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PubMed1999

N-terminal domains of fibrillin 1 and fibrillin 2 direct the formation of homodimers: a possible first step in microfibril assembly.

Aggregation of fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of fibrillin 1 and fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of fibrillin 1 and fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril.

Alkylation↗
PubMed1992

Characterization of an associated microfibril protein through recombinant DNA techniques.

The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.

Amino Acid Sequence↗
PubMed2004

The pattern of fibrillin deposition correlates with microfibril-associated glycoprotein 1 (MAGP-1) expression in cultured blood and lymphatic endothelial cells.

Fibrillins constitute the major structural components of 10-12nm microfibrils of the extracellular matrix of several elastic and non elastic tissues and of initial lymphatic vessel anchoring filaments. Microfibril-Associated Glycoprotein-1 (MAGP-1) binds fibrillin to tropoelastin during elastogenesis. We recently reported that cultured blood endothelial cells deposit fibrillin in a honeycomb pattern, whereas lymphatic endothelial cells form an irregular web. The aim of this immunohistochemical study was to verify whether the deposition pattern of fibrillin is related to the expression of MAGP-1 in confluent and postconfluent cultures of bovine aortic (AEC), pulmonary artery (PAEC) and lymphatic endothelial cells (LEC). In AEC and PAEC, MAGP-1 and fibrillin co-localized and their deposition increased with time in culture. In AEC, both proteins formed a honeycomb pattern. In LEC, MAGP-1 deposition was still negligible when fibrillin formed an irregular web covering the entire surface. PAEC, which in vivo are exposed to physiological conditions intermediate between AEC and LEC, had an intermediate pattern of deposition of fibrillin and MAGP-1. Assuming that early elastogenesis is an intrinsic functional need for the aorta, but not for the thoracic duct, we propose that delayed appearance of MAGP-1 in LEC may correlate with their irregular fibrillin deposition. Different fibrillin scaffolds could in turn account for the specificity of elastic fibers in compliance with the specific functional requirements of the tissue.

Animals↗
PubMed1988

Characterization of an antibody directed against a 128 kDa glycoprotein involved in the thrombogenicity of the elastin-associated microfibrils.

We have developed a monospecific antiserum directed against a major glycoprotein in the elastin-associated microfibrils with an apparent molecular mass of 128 kDa (GP 128). When immunoblotting or enzyme-linked immunosorbent microassay was used, its IgGs recognized thrombospondin in a platelet lysate, but did not react with several basement-membrane-derived macromolecules, nor with plasma fibronectin. Similar patterns of immunofluorescence and immunoperoxidase were found after incubation of endothelial cells with either anti-GP 128 or anti-(platelet thrombospondin) IgGs. Both antibodies inhibited the microfibrils- and thrombin-induced platelet aggregation, and were without effect on the aggregation by other inducers. These results confirm that there is an antigenic homology between GP 128 and thrombospondin.

Blood Coagulation↗
PubMed1981

Structure of rigid-layer of Rhizobium cell wall. I. Purification on the peptidoglycan from the cellulose microfibrils.

The bag shaped peptidoglycan layer of Rhizobium cell wall was isolated from intact cells after treatment with sodium dodecylsulfate and trypsin, chymotrypsin or pepsin digestion. Results of chemical analysis of acid hydrolyzed peptidoglycan revealed beside two amino sugars: glucosamine and muramic acid, three major amino acids; alanine, glutamic acid and 2,6-diaminopimelic acid and also significant amount of glucose. Evidence were provided that the polyglucose found in peptidoglycan preparations of three strains of Rhizobium trifolii, one of Rhizobium leguminosarum and one of Rhizobium meliloti consist of cellulose microfibrils. The content of cellulose present in Rhizobium peptidoglycans ranged from 60 to 80%. Methods of peptidoglycan purification from the cellulose microfibrils are described.

Amino Acids↗
PubMed1983

Structure of the rigid-layer of Rhizobium cell wall. III. Electron microscopic evidence for the cellulose microfibrils association with peptidoglycan sacculi.

