PubMed Health⌕ Search

Biomedical subjects

T Broekelmann

Publications and source records attributed to T Broekelmann.

10 recordsLinked to original sources
PubMed2001

Posttranslational modifications of microfibril associated glycoprotein-1 (MAGP-1).

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

Amino Acid Sequence↗
PubMed2000

The microfibrillar proteins MAGP-1 and fibrillin-1 form a ternary complex with the chondroitin sulfate proteoglycan decorin.

MAGP-1 and fibrillin-1, two protein components of extracellular microfibrils, were shown by immunoprecipitation studies to interact with the chondroitin sulfate proteoglycan decorin in the medium of cultured fetal bovine chondrocytes. Decorin interacted with each protein individually and with both proteins together to form a ternary complex. Expression of truncated fibrillin-1 proteins in Chinese hamster ovary cells localized proteoglycan binding to an amino-terminal region near the proline-rich domain. A spatially analogous fibrillin-2 truncated protein did not coprecipitate the same sulfated molecule, suggesting that chondroitin sulfate proteoglycan binding in this region is specific for fibrillin-1. An interaction between fibrillin and MAGP-1 was also observed under culture conditions that abrogated decorin secretion, suggesting that the two microfibrillar proteins can associate in the absence of the proteoglycan. Sulfation of matrix proteins is important for elastic fiber assembly because inhibition of sulfation was shown to prevent microfibrillar protein incorporation into the extracellular matrix of cultured cells.

Animals↗
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↗
PubMed1999

Processing of the fibrillin-1 carboxyl-terminal domain.

To investigate the processing and general properties of the fibrillin-1 carboxyl-terminal domain, three protein expression constructs have been developed as follows: one without the domain, one with the domain, and one with a mutation near the putative proteolytic processing site. The constructs have been expressed in two eukaryotic model systems, baculoviral and CHO-K1. Post-translational modifications that normally occur in fibrillin-1, including glycosylation, signal peptide cleavage, and carboxyl-terminal processing, occur in the three constructs in both cell systems. Amino-terminal sequencing of secreted protein revealed leader sequence processing at two sites, a primary site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28. Processing of the carboxyl-terminal domain could be observed by migration differences in SDS-polyacrylamide gel electrophoresis and was evident in both mammalian and insect cells. Immunological identification by Western blotting confirmed the loss of the expected region. The failure of both cell systems to process the mutant construct shows that the multi-basic sequence is the site of proteolytic processing. Cleavage of the fibrillin-1 carboxyl-terminal domain occurred intracellularly in CHO-K1 cells in an early secretory pathway compartment as demonstrated by studies with secretion blocking agents. This finding, taken with the multi-basic nature of the cleavage site and observed calcium sensitivity of cleavage, suggests that the processing enzyme is a secretory pathway resident furin-like protease.

Amidohydrolases↗
PubMed1996

Functional domains on elastin and microfibril-associated glycoprotein involved in elastic fibre assembly.

Studies in vitro suggest that the C-terminus of tropoelastin mediates elastin polymerization through an interaction with microfibril-associated proteins. In this study we have used cultured auricular chondrocytes as a model system to examine whether this interaction is critical for elastic fibre formation in vivo. Auricular chondrocytes, which deposit an abundant elastic fibre matrix, were cultured in the presence of Fab fragments of antibodies directed against the C-terminus (CTe) or an N-terminal domain (ATe) of tropoelastin. Immunofluorescent staining of the extracellular matrix deposited by the cells showed that the CTe antibody inhibited the deposition of elastin without affecting microfibril structure. Cells grown under identical conditions in the presence of ATe, however, formed fibres that stained normally for both elastin and microfibril proteins. Chondrocytes cultured in the presence of microfibril-associated glycoprotein (MAGP):21-35, an antibody directed against a domain near the N-terminus of MAGP, did not organize tropoelastin into fibres. However, immunostaining for MAGP and fibrillin revealed normal microfibrils. In agreement with the immunofluorescence staining patterns, fewer elastin-specific cross-links, indicative of insoluble elastin, were detected in the extracellular matrix of cells cultured in the presence of CTe. The medium from these cultures, however, contained more soluble elastin, consistent with an antibody-induced alteration of elastin assembly but not its synthesis. Northern analysis of antibody-treated and control cultures substantiated equivalent levels of tropoelastin mRNA. These results confirm that the C-terminus of tropoelastin interacts with microfibrils during the assembly of elastic fibres. Further, the results suggest that the interaction between tropoelastin and microfibrils might be mediated by a domain involving the N-terminal half of MAGP.

Animals↗
PubMed1996

Cell-type specific recognition of RGD- and non-RGD-containing cell binding domains in fibrillin-1.

The fibrillins are large glycoprotein components of 10-nm microfibrils found in the extracellular matrix of most tissues. Microfibrils play a role in elastic fiber assembly and serve to link cells to elastic fibers in the extracellular matrix. To determine whether fibrillin-1 specifically interacts with receptors on cells from fibrillin-rich tissues, we evaluated whether two cell types that produce different types of fibrillin can adhere to purified fibrillin-1 in cell adhesion assays. Our results indicate that both cell types attach and spread on fibrillin-1 and that the RGD sequence in the fourth 8-cysteine motif mediates this interaction. Fibroblast attachment to fibrillin-1 was sensitive to inhibition by antibodies to the alphavbeta3 receptor and by peptides encoding the RGD sequence in fibrillin-1 and the second RGD sequence in fibrillin-2. In contrast, adhesion of auricular chondroblasts to fibrillin-1 was only partially inhibited by these reagents, suggesting that some cell types recognize a second, non-RGD binding site within the fibrillin molecule. These findings confirm and extend ultrastructural studies that suggest a direct interaction between microfibrils and the cell surface and provide a functional explanation for how this association occurs.

Amino Acid Sequence↗
PubMed1995

Identification of an elastin cross-linking domain that joins three peptide chains. Possible role in nucleated assembly.

The alignment of elastin molecules in the mature elastic fiber was investigated by purifying and sequencing cross-link-containing peptides generated by proteolytic digestion incompletely cross-linked insoluble elastin. Peptides of interest were purified by reverse phase and size exclusion high performance liquid chromatography and characterized by amino acid analysis and protein sequencing. One peptide, consisting of the cross-linking domain encoded by exon 10, contained a modified lysine residue that had not condensed to form a polyfunctional cross-link. Although this domain contains the characteristic paired lysine residues found in other cross-linking domains of elastin, protein sequence analysis indicated that the first but not the second lysine had been oxidized by lysyl oxidase. This finding suggests that lysine residues in an individual cross-linking domain may not have equal susceptibility to oxidation by lysyl oxidase. In a second peptide, we found that a major cross-linking site in elastin is formed through the association of sequences encoded by exons 10, 19, and 25 and that the three chains are joined together by one desmosine and two lysinonorleucine cross-links. Past structural studies and computer modeling predict that domains 19 and 25 are linked by a desmosine cross-link, while domain 10 bridges domains 19 and 25 through lysinonorleucine cross-links. These findings, together with the high degree of sequence conservation for these three domains, suggest an important function for these regions of the molecule, possibly nucleating the aggregation and polymerization of tropoelastin monomers in the developing elastic fiber.

Amino Acid Sequence↗
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↗
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↗