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PubMed1998

The exon structure of the human MAGP-2 gene. Similarity with the MAGP-1 gene is confined to two exons encoding a cysteine-rich region.

A cDNA for human microfibril-associated glycoprotein-2 (MAGP-2) was used to screen a human leukocyte genomic DNA library in EMBL-3 vector. One clone, clone H (10 kilobase pairs (kbp)), was isolated that contained most of the MAGP-2 gene. The remainder of the 3' end of the gene was obtained by direct polymerase chain reaction amplification of genomic DNA. The human MAGP-2 gene was found to be about 11 kbp in size and to contain 10 evenly distributed exons. The internal exons range in size from 30 base pairs (bp) to 88 bp with exons 4 and 6 the only exons of equal size (45 bp). All internal intron:exon junctions are defined by canonical splice donor and acceptor sites. Each junction has a 1/2 codon split with the exception of the exon 8/9 junction, which has a 2/1 split. The translation initiation codon is in exon 2, and the final exon contains 110 bp of coding sequence, including 2 cysteine codons. Primer extension experiments identified only one major transcription initiation site, 213 bases upstream of the ATG site. Rapid analysis of cDNA ends-polymerase chain reaction analysis of the 5' end of MAGP-2 mRNA from placenta confirmed this result and did not detect any alternative splicing of transcripts. The putative promoter region of the MAGP-2 gene was found to be AT-rich and it lacked a TATA box and other common regulatory elements. However the sequence surrounding the transcription start site CTCA(+1)TTCC was similar to the consensus CTCA(+1)NTCT (N is any nucleoside) for an initiator element found in terminal deoxynucleotidyltransferase and a number of other highly regulated genes. Comparison with the previously characterized human MAGP-1 gene showed that structural similarity was largely confined to the exact size, sequence, and junction alignment of the two penultimate exons which encode the first six of the seven cysteine residues that are precisely spaced in both proteins. The findings are consistent with the growing evidence that, although MAGP-1 and MAGP-2 are both intimately involved in the biology of fibrillin-containing microfibrils, the MAGPs are structurally, functionally, and developmentally diverse proteins which share one characteristic cysteine-rich motif.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed2001

Protein interaction studies of MAGP-1 with tropoelastin and fibrillin-1.

Elastic fibers consist primarily of an amorphous elastin core associated with microfibrils, 10-12 nm in diameter, containing fibrillins and microfibril-associated glycoproteins (MAGPs). To investigate the interaction of MAGP-1 with tropoelastin and fibrillin-1, we expressed human MAGP-1 as a T7-tag fusion protein in Escherichia coli. Refolding of the purified protein produced a soluble form of MAGP-1 that displayed saturable binding to tropoelastin. Fragments of tropoelastin corresponding to the N-terminal, C-terminal, and central regions of the molecule were used to characterize the MAGP-1 binding site. Cleavage of tropoelastin with kallikrein, which cleaves after Arg(515) in the central region of the molecule, disrupted the interaction, suggesting that the separated N- and C-terminal fragments were insufficient to determine MAGP-1 binding to intact tropoelastin. In addition, no evidence of an interaction was observed between MAGP-1 and a tropoelastin construct consisting of domains 17-27 that brackets the kallikrein cleavage site, suggesting a complex mechanism of interaction between the two molecules. Binding of MAGP-1 was also tested with overlapping recombinant fibrillin-1 fragments. MAGP-1 bound to a region at the N terminus of fibrillin-1 in a calcium-dependent manner. In summary, these results suggest a model for the interaction of elastin with the microfibrillar scaffold.

Contractile Proteins↗
PubMed2001

Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly.

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg(45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Thr(1486)) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by beta-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.

Base Sequence↗
PubMed2002

Identification of a matrix-binding domain in MAGP1 and MAGP2 and intracellular localization of alternative splice forms.

