relative to their expression together as a single intact ligand domain. These results suggest that the EGF-like motifs 1 and 2 of PvMSP1 19 function as a core-binding portion in the attachment of PvMSP1 to erythrocytes.

Repression of the TMEFF2 Promoter by c-Myc

TMEFF2 is a novel transmembrane protein, containing two follistatin domains and an epidermal growth factor-like motif that is mainly expressed in the prostate and brain. Recently, we showed that expression of TMEFF2 could inhibit prostate cancer cell growth. In addition, the TMEFF2 gene is frequently hypermethylated in human tumor cells, suggesting that it might be a tumor suppressor gene. We cloned the 5′-flanking region of the human TMEFF2 gene and using a luciferase reporter assay showed that it contains a functional promoter. The 0.7 kb region upstream to the TMEFF2 transcription start site encompasses the minimal promoter required for TMEFF2 expression. Sequence analysis of the TMEFF2 promoter revealed potential binding sites for several transcription factors including Sp1 and an E-box that could be recognized by c-Myc. An inverse correlation between TMEFF2 and c-Myc expression was found in CWR22 prostate xenografts. Reporter gene and mobility shift assays demonstrated that c-Myc could repress TMEFF2 gene expression through its cognate site. In light of the probable role of TMEFF2 in inhibiting cell growth, its suppression may contribute to the oncogenic properties of c-Myc.

Genetic Heterogeneity of Cutis Laxa: A Heterozygous Tandem Duplication within the Fibulin-5 (FBLN5) Gene

Inherited cutis laxa is a connective tissue disorder characterized by loose skin and variable internal organ involvement, resulting from paucity of elastic fibers. Elsewhere, frameshift mutations in the elastin gene have been reported in three families with autosomal dominant inheritance, and a family with autosomal recessive cutis laxa was recently reported to have a homozygous missense mutation in the fibulin-5 gene. In the present study, we analyzed the gene expression of elastin and fibulins 1-5 in fibroblasts from five patients with cutis laxa. One patient was found to express both normal (2.2 kb) and mutant (2.7 kb) fibulin-5 mRNA transcripts. The larger transcript contains an internal duplication of 483 nucleotides, which resulted in the synthesis and secretion of a mutant fibulin-5 protein with four additional tandem calcium-binding epidermal growth factor-like motifs. The mutation arose from a 22-kb tandem gene duplication, encompassing the sequence from intron 4 to exon 9. No fibulin-5 or elastin mutations were detected in the other patients. The results demonstrate that a heterozygous mutation in fibulin-5 can cause cutis laxa and also suggest that fibulin-5 and elastin gene mutations are not the exclusive cause of the disease.

Molecular Determinants of the Sensory and Motor Neuron-derived Factor Insertion into Plasma Membrane

The sensory and motor neuron-derived factor (SMDF) is a type III neuregulin that regulates development and proliferation of Schwann cells. Although SMDF has been shown to be a type II protein, the molecular determinants of membrane biogenesis, insertion, and topology remain elusive. Here we used heterologous expression of a yellow fluorescent protein-SMDF fusion protein along with a stepwise deletion strategy to show that the apolar/uncharged segment (Ile(76)-Val(100)) acts as an internal, uncleaved membrane insertion signal that defines the topology of the protein. Unexpectedly, removal of the transmembrane segment (TM) did not eliminate completely membrane association of C-terminal fragments. TM-deleted fusion proteins, bearing the amino acid segment (Ser(283)-Glu(296)) located downstream to the epidermal growth factor-like motif, strongly interacted with plasma membrane fractions. However, synthetic peptides patterned after this segment did not insert into artificial lipid vesicles, suggesting that membrane interaction of the SMDF C terminus may be the result of a post-translational modification. Subcellular localization studies demonstrated that the 40-kDa form, but not the 83-kDa form, of SMDF was segregated into lipid rafts. Deletion of the N-terminal TM did not affect the interaction of the protein with these lipid microdomains. In contrast, association with membrane rafts was abolished completely by truncation of the protein C terminus. Collectively, these findings are consistent with a topological model for SMDF in which the protein associates with the plasma membrane through both the TM and the C-terminal end domains resembling the topology of other type III neuregulins. The TM defines its characteristic type II membrane topology, whereas the C terminus is a newly recognized anchoring motif that determines its compartmentalization into lipid rafts. The differential localization of the 40- and 83-kDa forms of the neuregulin into rafts and non-raft domains implies a central role in the protein biological activity.

