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Results for “Calcium-binding EGF-like domains”

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At least 19 recordsLinked to original sources

PubMed1996

Cloning and sequence analysis of human breast epithelial antigen BA46 reveals an RGD cell adhesion sequence presented on an epidermal growth factor-like domain.

The BA46 antigen of the human milk fat globule (HMFG) membrane is expressed in human breast carcinomas and has been used successfully as a target for experimental breast cancer radioimmunotherapy. To characterize this antigen further, we obtained the entire cDNA sequence and focused on its possible role in cell adhesion. The derived protein sequence of BA46 encodes a 387-residue precursor composed of a putative signal peptide, an amino-terminal epidermal growth factor (EGF)-like domain containing the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD), and human factor V and factor VIII C1/C2-like domains. The EGF-like domain of BA46 is similar to the calcium-binding EGF-like domains of several coagulation factors, but the BA46 domain lacks a residue required for calcium binding and the coagulation factor domains do not include an RGD sequence. Assuming that all EGF-like domains fold into a similar structure, the RGD-containing sequence in BA46 is inserted between two antiparallel beta strands. This positioning suggests a novel function for the EGF-like domain as a scaffold for RGD presentation.

Amino Acid Sequence↗

PubMed1991

Key residues involved in calcium-binding motifs in EGF-like domains.

Many extracellular proteins with diverse functions contain domains similar to epidermal growth factor (EGF), a number of which have a consensus Asp/Asn, Asp/Asn, Asp*/Asn*, Tyr/Phe (where the asterisk denotes a beta-hydroxylated residue). These include the coagulation factors IX and X, proteins with two EGF-like domains, the first of which contains the consensus residues. The first EGF-like domain of human factor IX contains a calcium-binding site, which is believed to be responsible for one of the high-affinity sites detected in this protein. Similar results have been obtained for bovine factor X. We have now used protein engineering and 1H-NMR techniques to investigate the importance of individual consensus residues for ligand binding. Measurement of a calcium-dependent Tyr 69 shift in the isolated first EGF-like domain from human factor IX demonstrates that Asp 47, Asp 49, and Asp 64 are directly involved in this binding. Gln 50, whose importance has previously been overlooked, is also involved in this binding. Two mutations in this domain, Asp 47----Glu, and Asp 64----Asn, present in patients with haemophilia B, reduce calcium binding to the domain greater than 4-fold and greater than 1,000-fold, respectively. Furthermore, the defective calcium binding of Asn 64 can be partially rescued by the compensatory mutation Gln 50----Glu. This latter mutation, when introduced singly more than doubles the affinity of the domain for calcium. This study thus defines residues involved in a new type of calcium-binding site and provides strong circumstantial evidence for calcium-binding motifs in many extracellular proteins, including the developmentally important proteins of Drosophila, notch, delta and crumbs.

Amino Acid Sequence↗

PubMed2025

Unraveling a novel FBN1 variant in Marfan syndrome with dilated aortic root manifestation.

BACKGROUND: Marfan syndrome (MFS) is a genetic disorder affecting connective tissue, with variable incidence rates. A significant portion of cases stems from novel genetic variants, while others inherit it from affected parents. OBJECTIVE: This study focuses on identifying the genetic cause of MFS in a specific family, using whole-exome sequencing (WES). METHODS: A 15-year-old male with confirmed MFS was examined, showing symptoms of palpitations and severe mitral valve regurgitation. WES was performed, followed by confirmation with Sanger sequencing. Variants were assessed for pathogenicity using bioinformatics tools and the American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: One potentially novel pathogenic variant was found in exon 14 of the FBN1 gene: c.1676delCinsAAT, p.Ala559GlufsTer21. In silico analysis suggested a deleterious impact on protein structure and function, supporting their pathogenic classification. CONCLUSION: The identification of this novel variant highlights the importance of the FBN1 gene in MFS, especially its cardiovascular manifestations. Early intervention can improve patient outcomes, while ongoing research holds promise for further advancements in treatment for Marfan syndrome.

Humans↗

PubMed1993

Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding.

A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115-residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.