MAGP1 is a small molecular mass protein associated with microfibrils in the extracellular matrix (ECM). To identify the molecular basis of its interaction with other microfibrillar proteins, deletion constructs of MAGP1 were expressed in a mammalian cell system that served as a model for microfibril assembly. This study identified a 54-amino acid sequence in the carboxyl-terminal region of the protein that defines a matrix-binding domain that is sufficient to target MAGP1 to the ECM. Site-directed mutagenesis demonstrated that binding activity is dependent on the presence of 7 cysteine residues in this sequence. MAGP2 contains a sequence similar to the matrix-binding domain of MAGP1, but could not associate with the ECM because of a single amino acid change. Two naturally occurring MAGP1 splice variants, MAGP1B (human-specific) and MAGP1D (found in mice), localized intracellularly when expressed as chimeric proteins with green fluorescent protein in rat lung fibroblasts. This suggests a second action site for MAGP1.

Alternative Splicing↗
PubMed2002

Effects on collagen VI mRNA stability and microfibrillar assembly of three COL6A2 mutations in two families with Ullrich congenital muscular dystrophy.

We recently reported a severe deficiency in collagen type VI, resulting from recessive mutations of the COL6A2 gene, in patients with Ullrich congenital muscular dystrophy. Their parents, who are all carriers of one mutant allele, are unaffected, although heterozygous mutations in collagen VI caused Bethlem myopathy. Here we investigated the consequences of three COL6A2 mutations in fibroblasts from patients and their parents in two Ullrich families. All three mutations lead to nonsense-mediated mRNA decay. However, very low levels of undegraded mutant mRNA remained in patient B with compound heterozygous mutations at the distal part of the triple-helical domain, resulting in deposition of abnormal microfibrils that cannot form extensive networks. This observation suggests that the C-terminal globular domain is not essential for triple-helix formation but is critical for microfibrillar assembly. In all parents, the COL6A2 mRNA levels are reduced to 57-73% of the control, but long term collagen VI matrix depositions are comparable with that of the control. The almost complete absence of abnormal protein and near-normal accumulation of microfibrils in the parents may account for their lack of myopathic symptoms.

Alleles↗
PubMed1994

Structure and expression of fibrillin-2, a novel microfibrillar component preferentially located in elastic matrices.

During the previous cloning of the fibrillin gene (FBN1), we isolated a partial cDNA coding for a fibrillin-like peptide and mapped the corresponding gene (FBN2) to human chromosome 5. (Lee, B., M. Godfrey, E. Vitale, H. Hori, M. G. Mattei, M. Sarfarazi, P. Tsipouras, F. Ramirez, and D. W. Hollister. 1991. Nature [Lond.]. 352:330-334). The study left, however, unresolved whether or not the FBN2 gene product is an extracellular component structurally related to fibrillin. Work presented in this report clarifies this important point. Determination of the entire primary structure of the FBN2 gene product demonstrated that this polypeptide is highly homologous to fibrillin. Immunoelectron microscopy localized both fibrillin proteins to elastin-associated extracellular microfibrils. Finally, immunohistochemistry revealed that the fibrillins co-distribute in elastic and non-elastic connective tissues of the developing embryo, with preferential accumulation of the FBN2 gene product in elastic fiber-rich matrices. These results support the original hypothesis that the fibrillins may have distinct but related functions in the formation and maintenance of extracellular microfibrils. Accordingly, we propose to classify the FBN1 and FBN2 gene products as a new family of extracellular proteins and to name its members fibrillin-1 and fibrillin-2, respectively.

Amino Acid Sequence↗
PubMed1962

An electron microscope study of radiation damage in the mouse oocyte.

Cytological changes occurring in young oocytes of the mouse, following whole-body x-irradiation, have been examined by light and electron microscopy. Two minutes after a dose of 200 r, a drop occurs in the number of mitochondria per oocyte. The normal number of mitochondria is restored in the next few minutes. Five to 6 hours after a dose of 7 r, and 30 to 45 minutes following a dose of 200 r, all the oocytes are markedly contracted. In the next 2 hours (at a dose of 7 r), some cells shrink further and become pycnotic, while others expand and show signs of karyolysis. Of the expanded cells, some (about 50 per cent of total young oocytes) become morphologically normal. In both pycnotic and karyolytic nuclei, the nucleolus was contracted but without loss of its fibrillar structure. The contracted nucleolus appeared similar to those seen in some nearly mature normal oocytes (these oocytes have a high natural death rate). No other cell type in the ovary was affected by doses of x-rays up to 200 r. Observations on the nucleus of the normal oocyte are included. In the dictyate stage of meiotic prophase the chromosomes were dispersed into bundles of 100 A microfibrils. The main component of the nucleolus was found to be tightly coiled fibrils of 60 to 100 A, which appear to have a close relationship with the microfibrils of the chromosomes.

