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M A Gibson

Publications and source records attributed to M A Gibson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Elastic fibre assembly: macromolecular interactions.

To investigate the mechanisms behind elastic fibre assembly, we studied the molecular interactions between elastin and microfibrillar components using solid-phase binding assays. Fibrillin 1, purified from tissue using reductive-saline extraction, showed no binding to microfibril-associated glycoprotein (MAGP) or tropoelastin. MAGP, however, was found to bind specifically to tropoelastin in a divalent-cation independent manner. Antibody inhibition studies indicated that the C-terminus of tropoelastin defined the interactive site with MAGP. MAGP and fibrillin were also substrates for transglutaminase, which may provide an important mechanism for stabilizing microfibrillar structure. In other studies we found that a major cross-linking region in elastin is formed through the association of domains encoded by exons 10, 19 and 25 of tropoelastin and that the three chains are joined together by one desmosine and two lysinonorleucine cross-links.

Amino Acid Sequence↗
PubMed1995

Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils.

Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of the guanidine-solubilized LTBP-2 appeared to be monomeric, indicating that it was not involved in disulfide-bonded aggregation either with itself or with latent TGF-beta. Additional LTBP-2 was resistant to solubilization with 6 M guanidine but was readily extracted with a reductive saline solution. This treatment is relatively specific for solubilization of microfibrillar constituents including fibrillin 1 and microfibril-associated glycoprotein. Therefore, it can be inferred that some LTBP-2 is bound covalently to the microfibrils by reducible disulfide linkages. The evidence suggests that LTBP-2 has a direct role in elastic fiber structure and assembly which may be independent of its growth factor-binding properties. Thus, LTBP-2 appears to share functional characteristics with both LTBP-1 and fibrillins.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Fibrillin-1 and fibrillin-2 show temporal and tissue-specific regulation of expression in developing elastic tissues.

The recent characterization of multiple fibrillin genes raises the question of whether each of the fibrillin proteins is a component of elastic fiber microfibrils and whether their expression during development of elastic tissues is consistent with a function associated with elastogenesis. To address these possibilities, the expression of fibrillin-1 and fibrillin-2 was compared with expression of MAGP and tropoelastin in two elastogenic tissues that undergo different developmental programs. For both fibrillins, the greatest increase in expression occurred during the last half of fetal development when elastin production is highest. In fetal bovine nuchal ligament, mRNA levels for fibrillin-1 and fibrillin-2 increased approximately threefold during this period, whereas tropoelastin increased 20-fold. Although the relative increase in expression of both fibrillins was equivalent, the basal level of fibrillin-1 expression was greater than fibrillin-2. In developing bovine aorta, fibrillin mRNA levels again paralleled tropoelastin expression although, compared to ligament, elastin synthesis began at an earlier fetal age in this tissue. Furthermore, the relative increase in aortic fibrillin-2 expression was greater than that for fibrillin-1 and the ratio of fibrillin-2 to fibrillin-1 was higher than in the ligament. In contrast to the fibrillins, MAGP expression in nuchal ligament and aorta remained at a constant high level throughout the fetal period. Indirect immunofluorescent staining and immunoelectron microscopy localized both fibrillins as well as MAGP to elastic fiber microfibrils in these developing tissues. The coordinate upregulation of fibrillin-1 and fibrillin-2 expression with the onset of tropoelastin production is consistent with a role in elastic fiber assembly. Our findings also suggest temporal and tissue-specific regulation for the fibrillins during development.

Age Factors↗
PubMed1994

Microfibril-associated glycoprotein binds to the carboxyl-terminal domain of tropoelastin and is a substrate for transglutaminase.