Amino Acid Sequence↗

PubMed1992

Ligand requirements for Ca2+ binding to EGF-like domains.

Site-specific mutagenesis studies of the first epidermal growth factor-like (EGF-like) domain of human clotting factor IX suggest that the calcium-binding site present in this domain (dissociation constant Kd = 1.8 mM at pH 7.5 and ionic strength I = 0.15) involved the carboxylate residues Asp47, Asp49 and Asp64. To further characterize the ligands required for calcium binding to EGF-like domains, two new mutations, Asp47----Asn and Asp49----Asn, were introduced into the domain by peptide synthesis. 1H-NMR spectroscopy was used to obtain the dissociation constants for calcium binding to these mutations. Calcium binding to the Asp49----Asn modified domain is only mildly affected (Kd = 6 mM, I = 0.15), whereas binding to the Asp47----Asn modified domain is severely reduced (Kd = 42 mM, I = 0.15). From these data, it is proposed that the anionic oxygen atoms of the side chains of residues 47 and 64 are essential for calcium binding, whereas the side chain ligand for calcium at residue 49 can be a carboxyamide oxygen. As a control, the introduction of the modification Glu78----Asp in a region of the domain not believed to be involved in calcium binding had very little effect on the Kd for calcium (Kd = 2.6 mM, I = 0.15). Finally, the effect of an Asp47----Gly substitution found in the natural haemophilia B mutant, factor IXAlabama, was investigated. This peptide has a markedly reduced affinity for calcium (Kd = 37 mM, I = 0.15), suggesting that the defect in factor IXAlabama is due to impaired calcium binding to its first EGF-like domain.

Amino Acid Sequence↗

PubMed1995

A Gly1127Ser mutation in an EGF-like domain of the fibrillin-1 gene is a risk factor for ascending aortic aneurysm and dissection.

Ascending aortic disease, ranging from mild aortic root enlargement to aneurysm and/or dissection, has been identified in 10 individuals of a kindred, none of whom had classical Marfan syndrome (MFS). Single-strand conformation analysis of the entire fibrillin-1 (FBN1) cDNA of an affected family member revealed a G-to-A transition at nucleotide 3379, predicting a Gly1127Ser substitution. The glycine in this position is highly conserved in EGF-like domains of FBN1 and other proteins. This mutation was present in 9 of 10 affected family members and in 1 young unaffected member but was not found in other unaffected members, in 168 chromosomes from normal controls, and in 188 chromosomes from other individuals with MFS or related phenotypes. FBN1 intragenic marker haplotypes ruled out the possibility that the other allele played a significant role in modulating the phenotype in this family. Pulse-chase studies revealed normal fibrillin synthesis but reduced fibrillin deposition into the extracellular matrix in cultured fibroblasts from a Gly1127Ser carrier. We postulate that the Gly1127Ser FBN1 mutation is responsible for reduced matrix deposition. We suggest that mutations such as this one may disrupt EGF-like domain folding less drastically than do substitutions of cysteine or of other amino acids important for calcium-binding that cause classical MFS. The Gly1127Ser mutation, therefore, produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes to ascending aortic aneurysm and dissection later in life.

Adult↗

PubMed1994

Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1.

Preadipocyte factor 1 (pref-1), a member of the EGF-like protein family, is a transmembrane protein with six tandem EGF-like repeats in the putative extracellular domain. Expression of pref-1 is abolished during the in vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of pref-1 in preadipocytes inhibits their differentiation [Smas, C.M., & Sul, H.S. (1993) Cell 73, 725-734]. In the present studies, we have isolated and characterized genomic clones for pref-1 and have identified multiple pref-1 transcripts generated by alternate splicing. The pref-1 gene consists of five exons and four introns spanning approximately 7.3 kb. By primer extension analysis, the transcription start site was determined to be 169 bp upstream from the translation initiation codon. We have identified functional promoter sequences by transient transfection using a 2.1 kb fragment of the pref-1 5' flanking region linked to a luciferase gene; the pref-1-luciferase fusion gene construct gave 20-fold higher promoter activity as compared to the promoterless vector. Analysis of exon-intron junctions reveals that unlike the majority of the mammalian EGF-like genes, EGF-like repeats of pref-1 are not encoded by discrete exons. Through RT-PCR and the isolation and analysis of multiple pref-1 cDNA clones, we have identified, in addition to full-length pref-1, five alternately spliced forms with various in-frame deletions of all or a part of the sixth EGF-like repeat, juxta-membrane, and predicted transmembrane domains.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1994

The Serrate locus of Drosophila and its role in morphogenesis of the wing imaginal discs: control of cell proliferation.