Animals↗
PubMed1956

A study of chromosomes with the electron microscope.

Amphibian lampbrush chromosomes and meiotic prophase chromosomes of various insects and plants consist of a bundle of microfibrils about 500 A thick. These fibrils are double, being made of two closely associated fibrils 200 A thick. Fragments of interphase nuclei contain a mass of fibrils 200 A thick. Ultrathin sections through nuclei in prophase or interphase show sections of these double or single fibrils cut at various angles. A comparison of sections with the methacrylate left in and sections that were shadowed after removing the methacrylate suggests that the OsO(4) reacts only with the outer part of the fibrils either because it does not penetrate, or as a result of a chemical difference of the inner core and the outside of the fibril. It is suggested that in analogy to the structure of the tobacco mosaic virus the chromosomal microfibril may have an inner core of DNA surrounded by a shell of protein.

Cell Nucleus↗
PubMed1964

DNA AND THE FINE STRUCTURE OF SYNAPTIC CHROMOSOMES IN THE DOMESTIC ROOSTER (GALLUS DOMESTICUS).

The indium trichloride method of Watson and Aldridge (38) for staining nucleic acids for electron microscopy was employed to study the relationship of DNA to the structure of the synaptinemal complex in meiotic prophase chromosomes of the domestic rooster. The selectivity of the method was demonstrated in untreated and DNase-digested testis material by comparing the distribution of indium staining in the electron microscope to Feulgen staining and ultraviolet absorption in thicker sections seen with the light microscope. Following staining by indium, DNA was found mainly in the microfibril component of the synaptinemal complex. When DNA was known to have been removed from aldehyde-fixed material by digestion with DNase, indium stainability was also lost. However, staining of the digested material with non-selective heavy metal techniques demonstrated the presence of material other than DNA in the microfibrils and showed that little alteration in appearance of the chromosome resulted from DNA removal. The two dense lateral axial elements of the synaptinemal complex, but not the central one to any extent, also contained DNA, together with non-DNA material.

Animals↗
PubMed1970

Scale formation in chrysophycean algae. I. Cellulosic and noncellulosic wall components made by the Golgi apparatus.

The cell wall of the marine chrysophycean alga Pleurochrysis scherfellii is composed of distinct wall fragments embedded in a gelatinous mass. The latter is a polysaccharide of pectic character which is rich in galactose and ribose. These wall fragments are identified as scales. They have been isolated and purified from the vegetative mother cell walls after zoospore formation. Their ultrastructure is described in an electron microscope study combining sectioning, freeze-etch, and negative staining techniques. The scales consist of a layer of concentrically arranged microfibrils (ribbons with cross-sections of 12 to 25 x 25 to 40 A) and underlying radial fibrils of similar dimensions. Such a network-plate is densely coated with particles which are assumed to be identical to the pectic component. The microfibrils are resistant to strong alkaline treatment and have been identified as cellulose by different methods, including sugar analysis after total hydrolysis, proton resonance spectroscopical examination (NMR spectroscopy) of the benzoylated product, and diverse histochemical tests. The formation and secretion of the scales can be followed along the maturing Golgi cisternae starting from a pronounced dilated "polymerization center" as a completely intracisternal process which ends in the exocytotic extrusion of the scales. The scales reveal the very same ultrastructure within the Golgi cisternae as they do in the cell wall. The present finding represents the first evidence on cellulose formation by the Golgi apparatus and is discussed in relation to a basic scheme for cellulose synthesis in plant cells in general.

Cell Wall↗
PubMed1971

The fine structure of bovine nasal cartilage. Extraction as a technique to study proteoglycans and collagen in cartilage matrix.