Molecular cloning and expression study of Xenopus latent TGF-β binding protein-1 (LTBP-1)

Latent transforming growth factor β binding protein-1 (LTBP-1) is important in regulating the localization and activation of transforming growth factor β. In this paper is reported the isolation of the full-length Xenopus LTBP-1 cDNA from screening a neurula embryo cDNA library. Sequence analysis of XLTBP-1 cDNA revealed an open reading frame of 4518 bp encoding a 1398 amino acid protein with a molecular mass of 154.1 kDa and an isoelectric point of 4.65. The Xenopus XLTBP-1 shares 61 and 65% amino acid identity with the mouse and human LTBP-1, respectively. It contains 17 epidermal growth factor-like motifs and four eight-cysteine repeats (8-Cys). RNase protection assay revealed that XLTBP-1 is a maternal and zygotic gene, while whole-mount in situ hybridization analysis performed on embryos at different stages showed that during early Xenopus development, XLTBP-1 mRNA is expressed in the Spemann organizer, prechordal and chordal mesoderm, and later on in the organizer derived tissues. These findings suggest an important role for XLTBP-1 in embryo axis formation.

Versican Modulates Embryonic Chondrocyte Morphology via the Epidermal Growth Factor-like Motifs in G3

This investigation was designed to characterize the effect of the extracellular matrix molecule versican on chondrocyte morphology, using the well-studied chondrocyte cell culture system. When cultured chondrocytes reverted or “dedifferentiated” to a fibroblast-like morphology, we found that versican expression was significantly enhanced. Transfection of chondrocytes, isolated from embryonic chicken sterna, with a chicken miniversican construct accelerated the reversionprocess, while expression of an antisense construct inhibited it. A mutant miniversican lacking two epidermal growth factor-like motifs (versicanΔEGF) promoted differentiation, as shown by morphological changes and changes in the expression of other extracellular matrix molecules. A truncated versican mutant, the G3ΔEGF, a G3 domain lacking its two epidermal growth factor-like motifs, also enhanced differentiation. This effect is related to G3ΔEGF-induced change in cytoskeleton, since transfected cells exhibited misassembly of actin filaments. This article thus provides the first evidence that versican modulates chondrocyte morphology via changes in cytoskeletal structure, and may imply that extracellular matrix molecules play an important role in cell differentiation.

Associations of distinct variants of the intestinal mucin gene MUC3A with ulcerative colitis and Crohn's disease.

Ulcerative colitis (UC) and Crohn's disease (CD), the major forms of inflammatory bowel diseases (IBDs). are multifactorial disorders of unknown etiology. We reported a possible association of rare variable number of tandem repeat (VNTR) alleles of the "MUC3" gene with a susceptibility to UC. However, an entire structure of "MUC3" is still unknown because the long stretches of tandem repeats in this "gene" make its cloning extraordinarily difficult. In this study, we report evidence that "MUC3" consists of two genes, MUC3A and MUC3B, both of which encode membrane-bound mucins with two epidermal growth factor-like motifs, and we describe the complete 3'-terminal structures of these two genes. We have also analyzed the single nucleotide polymorphisms (SNPs) in the exonic sequences of the 3' portions of these two genes to investigate whether sequence variations in these regions can cause person-to-person differences in the susceptibility to IBDs, and report here that non-synonymous SNPs of MUC3A, involving a tyrosine residue with a proposed role in cell signaling, may confer genetic predisposition to CD (P = 0.0132). Our findings suggest that variants of MUC3A may be involved in the occurrence of UC and CD in distinct manners.

Conservative Structure of the Plaque Matrix Protein of Mussels in the Genus Mytilus.