The Drosophila gene Serrate encodes a transmembrane protein with 14 EGF-like repeats in its extracellular domain. Here we show that loss-of-function mutations in this gene lead to larval lethality. Homozygous mutant larvae fail to differentiate the anterior spiracles, exhibit poorly developed mouth-hooks and show a severe reduction in the size of the wing and haltere primordia, which is not due to cell death. The few homozygous mutant escapers that pupariate develop into pharate adults that almost completely lack wings and halteres. Clonal analysis in the adult epidermis demonstrates a requirement for Serrate during wing and haltere development. Targeted ectopic expression of Serrate in the imaginal discs using the yeast transcriptional activator Gal4 results in regionally restricted induction of cell proliferation, e.g. the ventral tissues in the case of the wings and halteres. The results suggest that the wild-type function of Serrate is required for the control of position-specific cell proliferation during development of meso- and metathoracic dorsal discs, which in turn exerts a direct effect on morphogenesis.

Animals↗

PubMed1990

Fibulin is an extracellular matrix and plasma glycoprotein with repeated domain structure.

We have studied the expression of fibulin in cultured fibroblasts and determined its primary structure by cDNA cloning. Our results show that fibulin is a secreted glycoprotein that becomes incorporated into a fibrillar extracellular matrix when expressed by cultured cells or added exogenously to cell monolayers. In addition, we find that fibulin is present in plasma at a level of 33 +/- 3 micrograms/ml. Sequencing of multiple fibulin cDNAs indicates that a process of alternative splicing results in the expression of three fibulin transcripts. The transcripts encode overlapping polypeptides differing only in carboxy-terminal segments. Common to the three predicted forms of fibulin is a unique 537-amino acid-long cysteine-rich polypeptide and a 29-residue signal peptide. The amino-terminal portion of fibulin contains a repeated element with potential disulfide loop structure resembling that of the complement component anaphylatoxins C3a, C4a, and C5a as well as proteins of the albumin gene family. The bulk of the remaining portion of the molecule is a series of nine EGF-like repeats.

Amino Acid Sequence↗

PubMed1994

Fibulin-2 (FBLN2): human cDNA sequence, mRNA expression, and mapping of the gene on human and mouse chromosomes.

Fibulin-2 is a new extracellular matrix protein that we recently identified by characterizing mouse cDNA clones. Fibulin-2 mRNA is prominently expressed in mouse heart tissue and is present in low amounts in other tissues. In this study, we isolated and sequenced a 4.1-kb human fibulin-2 cDNA, which encoded a mature protein of 1157 amino acids preceded by a 27-residue signal sequence. The predicted polypeptide contains three consecutive anaphylatoxin-related segments (domain I) in its central region followed by 10 EGF-like repeats (domain II), 9 of which have a consensus sequence for calcium binding. The 408-residue N-terminal region consists of two separate subdomains, a cysteine-rich segment of 150 residues (Na subdomain) and a cysteine-free segment with a stretch of acidic amino acids (Nb subdomain). The 115-residue C-terminal segment (domain III) is similar to the C variant of fibulin-1. The amino acid sequences of the human and mouse fibulin-2 share approximately 90% identity in domains Na, I, II, and III but only 62% identity in domain Nb. The human cDNA lacks an EGF-like repeat, which is alternatively spliced in the mouse cDNA clones, and a potential cell-binding Arg-Gly-Asp sequence found in the Nb domain of the mouse counterpart. Northern blot analysis of mRNA from various human tissues reveals an abundant 4.5-kb transcript in heart, placenta, and ovary tissue. The expression pattern differs from that of fibulin-1. The fibulin-2 gene was localized by in situ hybridization to the p24-p25 region of human chromosome 3 and to the band D-E of mouse chromosome 6.