Bovine nasal cartilage was studied by electron microscopy before and after extraction with 4 M guanidinium chloride or 1.9 M CaCl(2). These solvents removed matrix granules, basophilia, and 85% of the proteoglycan complex, measured as hexuronate. Simultaneously, many collagen fibrils were disaggregated into component microfibrils (approximately 40 A thick). In contrast to the above solvents, exhaustive extraction with 0.5 M guanidinium chloride removed 20% of the proteoglycan complex, and matrix granules were reduced in size but not in number. Extraction with 4 M CaCl(2) removed only 10% of the proteoglycan complex, did not remove matrix granules, and caused the normal banding pattern of collagen to disappear. The banding was restored by further treatment with trypsin. Trypsin, before or after 4 M CaCl(2), removed matrix granules and 90% of the proteoglycan complex. We conclude that matrix granules are an electron microscopic representation of the proteoglycan complex, and consist of more than one proteoglycan macromolecule. It would appear that 4 M guanidinium chloride and 1.9 M CaCl(2), in addition to removing most of the proteoglycan complex, also disaggregate some of the collagen fibrils into their component microfibrils.

Animals↗
PubMed1971

The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers.

Smooth muscle derived from the inner media and intima of immature guinea pig aorta were grown for up to 8 wk in cell culture. The cells maintained the morphology of smooth muscle at all phases of their growth in culture. After growing to confluency, they grew in multiple overlapping layers. By 4 wk in culture, microfibrils (110 A) appeared within the spaces between the layers of cells. Basement membrane-like material also appeared adjacent to the cells. Analysis of the microfibrils showed that they have an amino acid composition similar to that of the microfibrillar protein of the intact elastic fiber. These investigations coupled with the radioautographic observations of the ability of aortic smooth muscle to synthesize and secrete extracellular proteins demonstrate that this cell is a connective tissue synthetic cell.

Amino Acids↗
PubMed1973

Structures linking the myonemes, endoplasmic reticulum, and surface membranes in the contractile ciliate Vorticella.

An electron microscope investigation of the interface between the myonemes of Vorticella convallaria and their associated endoplasmic reticulum (ER) has revealed structures of a complex morphology linking these two organelles. These structures are named "linkage complexes". Each complex contains a spindle-shaped midpiece which lies in a groove of the ER membrane. Microfilaments splay out from the tips of the midpiece and may come in contact with the inner alveolar sac membrane. Three to six raillike structures lie on each side of the midpiece and parallel it. The ER membrane appears to pass through the sides of the rails. In the lumen of the ER these rails are associated with a meshwork of filaments. A cradle of five rods lies within the groove under the midpiece. The ER membrane also passes through these rods which contact the same meshwork. In the scopular region and in the stalk the microfilaments from the midpiece form a bundle which passes into the lumen of modified basal bodies. These basal bodies are connected to the alveolar sac which, in the stalk, passes as a flattened tube along its length. The parts of the dissociated linkage complex are scattered throughout the spasmoneme of the stalk along membranes of the intraspasmonemal tubules. Thus, both stalk and body contractile bundles have linkage complexes that link their associated membrane systems to the microfibrils and, in turn, connect this membrane-microfibrillar interface to the pellicular membranes. The arrangement of the linkage complex suggests an involvement in the control of the transport of calcium ions between ER and microfibrils, and possibly the transfer of a message from the surface membranes to the sites of calcium release to trigger myonemal contraction.

Animals↗
PubMed1976

Synthesis of basement membrane collagen by cultured human endothelial cells.

Studies were performed to determine if cultured human endothelial cells synthesized basement membrane collagen. In culture, endothelial cells were attached to grossly visible membranous structures which on light microscopy were composed of ribbons of dense, amorphous material. On transmission electron microscopy, these membranous structures consisted of amorphous basement membrane, and material morphologically similar to microfibrils and elastic fibers. By immunofluorescence microscopy, these membranous structures stained brightly with antisera to human glomerular basement membrane. Cultured endothelial cells incorporated [3H]proline into protein; 18% of the incorporated [3H]proline was solubilized by purified collagenase. When endothelial cells were cultured with [14C]proline, 7.1% of the incorporated counts were present as [14C]hydroxyproline. Cultured endothelial cells were labeled with [3H]glycine and [3H]proline and digested with pepsin. The resulting fractions on analysis by SDS-polyacrylamide gel electrophoresis contained two radioactive protein peaks of mol wt 94,200 and 120,500. Both these peaks disappeared after digestion with purified collagenase. The peak of mol wt 120,500 corresponds to that of alpha1 (IV) collagen; the peak of the mol wt 94,200 probably corresponds to that of alpha1 (III) collagen. Thus, cultured human endothelial cells synthesize material which is morphologically and immunologically like amorphous basement membrane and biochemically like basement membrane collagen. Cultured endothelial cells probably also synthesize material which is morphologically similar to microfibrils and elastic fibers.