The complementary DNA encoding the byssal plaque matrix protein (fp-2) of the mussel Mytilus coruscus was isolated. The predicted amino acid sequence (474 amino acids) consists of four parts: the signal peptide, the amino-terminal nonrepetitive domain, the central repetitive domain containing 11 repeats of an epidermal growth factor-like motif, and the carboxy-terminal nonrepetitive domain. The amino acid sequence is 82.7%, similar to that of fp-2 of Mytilus galloprovincialis, and the basic structure including number and motif of repeats is highly conservative. Amino acid substitutions are less frequent in "consensus positions" of the central repetitive domain (13.1%), and most of them are changes from irregular amino acids to regular ones. Thus, the structure of fp-2 was found to be conservative between species. It was presumed that the basic structure of fp-2 is unchangeable to maintain the flexible and durable matrix structure and that variation is not required because fp-2 is protected by other surface proteins.

Structure, Chromosomal Localization, and Promoter Analysis of the Human Elastin MicrofibrilInterfase Located proteIN (EMILIN) Gene

Elastin microfibril interfase-located protein (EMILIN) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as the blood vessels, skin, heart, and lung. It occurs with elastic fibers at the interface between amorphous elastin and microfibrils. In vitro experiments suggested a role for EMILIN in the process of elastin deposition. This multimodular protein consists of 995 amino acids; the domain organization includes a C1q-like globular domain at the C terminus, a short collagenous stalk, a region containing two leucine zippers, and at least four heptad repeats with a high potential for forming coiled-coil alpha-helices and, at the N terminus, a cysteine-rich sequence characterized by a partial epidermal growth factor-like motif and homologous to a region of multimerin. Here we report the complete characterization of the human and murine EMILIN gene, their chromosomal assignment, and preliminary functional data of the human promoter. A cDNA probe corresponding to the C terminus of EMILIN was used to isolate two genomic clones from a human BAC library. Sequencing of several derived subclones allowed the characterization of the whole gene that was found to be about 8 kilobases in size and to contain 8 exons and 7 introns. The internal exons range in size from 17 base pairs to 1929 base pairs. All internal intron/exon junctions are defined by canonical splice donor and acceptor sites, and the different domains potentially involved in the formation of a coiled-coil structure are clustered in the largest exon. The 3'-end of the EMILIN gene overlaps with the 5'-end of the promoter region of the ketohexokinase gene, whose chromosomal position is between markers D2S305 and D2S165 on chromosome 2. A 1600-base pair-long sequence upstream of the translation starting point was evaluated for its promoter activity; five deletion constructs were assayed after transfection in primary chicken fibroblasts and in a human rhabdomyosarcoma cell line. This analysis indicates the existence of two contiguous regions able to modulate luciferase expression in both cell types used, one with a strong activatory function, ranging from positions -204 to -503, and the other, ranging from positions -504 to -683, with a strong inhibitory function.

Mouse fibulin-2 gene. Complete exon-intron organization and promoter characterization.

Fibulin-2, an extracellular matrix protein containing tandem arrays of calcium-binding epidermal growth factor-like motifs, is present in the basement membrane and stroma of many tissues. Its expression pattern suggested an essential role in organogenesis, particularly in embryonic heart development. In this study, we cloned the extreme 5' end of the mouse fibulin-2 cDNA, isolated phage and cosmid clones encoding the entire gene, and functionally characterized the promoter. The gene was found to consist of 18 exons spanning 55 kb of DNA. The exon-intron organization reflected the modular structure of the protein. Exon 9 was subjected to alternative splicing. All splice junctions conformed to the GT/AG rule, except that GC instead of GT was found in the splice donor site of exon 4. The gene lacked TATA and CAAT boxes but contained an initiator element (Inr) and several consensus Sp1 binding sites surrounding the transcription start sites. By transient transfection of promoter deletion constructs, a 0.46-kb region containing the clustered Sp1 sites was found to confer a high promoter activity.