Microfibril-associated glycoprotein (MAGP) is an integral component of microfibrillar structures that play a critical role in the organization of elastic fibers in the extracellular matrix. To study possible molecular interactions between MAGP and other elastic fiber components, we have generated native MAGP using a baculovirus expression system and tested its ability to associate with tropoelastin and fibrillin. MAGP produced by SF9 cells underwent processing similar to the mammalian protein, including correct cleavage of the signal peptide and sulfation of tyrosine residues. When tested in solid-phase binding assays, native MAGP specifically bound to tropoelastin but not fibrillin-1. Binding to tropoelastin was divalent cation-independent and was completely blocked by reduction and alkylation of either protein. Antibody inhibition studies indicated that the carboxyl terminus of tropoelastin mediated its interaction with MAGP. In addition to binding to elastin, MAGP was also a substrate for transglutaminase, which might explain its propensity to form high molecular weight aggregates that cannot be dissociated with reduction or denaturation. Together, the results of this study provide new insights into the functional relationship between microfibrillar proteins and have important implications for understanding elastic fiber assembly.

Amino Acid Sequence↗
PubMed1991

Complementary DNA cloning establishes microfibril-associated glycoprotein (MAGP) to be a discrete component of the elastin-associated microfibrils.

Affinity-purified antibodies to microfibril-associated glycoprotein (MAGP) were used to screen a random-primed, bovine nuchal ligament cDNA library in lambda gt11. A 303-base pair clone, cM5, was isolated which encoded an amino acid sequence homologous with that determined directly from a Lys-C peptide of MAGP. A 936-base pair cDNA clone, cM32, was identified in an oligo(dT)-primed cDNA library using plaque hybridization with clone cM5. Clone cM32 encoded amino acid sequences corresponding to sequences obtained from three Lys-C peptides of MAGP, indicating that the clone was an authentic cDNA for the glycoprotein. The cDNA coded for the entire MAGP polypeptide (21 kDa) of 183 amino acids including a putative signal peptide of 17-19 amino acids. This was confirmed by in vitro translation of synthetic mRNAs transcribed from cM32. The amino acid composition of the encoded protein was virtually identical to that previously published for MAGP. DNA sequence analysis of cM32 indicated that MAGP contains two structurally dissimilar regions, an amino-terminal domain containing high levels of glutamine, proline, and acidic amino acids and a carboxyl-terminal domain containing all 13 of the cysteine residues and most of the basic amino acids. Northern blot hybridization of poly(A+) RNA from fetal nuchal ligament with clone cM32 identified a single mRNA species for MAGP of approximately 1.1 kilobases. The evidence indicates that MAGP is a distinct component of 12-nm microfibrils and that it is not derived from a larger microfibrillar glycopolypeptide.

Actin Cytoskeleton↗
PubMed1989

The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues.

A procedure has been developed which is much more specific for the solubilization of the elastin-associated microfibrils from fetal bovine nuchal ligament using treatment with reductive saline in place of reductive guanidine hydrochloride buffer. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reductive saline extracts were shown to contain only five major protein bands with Mrs of 340,000, 78,000, 70,000, 31,000, and 25,000. The 31-kDa species was identified immunologically as the previously described macromolecule named microfibril-associated glycoprotein (MAGP) (Gibson, M. A., Hughes, J. L., Fanning, J. C., and Cleary, E. G. (1986) J. Biol. Chem. 261, 11429-11436). The proteins were purified by gel permeation, ion exchange, and affinity chromatography. Amino acid analyses showed that each protein had a profile which was distinct from that of MAGP although each was also high in acidic amino acids and cystine. The 340- and 78-kDa species were each demonstrated by immunoelectron microscopy with affinity-purified antibodies to be derived from the elastin-associated microfibris, and these were provisionally named microfibrillar protein 340 (MP340) and microfibrillar protein 78 (MP78), respectively. Each of the above antibodies gave a tissue distribution identical to that of anti-MAGP antibodies, and thus MP340 and MP78 also were identified with the 12-nm microfibrils of nonelastic tissues. MP340 was shown to absorb out completely the microfibrillar immunoreactivity of anti-(reductive guanidine hydrochloride extract) antibodies, indicating that MP340 was (a) the major microfibrillar constituent in these extracts and (b) the second unidentified microfibrillar antigen described previously. The relationship of the 70- and 25-kDa proteins to microfibrils is yet to be established. Immunoblot and immunoabsorption studies showed that MAGP and MP78 were immunologically related to MP340 but not to each other. Cyanogen bromide peptide mapping indicated that MAGP was structurally related to MP340. It is postulated that MAGP and MP78 are constituents of MP340 which in turn is the subunit of which the 12-nm microfibrils are composed.