The morphology of peptidoglycan layer of Rhizobium cell wall was examined by transmission electron microscopy. Peptidoglycans were isolated from intact cells after treatment with sodium dodecyl sulfate, extraction with aqueous 45% phenol and then with a mixture of chloroform-methanol. Finally rigid layers were digested with trypsin and chymotrypsin. The results indicate the presence of lump or bar-like structures on the surface of the cell shaped peptidoglycan sacculi. Evidence is provided suggesting that the cellulose microfibrils arise directly from these excrescences found on the peptidoglycan surface. Digestion with cellulase removed all cellulose microfibrils whereas the lumps and bars remained as an integral part of the Rhizobium peptidoglycan.

Cell Wall↗
PubMed1981

Immunohistochemical comparison of ocular zonules and the microfibrils of elastic tissue.

Antibody to bovine zonules raised in rabbits gave diffuse staining of zonular fibrils with prominent accumulations at 35 to 45 nm sites of overbanding by means of the indirect immunoperoxidase technique. Antibody binding occurred in both fresh and paraffin-embedded material and was not species specific. The same pattern of heavy antibody binding and 35 to 45 nm periodicity was found on the microfibrils of elastic tissue in human and bovine ciliary body, calf ligamentum nuchae, and chick aorta. This cross reaction is the first direct evidence of a structural similarity or identity between components of the zonules and the microfibrils of elastic tissue, correlating with similarities in their morphology and amino acid profiles. Interstitial collagen fibers appear to have a minimal quantity of antigenically similar material at cross-banding sites. These relationships may hold important clues to the site of systemic involvement in connective tissue diseases associated with lens dislocation. Zonular antibody should prove useful in investigating abnormalities of the zonular-elastic microfibrillar system and cross-banding sites.

Animals↗
PubMed2005

36-kDa microfibril-associated glycoprotein (MAGP-36) is an elastin-binding protein increased in chick aortae during development and growth.

MAGP-36 was discovered in porcine aorta in 1989 and is thought to be one of the microfibril-associated proteins. MAGP-36 has been localized on the surface of elastic fibers or laminae in immunohistochemical studies. However, its functional role in the aorta is obscure. Herein, we report on the binding activity of MAGP-36 to components of the aortic wall and its accumulation pattern in the aorta during development and growth. In vitro, MAGP-36 bound to elastin and collagen in a Ca(2+)-dependent manner, and mediated the adhesion of human aortic smooth muscle cells. This cell adhesion mostly depended on the RGD-containing domain of MAGP-36. We examined the accumulation of MAGP-36 with quantitative Western blot analysis and immunoelectron microscopy in chick aortae during development and growth. The amount of MAGP-36 increased on the surface of elastic fibers or laminae between days 14 and 34 after the start of incubation, and reached a plateau at about 53 days. This accumulation of MAGP-36 roughly correlated with an increase in blood pressure for this period. Thus, MAGP-36 might be a bridging protein that connects elastin to other components of the aortic wall and might play a role in maintaining the integrity of the aortic structure under arterial pressure.

Animals↗
PubMed2004

Differential expression of fibrillin-3 adds to microfibril variety in human and avian, but not rodent, connective tissues.

The human genome contains three fibrillins: FBN1 and FBN2, both well characterized, and FBN3, reported only as a cDNA sequence. Like FBN2, the highest expression levels of FBN3 were found in fetal tissues, with only low levels in postnatal tissues. Immunolocalization demonstrated fibrillin-3 in extracellular microfibrils abundant in developing skeletal elements, skin, lung, kidney, and skeletal muscle. Unlike the other two fibrillins, FBN3 expression is high in brain, and FBN3 is alternatively spliced, removing the exon encoding cbEGF2. Like FBN1, FBN3 contains three alternate exons in the 5' UTR. While FBN3 orthologs were identified in cow and chicken, Fbn3 appears to have been inactivated in the mouse genome, perhaps during chromosome fission events. Located on chromosome 19p13.3-13.2, FBN3 is a candidate gene for Weill-Marchesani syndrome.

Alternative Splicing↗
PubMed2003

Discrete integration of collagen XVI into tissue-specific collagen fibrils or beaded microfibrils.