Amino Acid Sequence↗

PubMed1995

Structural characterization of two variants of fibulin-1 that differ in nidogen affinity.

Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains 1 (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed KD = 60 nM for variant C and KD > 1 microM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.

Amino Acid Sequence↗

PubMed1993

The evolution of the thrombospondin gene family.

Thrombospondin-1 is an adhesive glycoprotein that is involved in cellular attachment, spreading, migration, and proliferation. To date, four genes have been identified that encode for the members of the thrombospondin gene family. These four genes are homologous to each other in the EGF-like (type 2) repeats, the calcium-binding (type 3) motifs, and the COOH-terminal. The latter has been reported to be a cell-binding domain in thrombospondin-1. Phylogenetic trees have been constructed from the multisequence alignment of thrombospondin sequences from human, mouse, chicken, and frog. Two different algorithms generate comparable results in terms of the topology and the branch lengths. The analysis indicates that an early form of the thrombospondin gene duplicated about 925 million years ago. The gene duplication that produced the thrombospondin-1 and -2 branches of the family is predicted to have occurred 583 million years ago, whereas the gene duplication that produced the thrombospondin-3 and -4 branches of the family is predicted to have occurred 644 million years ago. These results indicate that the members of the thrombospondin gene family have existed throughout the evolution of the animal kingdom and thus probably participate in functions that are common to most of its members.

Amino Acid Sequence↗

PubMed1993

The role of calcium in the organization of fibrillin microfibrils.

The microfibrillar glycoprotein fibrillin has a multidomain structure which contains forty-three epidermal growth factor-like motifs with calcium-binding consensus sequences. We have utilized intact microfibrils isolated from human dermal fibroblast cultures to investigate the putative influence of bound calcium on microfibrillar organization and integrity. Incubation with EDTA or EGTA rapidly resulted in gross disruption of microfibril morphology. The treatment induced disorganization of the interbead domains although the regular beaded arrangement was always apparent. These changes were readily reversible on replacing calcium, indicating that the treatment had not compromised microfibrillar integrity. The data localize calcium binding EGF-like repeats to the interbead domains and indicate that lateral packing of fibrillin monomers is calcium-dependent. This arrangement suggests how mutations in epidermal growth factor-like domains of fibrillin might cause the disruption in microfibril organization and interactions which underlies the clinical symptoms of some Marfan syndrome patients.

Adult↗

PubMed1995

EMR1, an unusual member in the family of hormone receptors with seven transmembrane segments.

Proteins with seven transmembrane segments (7TM) define a superfamily of receptors (7TM receptors) sharing the same topology: an extracellular N-terminus, three extramembranous loops on either side of the plasma membrane, and a cytoplasmic C-terminal tail. Upon ligand binding, cytoplasmic portions of the activated receptor interact with heterotrimeric G-coupled proteins to induce various second messengers. A small group, recently recognized on the basis of homologous primary amino acid sequences, comprises receptors to hormones of the secretin/vasoactive intestinal peptide/glucagon family, parathyroid hormone and parathyroid hormone-related peptides, growth hormone-releasing factor, corticotropin-releasing factor, and calcitonin. A cDNA, extracted from a neuroectodermal cDNA library, was predicted to encode a new 886-amino-acid protein with three distinct domains. The C-terminal third contains the seven hydrophobic segments and characteristic residues that allow the protein to be readily aligned with the various hormone receptors in the family. Six egf-like modules, at the N-terminus of the predicted mature protein, are separated from the transmembrane segments by a serine/threonine-rich domain, a feature reminiscent of mucin-like, single-span, integral membrane glycoproteins with adhesive properties. Because of its unique characteristics, this putative egf module-containing, mucin-like hormone receptor has been named EMR1. Southern analysis of a panel of somatic cell hybrids and fluorescence in situ hybridization have assigned the EMR1 gene to human chromosome 19p13.3.

Amino Acid Sequence↗

PubMed1990

The gene Serrate encodes a putative EGF-like transmembrane protein essential for proper ectodermal development in Drosophila melanogaster.