Amino Acids↗
PubMed1989

The tissue distribution of microfibrils reacting with a monospecific antibody to MAGP, the major glycoprotein antigen of elastin-associated microfibrils.

Elastic tissue, when viewed in the electron microscope, consists of an amorphous component that is immunoreactive with anti-tropoelastin (TE) antibodies and microfibrils, that react with monospecific antibodies against a 31 kDa microfibrillar glycoprotein constituent, called MAGP. A detailed study of the tissue distribution of microfibrils and of the two elastic tissue antibodies has been carried out, using single and double-labeled immunogold techniques in high resolution electron microscopy. Microfibrils similar in appearance to those associated with elastic tissue and immunoreactive with the anti-MAGP antibody, have been demonstrated in many tissues in the absence of amorphous elastic tissue. In the majority of these tissues, specific anti-TE antibody localization was demonstrated in the immediate vicinity of the microfibrils, or alternatively, the microfibrils were shown to be in direct continuity with microfibrils of similar morphology, which were associated with material immunoreactive with anti-TE antibody. The diameter of these microfibrils varied between 8 nm and 16 nm. They were unbranched structures of indefinite length, with a tubular profile on cross section and periodic staining in longitudinal section. In some tissues, notably in the ciliary zonule and in the mesangial region of the renal glomerulus, microfibrils of similar morphology were demonstrated which were immunoreactive with anti-MAGP antibody, but which were unrelated to amorphous elastic tissue and with which anti-TE antibody localization could not be demonstrated. The evidence available supports the conclusion that all these microfibrils are members of a single class of structures, which are widely distributed in the tissues and which are secreted by a range of cell types. Attention is directed to the close relationship between these microfibrils and the basement membrane of the glomerulus, of uterine smooth muscle, of the basal cells of the epidermis and of the reticulum cells of the spleen.

Animals↗
PubMed1988

Cell growth on metallic glasses: the interaction of amorphous metal alloys with cultured neuronal, osteoblast, endothelial, and fibroblast cells.

Ferrous based, corrosion resistant amorphous alloys supported the adhesion and growth of cultured chick neuronal cells, human marrow stromal cells (presumptive osteoblasts), bovine aortal endothelial cells, and hamster kidney fibroblasts. Alloys of compositions Fe60Ni10Cr10P13C7, Fe70Cr10P13C7, and Fe70Cr10P13B7 were found to be suitable. In contrast the crystalline form of these alloys was markedly less effective. Outgrowth of neurites from neuronal cells was promoted by precoating the metal surface with either laminin or neurite promotion factor. The adhesion of osteoblasts and fibroblasts suggests that corrosion resistant metal glasses should be considered as biomaterials useful for orthopedic applications. The adhesion of neuronal cells accompanied by neurite outgrowth indicates that the system might provide a functional interface between the neuromuscular system and an electromagnetic material that could be useful in bionic engineering.

Alloys↗
PubMed1988

Post-embedding methods for immunolocalization of elastin and related components in tissues.

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.

Animals↗
PubMed1988

Punjabi orchard farmers: an immigrant enclave in rural California.

Examination of the adaptation patterns of Punjabi Sikh orchard farmers in rural California provides further evidence of ways in which strong ethnic enclaves help to promote rapid economic self-sufficiency among immigrant groups. The discussion emphasizes the need to consider not only the cultural traditions of group members, but also the historical context of immigration and the immigrants' perceptions of their particular situation. The Punjabi case indicates, too, how the 1965 Immigration Act, with its preference for family reunification, has promoted the formation of immigrant enclave.