The structural and functional diversity of extracellular matrices is determined, not only by individual macromolecules, but even more decisively, by the alloyed aggregates they form. Although quantitatively major matrix molecules can occur ubiquitously, their organization varies from one tissue to another due to their amalgamation with specific sets of minor components. Here, we show that the fibril-associated collagen with interrupted triple helices collagen XVI is unique in that, depending on the tissue context, it can be incorporated into distinct suprastructural aggregates. In papillary dermis, the protein unexpectedly does not occur in banded collagen fibrils, but rather, is a component of specialized fibrillin-1-containing microfibrils. In territorial cartilage matrix, however, collagen XVI is not a component of aggregates containing fibrillin-1. Instead, the protein resides in a discrete population of thin, weakly banded collagen fibrils also containing collagens II and XI. Collagen IX also occurs in this population of fibrils, but at longitudinal locations discrete from those of collagen XVI. This suprastructural versatility of a collagen is without precedent and highlights pivotal differences in the tissue-specific organization of matrix aggregate structures.

Adult↗
PubMed2003

A sulphur matrix complex, elastic fibril composed of a fine core of amorphous elastin and microfibrils was largely accumulated in the aortic intima of aged rats.

We examined elastic fibrils in the aortic intima of aged rats with elastin staining, elastase digestion and X-ray microanalysis. The innermost elastic lamina, heavily stained by a brief treatment with azure II-toluidine blue without heating, was thin and fragmented, and an amorphous substance that stained weakly to moderately was accumulated in the thick subendothelial space of 26-28-month-old rat aortas. The substance was always present in the intimas; to a large extent in the aged rats but rarely in 5-month-old rats, and disappeared after digestion with elastase. The amorphous substance was identified as elastic fibrils approximately 0.1-0.2 microm in diameter under an electron microscope with tannic acid-uranyl acetate/lead citrate staining. Elastase digestion revealed that elastic fibrils were composed of a number of microfibrils, which were 10-12 nm in diameter, in and around a fine core of amorphous elastin. X-ray microanalysis revealed a clear peak of sulphur in the elastic fibrils.

Age Factors↗
PubMed2005

Polymorphisms in cinnamoyl CoA reductase (CCR) are associated with variation in microfibril angle in Eucalyptus spp.

Linkage disequilibrium (LD) mapping using natural populations results in higher resolution of marker-trait associations compared to family-based quantitative trait locus (QTL) studies. Depending on the extent of LD, it is possible to identify alleles within candidate genes associated with a trait. Analysis of a natural mutant in Arabidopsis has shown that mutations in cinnamoyl CoA reductase (CCR), a key lignin gene, affect physical properties of the secondary cell wall such as stiffness and strength. Using this gene, we tested whether LD mapping could identify alleles associated with microfibril angle (MFA), a wood quality trait affecting stiffness and strength of wood. We identified 25 common single-nucleotide polymorphism (SNP) markers in the CCR gene in Eucalyptus nitens. Using single-marker and haplotype analyses in 290 trees from a E. nitens natural population, two haplotypes significantly associated with MFA were found. These results were confirmed in two full-sib families of E. nitens and Eucalyptus globulus. In an effort to understand the functional significance of the SNP markers, we sequenced the cDNA clones and identified an alternatively spliced variant from the significant haplotype region. This study demonstrates that LD mapping can be used to identify alleles associated with wood quality traits in natural populations of trees.

Aldehyde Oxidoreductases↗
PubMed1982

Microfibrils in glomerulopathies of children: an ultrastructural study.

Extracellular microfibrils were found in 47 per cent. of 360 renal biopsies performed in children with a variety of nephropathies. They were observed in the mesangial matrix and basal lamina. They are 17 to 35 nm in diameter and a few show cross-striations with a periodicity of about 10 nm. They are different from fibrin or interstitial collagen. Their close association with thinner filaments in the mesangial matrix and basal lamina suggests that they are derived from such filaments.

Adolescent↗
PubMed1994

Isolation and characterization of a 36-kDa microfibril-associated glycoprotein by the newly synthesized isoquinolinesulfonamide affinity chromatography.

We have reported here the biochemical characterization of a newly identified microfibril-associated protein of 36-kDa (36-kDa MAP) from bovine aorta. Using Ca(2+)-dependent affinity chromatography on an isoquinolinesulfonamide derivative (CKA-1303)-coupled Sepharose, we obtained a pure form of 36-kDa MAP. This compound should serve as a useful tool for clarifying the physiological roles of 36-kDa MAP. In addition, 45Ca-autoradiography clearly indicated that 36-kDa MAP binds Ca2+. Partial amino acid sequence of 36-kDa MAP was determined. Our search of that published sequence against a 36-kDa MAP sequence revealed the resemblance to fibrinogen-related proteins of sea cucumber, cytotactin and tenascin which are substrate-adhesion proteins made at critical stages of histogenesis.