Mutations in the third chromosome gene Serrate are shown to display genetic interactions with specific alleles of the neurogenic locus Notch, which encodes a transmembrane protein with epidermal growth factor (EGF) homology. Embryonic lethal Serrate mutations exhibit epidermal and neuronal defects, which are reminiscent of those produced by mutations in the Drosophila EGF receptor homolog gene. We present the molecular cloning of Serrate and show that it encodes two coordinately expressed transcripts from a genomic interval greater than 30 kb in length. The deduced protein product of 1404 amino acids contains a single transmembrane domain and 14 EGF-like repeats. Thus, Serrate represents another member of the group of EGF-containing loci in Drosophila. Whole-mount in situ hybridization analysis reveals complex temporal and spatial patterns of RNA expression consistent with the epidermal and neuronal defects observed in mutant embryos. Finally, we discuss the implications of Serrate function within the context of other cell-surface molecules known to be involved in the differentiation of ectodermally derived tissues.

Alleles↗

PubMed1990

Entactin: structure and function.

Entactin is an integral and ubiquitous component of the basement membrane. The amino acid sequences of the mouse and human molecules have been determined and exhibit 85% sequence identity. The molecule is organized into three structural domains, an N-terminal globule (I) is linked to a smaller C-terminal globule (III) by a rigid stalk (II) largely consisting of cysteine-rich EGF-like homology repeats and a cysteine-rich thyroglobulin homology repeat. The molecule binds calcium ions and supports cell adhesion. However, its major function may be the assembly of the basement membrane. The carboxyl globule binds tightly to one of the short arms of laminin at the inner rodlike segment. This same region is also believed to be responsible for the attachment of entactin to type IV collagen at approximately 80 nm from its carboxyl noncollagenous end. Entactin therefore could serve as a bridge between the two most abundant molecules in the basement membrane. Supporting evidence for this role has been obtained from transfection of human choriocarcinoma, JAR, cells with the entactin gene. JAR cells synthesize laminin and type IV collagen but not entactin. Transfection of entactin into the cells stimulated incorporation of laminin and type IV collagen along with entactin into the extracellular matrix and into structures resembling focal contacts. The calcium-binding activity of entactin may play a role in the matrix assembly process. The protease sensitivity of entactin suggests that it may be a target for proteolytic activity during tissue remodeling, metastasis, and other events requiring the turnover of the basement membrane.

Amino Acid Sequence↗

PubMed1992

How an epidermal growth factor (EGF)-like domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X.

Domains homologous to the epidermal growth factor (EGF) are important building blocks for extracellular proteins. Proteins containing these domains have been shown to function in such diverse biological processes as blood coagulation, complement activation, and the developmental determination of embryonic cell fates. Many of these proteins require calcium for their biological function. In the case of coagulation factors IX and X and anticoagulants proteins C and S, calcium has been found to bind to the EGF-like domains. We have now determined the three-dimensional structure of the calcium-bound form of the NH2-terminal EGF-like domain in coagulation factor X by two-dimensional NMR and simulated folding. Ligands to the calcium ion are the two backbone carbonyls in Gly-47 and Gly-64, as well as the side chains in Gln-49, erythro-beta-hydroxyaspartic acid (Hya) 63, and possibly Asp-46. The conserved Asp-48 is not a ligand in our present structures. The remaining ligands are assumed to be solvent molecules or, in the intact protein, ligands from neighboring domains. Other proteins interacting in a calcium-dependent manner may also contribute ligands. A comparison with the calcium-free form shows that calcium binding induces strictly local structural changes in the domain. Residues corresponding to the side chain ligands in factor X are conserved in many other proteins, such as the integral membrane protein TAN-1 of human lymphocytes and its developmentally important homolog, Notch, in Drosophila. Calcium binding to EGF-like domains may be crucial for numerous protein-protein interactions involving EGF-like domains in coagulation factors, plasma proteins, and membrane proteins. Therefore, there is reason to believe that this novel calcium site plays an important role in the biochemistry of extracellular proteins.

Amino Acid Sequence↗

PubMed1990

Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S.

Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.

Amino Acid Sequence↗