Agriculture↗
PubMed1987

The immunohistochemical localisation of microfibril-associated glycoprotein (MAGP) in elastic and non-elastic tissues.

We have previously identified the major antigen of elastin-associated microfibrils as a 31kD glycoprotein which we named microfibril-associated glycoprotein or MAGP. Affinity-purified antibodies to MAGP were shown to localise specifically to elastin-associated microfibrils in sections of bovine foetal nuchal ligament. In the present paper we compare the localisation of anti-MAGP antibodies and anti-tropoelastin antibodies in a range of bovine elastic and non-elastic tissues. The results show that anti-MAGP antibodies invariably localised to immuno-reactive elastic fibres, wherever they occurred. Extensive additional localisation was observed in a number of tissues. This extra distribution of anti-MAGP antibodies was found to correspond to those structures exhibiting the oxytalan histochemical staining reaction in tissues such as skin, periodontal ligament and ocular zonule. Since these oxytalan fibres have been shown to consist of 12 nm microfibrils which are morphologically similar to those of elastic fibres (and unpublished data from this laboratory confirm this conclusion), the results suggest that MAGP is a component of 12 nm microfibrils in both elastic and non-elastic tissues. Anti-tropoelastin antibodies did not localise to these oxytalan fibres, suggesting that tropoelastin is not a component of 12 nm microfibrils. MAGP was also detected in extracellular matrix regions of tissues such as skeletal muscle, Achilles tendon and spleen, suggesting that 12 nm microfibrils, containing one or more macromolecular constituents in common, make up an important structural system within the extracellular matrix in a wide range of elastic and non-elastic tissues.

Animals↗
PubMed1986

The major antigen of elastin-associated microfibrils is a 31-kDa glycoprotein.

The major antigen derived from elastic fiber microfibrils was identified as a Mr = 31,000 glycoprotein, using immunoblotting and immunohistochemical techniques with antisera raised to "reductive guanidine extracts" of fetal bovine nuchal ligament, and to subfractions of these. A second, elastic fiber-derived, but unidentified, antigen of large molecular size (Mr greater than 200,000) was present in these extracts. Antisera raised to the purified 31-kDa glycoprotein were shown, by immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. Thus, the macro-molecule was called "microfibril-associated glycoprotein" or MAGP. MAGP is an acidic glycoprotein with a distinctive amino acid composition, being exceptionally rich in glutamic acid, rich in cystine, and low in glycine. MAGP was extractable from tissue homogenates using NaCl, urea, or guanidine hydrochloride solutions, only if a strong reducing agent was present. Thus, disulfide bonding is important for the strong association of MAGP with elastic fibers. Immunoblotting with anti-MAGP antiserum identified two additional reactive species, of Mr = 60,000 and Mr approximately 300,000, in tissue extracts. As only the 31-kDa species was detected in fibroblast culture medium, these additional species were probably aggregates, rather than precursors. MAGP did not react with antilysyl oxidase antiserum on immunoblots or by enzyme-linked immunosorbent assay. MAGP is the first macromolecule to have been established to be a constituent of elastin-associated microfibrils in both developing and mature elastic tissues.

Amino Acids↗
PubMed1985

CL glycoprotein is the tissue form of type VI collagen.