Basement Membrane↗
PubMedDate not supplied

Fibrillin-1 misfolding and disease.

Fibrillin-1 is a 350 kDa calcium-binding protein which assembles to form 10-12 nm microfibrils in the extracellular matrix (ECM). The structure of fibrillin-1 is dominated by two types of disulfide-rich motifs, the calcium- binding epidermal growth factor-like (cbEGF) and transforming growth factor beta binding protein-like (TB) domains. Disruption of fibrillin-1 domain structure and function contributes to the pathogenic mechanisms underlying two inherited diseases with very different etiologies: Marfan syndrome (MFS) and homocystinuria (HC). MFS is a connective tissue disease caused by mutations in the fibrillin-1 gene FBN1. Many missense mutations cause variable degrees of fibrillin-1 domain misfolding, which may affect the delivery of fibrillin-1 to the ECM and/or its assembly into microfibrils. HC is a metabolic disorder which affects methionine metabolism and results in raised serum levels of the highly reactive thiol-containing amino acid homocysteine. Patients with HC often exhibit ocular and skeletal defects resembling the MFS phenotype, suggesting that elevated homocysteine levels may lead to chemical reduction of disulfide bonds within fibrillin-1 domains resulting in the loss of native structure. Protein misfolding therefore is implicated in pathogenic mechanisms underlying MFS and HC.

Amino Acid Sequence↗
PubMed1993

The molecular basis of Marfan syndrome.

The Marfan syndrome is an inherited, autosomal dominant disorder that affects the skeletal, ocular, and cardiovascular systems. Recent biochemical and genetic studies have demonstrated that this deadly genetic disorder arises from defects in the connective tissue protein fibrillin. Fibrillin is a component of microfibrils, structures found in the extracellular matrices of most tissues, including those affected in Marfan patients. The appearance of microfibrils in the matrix produced by Marfan patient fibroblasts is different from that of normal cells. Genetic linkage between the fibrillin gene and the Marfan phenotype has been established and the gene mapped to the same chromosomal position as the disease locus. In several instances, the disease has been associated with mutations in the fibrillin gene, confirming that defects in fibrillin cause the Marfan syndrome.

Amino Acid Sequence↗
PubMed2001

Mutation of the gene encoding fibrillin-2 results in syndactyly in mice.

Fibrillins are large, cysteine-rich glycoproteins that form microfibrils and play a central role in elastic fibrillogenesis. Fibrillin-1 and fibrillin-2, encoded by FBN1 on chromosome 15q21.1 and FBN2 on chromosome 5q23-q31, are highly similar proteins. The finding of mutations in FBN1 and FBN2 in the autosomal dominant microfibrillopathies Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), respectively, has highlighted their essential role in the development and homeostasis of elastic fibres. MFS is characterized by cardiovascular, skeletal and ocular abnormalities, and CCA by long, thin, flexed digits, crumpled ears and mild joint contractures. Although mutations arise throughout FBN1, those clustering within exons 24-32 are associated with the most severe form of MFS, so-called neonatal MFS. All the mutations described in CCA occur in the "neonatal region" of FBN2. Both MFS and CCA are thought to arise via a dominant negative mechanism. The analysis of mouse mutations has demonstrated that fibrillin-1 microfibrils are mainly engaged in tissue homeostasis rather than elastic matrix assembly. In the current investigation, we have analysed the classical mouse mutant shaker-with-syndactylism using a positional candidate approach and demonstrated that loss-of-function mutations outside the "neonatal region" of Fbn2 cause syndactyly in mice. These results suggest that phenotypes distinct from CCA may result in man as a consequence of mutations outside the "neonatal region" of FBN2.

Amino Acid Sequence↗