Amino Acid Sequence↗
PubMed1998

X-Ray diffraction studies of fibrillin-rich microfibrils: effects of tissue extension on axial and lateral packing.

X-ray diffraction of hydrated fibrillin rich microfibrils, in the form of zonular filaments from bovine eyes, demonstrated meridional diffraction peaks indexing on a fundamental periodicity of approximately 56 nm in the relaxed state. The effect of sample extensions of up to 50% in length produced an increase in the axial periodicity of only 4% as judged by alteration of the diffraction peak position of the third meridional order. This effect was shown to be reversible. Further extension to 100% of the tissue rest length caused extensive deterioration in the quality of the diffraction and resulted in a more complex meridional diffraction series, where the fundamental axial periodicity also changed to a length of approximately 80 nm. The fibrillin diffraction image also contains an equatorial diffraction peak that is enhanced upon tissue extension. The measurement of the molecular spacing from the equatorial diffraction profile indicated that the closest approach of molecules gave a broad interference peak of spacing 28 nm, this is nearly twice the molecular diameter as estimated from electron microscopy of dehydrated samples.

Animals↗
PubMed2000

Organization of the mouse microfibril-associated glycoprotein-2 (MAGP-2) gene.

A 1.4-kb EST clone encoding mouse microfibril-associated glycoprotein-2 (MAGP-2), identified by its similarity with the reported human cDNA, was used to screen a mouse 129 genomic bacterial artificial chromosome (BAC) library. The mouse gene contains 10 exons spanning 16 kb, located on the distal region of Chromosome (Chr) 6. The exons range in size from 24 to 963 bp, with the ATG located in exon 2. The tenth and largest exon contains 817 bp of 3' untranslated sequence, including a B2 repetitive element. Northern analysis demonstrates abundant expression of MAGP-2 mRNA in skeletal muscle, lung, and heart. Sequence analysis of additional cDNA clones suggests that the two mRNA forms of MAGP-2 in the mouse arise from alternative polyadenylation site usage. The promoter does not contain an obvious TATA box, and the sequence surrounding the start site does not conform to the consensus for an initiator promoter element. Additionally, the mouse promoter contains 22 copies of a CT dinucleotide repeat sequence located approximately 155 bp 5' to exon 1. MAGP-2 gene and compared it with that of the human gene (Hatzinikolas and Gibson 1998). While the mouse and human MAGP-2 proteins are similar in sequence, the promoters for the two genes share little in common. The presence of two mRNA species for MAGP-2 in the mouse raised the possibility that more than one isoform of the protein might be synthesized. We have characterized both mRNA species and determined that they do not code for different variants of the protein.

Alternative Splicing↗
PubMed1979

The limiting collagen microfibril. The minimum structure demonstrating native axial periodicity.

Collagen fibers were grown from solutions of acid-soluble or neutral salt-soluble collagen in 0.5 M acetic acid by rapid dialysis. The collagen was obtained under conditions where protease inhibitors were present at every stage of extraction and purification. Under the conditions used, length-wise but not lateral filament growth proceeded rapidly and gel-like networks were formed, Water readily exuded from the networks. The networks were stretched to fibrous form during drying. Small-angle X-ray diffraction showed the stretched fibrils to be highly ordered, showing up to 20 orders of the 670 A meridional periodicity. Intermediate- and wide-angle photographs show equatorial reflections at a spacing corresponding to approximately 12.5 A which is related to the intermolecular distance but none related to a microfibrillar packing at the 35-40 A level. Electron microscopy of the gel networks before stretching shows the presence of thin filaments with diameters predominantly in the 35-40 A range. No cross-striated fibrils are seen in electron micrographs of either stretched fibers or unstretched fibers. Thus, intermolecular packing in accord with the 670 A axial periodicity can take place within approximately 40 A diameter thin filaments. These correspond to the structures previously postulated to be collagen 'microfibrils'.

Animals↗