CL glycoprotein (CLGP), the 140,000-dalton collagenous glycoprotein, has been isolated from fetal bovine aorta and nuchal ligament, in milligram amounts in its reduced and alkylated form, using a multistage procedure. This material exhibited a characteristic amino acid composition with a consistent ratio of hydroxylysine to hydroxyproline (approximately 1:1). Digestion of CLGP with bacterial collagenase yielded three discrete noncollagenous fragments. Monospecific anti-CLGP antiserum exhibited strong cross-reactivity with the pepsin-resistant polypeptides of type VI collagen. CLGP was also prepared in the unreduced disulfide-bonded form and in a partially reduced form, using brief treatment with cysteine. On treatment with pepsin these preparations yielded resistant peptides corresponding in size to the longer and shorter forms, respectively, of type VI collagen. A slightly larger, soluble form of CLGP (Mr = 150,000) was detected in the media from cultures of aortic smooth muscle cells and nuchal ligament fibroblasts. The evidence indicates that CLGP is the native form in which type VI collagen is present in the tissues and that it consists of three structurally distinct polypeptide chains, each about 140 kDa in mass, which are disulfide bonded into a triple-helical molecule. The CLGP molecules appear to be present in the tissues as dimers and larger aggregates, stabilized by intermolecular disulfide bonding. The distribution of type VI collagen will thus be as described in our earlier immunofluorescence studies with anti-CLGP antiserum (Gibson, M.A., and Cleary, E.G. (1983) Collagen Relat. Res. 3, 469-488).

Amino Acids↗
PubMed1984

Microfibrillar protein from elastic tissue: a critical evaluation.

Many workers have claimed to have isolated proteins which have been derived from the microfibrillar components of elastic tissue. Virtually all of these preparations have been derived from extracts made with strong solutions of guanidinium chloride (GuHCl) under reducing conditions following Ross and Bornstein (1969). The products have ranged from heterogeneous mixtures of proteins to discrete glycoproteins. In no case has identity between an individual protein and the elastin-associated microfibrils been confirmed by immunoelectron microscopy. We have undertaken a detailed re-examination of the extractability of elastin-associated microfibrils and of the composition of the extracts from foetal bovine nuchal ligament. Finely homogenized samples were subjected to a series of extractions (including cyclical treatments with GuHCl and purified bacterial collagenase) in the presence of inhibitors of protease activity. Under these conditions it has been shown that--(i) microfibrils were removed progressively by GuHCl, throughout the extraction schedule, without the need for reduction; (ii) all remaining microfibrils were removed by reductive GuHCl extraction; (iii) the product from this reductive extraction consisted of a heterogeneous mixture of proteins including several glycoproteins; (iv) a major antigenic constituent of the mixture of proteins localized to elastin-associated microfibrils, as shown by immunoelectron microscopy. It is concluded that, while reductive GuHCl extracts do contain components with antigenic activity that is localized on elastin-associated microfibrils, they have many non-microfibrillar components. We stress that claims that a macromolecule is microfibrillar must be substantiated by immunoelectron microscopy.

Animals↗
PubMed1983

Distribution of CL glycoprotein in tissues: an immunohistochemical study.

CL glycoprotein is a collagen-like glycoprotein which we have recently isolated from rapidly growing fetal bovine, elastin-rich tissues. This protein has a molecular weight of approximately 140,000 daltons, contains hydroxyproline and hydroxylysine and is digested by highly purified collagenase to yield three large polypeptides. A specific antibody has been developed against this protein and has been used for immunofluorescence microscopy to study the distribution of CL glycoprotein in a range of tissues. It has been shown that the antibody localized in the intercellular matrix of nuchal ligament and aorta, of the non-elastic Achilles tendon and in complex tissues such as kidney, lung, skin and spleen. The antibody also localized to the surface of aortic smooth muscle cells-presumably to the basement membrane, but did not bind to other basement membranes, including the vascular subendothelial basement membrane. The pattern of distribution was similar in adult bovine tissues. As this antibody showed no avidity for elastic tissue elements, it is most unlikely that CL glycoprotein is a constituent of elastin-associated microfibrils. When the pattern of the CL glycoprotein distribution within the tissues was studied, it was found that, apart from its concentration around vascular smooth muscle cells, CL glycoprotein exhibited considerable overlap in distribution with the interstitial collagens. On the basis of these observations and having regard to its biochemical characteristics, it is proposed that CL glycoprotein has a structural role inter-linking interstitial components to one another and to vascular smooth muscle cells.

Animals↗