Mammalian Homologues of the Drosophila Slit Protein Are Ligands of the Heparan Sulfate Proteoglycan Glypican-1 in Brain

Using an affinity matrix in which a recombinant glypican-Fc fusion protein expressed in 293 cells was coupled to protein A-Sepharose, we have isolated from rat brain at least two proteins that were detected by SDS-polyacrylamide gel electrophoresis as a single 200-kDa silver-stained band, from which 16 partial peptide sequences were obtained by nano-electrospray tandem mass spectrometry. Mouse expressed sequence tags containing two of these peptides were employed for oligonucleotide design and synthesis of probes by polymerase chain reaction and enabled us to isolate from a rat brain cDNA library a 4.1-kilobase clone that encoded two of our peptide sequences and represented the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. Comparisons with recently published sequences also showed that our peptides were derived from proteins that are members of the Slit/MEGF protein family, which share a number of structural features such as N-terminal leucine-rich repeats and C-terminal epidermal growth factor-like motifs, and in Drosophila Slit is necessary for the development of midline glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523-1534 amino acids, and our peptide sequences correspond best to those present in human Slit-1 and Slit-2. Binding of these ligands to the glypican-Fc fusion protein requires the presence of the heparan sulfate chains, but the interaction appears to be relatively specific for glypican-1 insofar as no other identified heparin-binding proteins were isolated using our affinity matrix. Northern analysis demonstrated the presence of two mRNA species of 8. 6 and 7.5 kilobase pairs using probes based on both N- and C-terminal sequences, and in situ hybridization histochemistry showed that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where we have previously also demonstrated glypican-1 mRNA and immunoreactivity. Our results therefore indicate that Slit family proteins are functional ligands of glypican-1 in nervous tissue and suggest that their interactions may be critical for certain stages of central nervous system histogenesis.

EMILIN, a Component of the Elastic Fiber and a New Member of the C1q/Tumor Necrosis Factor Superfamily of Proteins

EMILIN (elastin microfibril interface located protein) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-day-old embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMILIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil alpha helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of beta-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative.

Phylogenetic Analysis of Vertebrate and Invertebrate Delta/Serrate/LAG-2 (DSL) Proteins

Delta/Serrate/LAG-2 (DSL) proteins are putative transmembrane signaling molecules that regulate cell differentiation in metazoans. DSL proteins are characterized by the presence of a motif unique to these proteins, the DSL motif, and a variable number of tandemly repeated copies of an epidermal growth factor-like (EGF) motif. We have completed a phylogenetic analysis of 15 DSL proteins from eight species. Our findings reveal that at least one gene duplication occurred prior to the divergence of theDrosophila melanogasterand vertebrate lineages, with subsequent duplications in vertebrates. The three knownCaenorhabditis elegansproteins likely arose by two independent duplications in the nematode lineage. Analysis of EGF repeats suggests that EGF 2 has been conserved among DSL proteins in vertebrates andD. melanogaster.The sequences of two EGF repeats have been perfectly conserved in vertebrate orthologs: EGF 2 in Delta and EGF 15 in Jagged/Serrate. Finally, the linear order of EGF repeats has been conserved in the vertebrate Jagged/Serrate orthologs and vertebrate Delta orthologs.

Immunolocalization of latent transforming growth factor-beta binding protein-1 (LTBP1) during mouse development: possible roles in epithelial and mesenchymal cytodifferentiation.

Latent transforming growth factor-beta binding protein-1 (LTBP1) is a member of the fibrillin family; it is a glycoprotein of more than 190 kDa that is characterized by its possession of 16-18 epidermal growth factor-like motifs and 8 cysteine residues. The secretion of transforming growth factor-beta involves its release from cells in a large latent complex containing LTBP1, a latency-associated peptide, and the mature region of the growth factor. Using a polyclonal antibody specific for LTBP1 (Ab39), we examined the immunohistochemical localization of this molecule during mouse embryogenesis between 8.5 and 13.5 embryonic days. An extracellular fibrillar structure containing LTBP1 was found in both the basement membrane of epithelia and mesenchymal tissue in which extensive tissue remodeling is carried out. Immunoelectron microscopy revealed Ab39 immunoreactivity on a 5- to 10-nm microfibrillar component of these basement membranes as well as in mesenchymal tissue. These results suggest that LTBP1 is one of the extracellular microfibrillar components of the basement membrane and of mesenchymal tissue, and that it may play an important role in the regulation of developmental phenomena involved in epithelial-mesenchymal interaction and epithelial differentiation, processes in which transforming growth factor-beta is required for the control of cellular differentiation.

Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae.

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis. We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display. We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development. This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)-like repeats and three novel cysteine-rich repeats. Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo. As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis.