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David J McQuillan

Publications and source records attributed to David J McQuillan.

12 recordsLinked to original sources
PubMed2006

Decorin modulates fibrin assembly and structure.

Emerging evidence indicates that fibrin clotting is regulated by different external factors. We demonstrated recently that decorin, a regulator of collagen fibrillogenesis and transforming growth factor-beta activity, binds to the D regions of fibrinogen (Dugan, T.A., Yang, V. W.-C., McQuillan, D.J., and Höök, M. (2003) J. Biol. Chem. 278, 13655-13662). We now report that the decorin-fibrinogen interaction alters the assembly, structure, and clearance of fibrin fibers. Relative to fibrinogen, substoichiometric amounts of decorin core protein modulated clotting, whereas an excess of an active decorin peptide was necessary for similar activity. These concentration-dependent effects suggest that decorin bound to the D regions sterically modulates fibrin assembly. Scanning electron microscopy images of fibrin clotted in the presence of increasing concentrations of decorin core protein showed progressively decreasing fiber diameter. The sequestration of Zn(2+) ions from the N-terminal fibrinogen-binding region abrogated decorin incorporation into the fibrin network. Compared with linear thicker fibrin fibers, the curving thin fibers formed with decorin underwent accelerated tissue-type plasminogen activator-dependent fibrinolysis. Collectively, these data demonstrate that decorin can regulate fibrin organization and reveal a novel mechanism by which extracellular matrix components can participate in hemostasis, thrombosis, and wound repair.

Binding Sites↗
PubMed2006

Decorin protein core inhibits in vivo cancer growth and metabolism by hindering epidermal growth factor receptor function and triggering apoptosis via caspase-3 activation.

Decorin is not only a regulator of matrix assembly but also a key signaling molecule that modulates the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR). Decorin evokes protracted internalization of the EGFR via a caveolar-mediated endocytosis, which leads to EGFR degradation and attenuation of its signaling pathway. In this study, we tested if systemic delivery of decorin protein core would affect the biology of an orthotopic squamous carcinoma xenograft. After tumor engraftment, the animals were given intraperitoneal injections of either vehicle or decorin protein core (2.5-10 mg kg(-1)) every 2 days for 18-38 days. This regimen caused a significant and dose-dependent inhibition of the tumor xenograft growth, with a concurrent decrease in mitotic index and a significant increase in apoptosis. Positron emission tomography showed that the metabolic activity of the tumor xenografts was significantly reduced by decorin treatment. Decorin protein core specifically targeted the tumor cells enriched in EGFR and caused a significant down-regulation of EGFR and attenuation of its activity. In vitro studies showed that the uptake of decorin by the A431 cells was rapid and caused a protracted down-regulation of the EGFR to levels similar to those observed in the tumor xenografts. Furthermore, decorin induced apoptosis via activation of caspase-3. This could represent an additional mechanism whereby decorin might influence cell growth and survival.

Animals↗
PubMed2006

Biglycan regulates the expression and sarcolemmal localization of dystrobrevin, syntrophin, and nNOS.

The dystrophin-associated protein complex (DAPC) provides a linkage between the cytoskeleton and the extracellular matrix (ECM) and is also a scaffold for a host of signaling molecules. The constituents of the DAPC must be targeted to the sarcolemma in order to properly function. Biglycan is an ECM molecule that associates with the DAPC. Here, we show that biglycan null mice exhibit a mild dystrophic phenotype and display a selective reduction in the localization of alpha-dystrobrevin-1 and -2, alpha- and beta1-syntrophin, and nNOS at the sarcolemma. Purified biglycan induces nNOS redistribution to the plasma membrane in cultured muscle cells. Biglycan protein injected into muscle becomes stably associated with the sarcolemma and ECM for at least 2 wk. This injected biglycan restores the sarcolemmal expression of alpha-dystrobrevin-1 and -2, and beta1- and beta2-syntrophin in biglycan null mice. We conclude that biglycan is important for the maintenance of muscle cell integrity and plays a direct role in regulating the expression and sarcolemmal localization of the intracellular signaling proteins dystrobrevin-1 and -2, alpha- and beta1-syntrophin and nNOS.

Animals↗
PubMed2006

Biglycan binds to alpha- and gamma-sarcoglycan and regulates their expression during development.

The dystrophin-associated protein complex (DAPC), which links the cytoskeleton to the extracellular matrix, is essential for muscle cell survival, and is defective in a wide range of muscular dystrophies. The DAPC contains two transmembrane subcomplexes-the dystroglycans and the sarcoglycans. Although several extracellular binding partners have been identified for the dystroglycans, none have been described for the sarcoglycan subcomplex. Here we show that the small leucine-rich repeat (LRR) proteoglycan biglycan binds to alpha- and gamma-sarcoglycan as judged by ligand blot overlay and co-immunoprecipitation assays. Our studies with biglycan-decorin chimeras show that alpha- and gamma-sarcoglycan bind to distinct sites on the polypeptide core of biglycan. Both biglycan proteoglycan as well as biglycan polypeptide lacking glycosaminoglycan (GAG) side chains are components of the dystrophin glycoprotein complex isolated from adult skeletal muscle membranes. Finally, our immunohistochemical and biochemical studies with biglycan null mice show that the expression of alpha- and gamma-sarcoglycan is selectively reduced in muscle from young (P14-P21) animals, while levels in adult muscle (> or = P35) are unchanged. We conclude that biglycan is a ligand for two members of the sarcoglycan complex and regulates their expression at discrete developmental ages.

Animals↗
PubMed2005

Decorin core protein secretion is regulated by N-linked oligosaccharide and glycosaminoglycan additions.

Expression of decorin using the vaccinia virus/T7 expression system resulted in secretion of two distinct glycoforms: a proteoglycan substituted with a single chondroitin sulfate chain and N-linked oligosaccharides and a core protein glycoform substituted with N-linked glycans but without a glycosaminoglycan chain. In this report, we have addressed two distinct questions. What is the rate-limiting step in glycosaminoglycan synthesis? Is glycosylation with either N-linked oligosaccharides or glycosaminoglycan required for secretion of decorin? N-terminal sequencing of the core protein glycoform, the addition of benzyl-beta-d-xyloside, and a UDP-xylose: core protein beta-d-xylosyltransferase activity assay show that xylosylation is a rate-limiting step in chondroitin sulfate biosynthesis. Decorin can be efficiently secreted with N-linked oligosaccharides alone or with a single chondroitin sulfate chain alone; however, there is severely impaired secretion of core protein devoid of any glycosylation. A decorin core protein mutant devoid of N-linked oligosaccharide attachment sites will not be secreted by Chinese hamster ovary cells deficient in xylosyltransferase or by parental Chinese hamster ovary wild type cells if the xylosyltransferase recognition sequence is disrupted. This finding suggests that quality control mechanisms sensitive to an absence of N-linked oligosaccharides can be abrogated by interaction of the core protein with the glycosaminoglycan synthetic machinery. We propose a model of regulation of decorin secretion that has several components, including appropriate substitution with N-linked oligosaccharides and factors involved in glycosaminoglycan synthesis.

Animals↗
PubMed2005

Interactions of the extracellular matrix proteoglycans decorin and biglycan with C1q and collectins.

Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.

Amino Acid Sequence↗
PubMed2005

Decorin evokes protracted internalization and degradation of the epidermal growth factor receptor via caveolar endocytosis.

Decorin inhibits the epidermal growth factor receptor (EGFR) by down-regulating its tyrosine kinase activity, thereby blocking the growth of a variety of transformed cells and tumor xenografts. In this study we provide evidence that decorin directly binds to the EGFR causing its dimerization, internalization, and ultimately its degradation. Using various pharmacological agents to disrupt clathrin-dependent and -independent endocytosis, we demonstrate that decorin evokes a protracted internalization of the EGFR primarily via caveolar-mediated endocytosis. In contrast to EGF, decorin targets the EGFR to caveolae, but not to early or recycling endosomes. Ultimately, however, both EGF- and decorin-induced pathways converge into late endosomes/lysosomes for final degradation. Thus, we have discovered a novel biological mechanism for decorin that could explain its anti-proliferative and anti-oncogenic mode of action.

Antineoplastic Agents↗
PubMed2005

N-linked keratan sulfate in the aggrecan interglobular domain potentiates aggrecanase activity.

Keratan sulfate is thought to influence the cleavage of aggrecan by metalloenzymes. We have therefore produced a recombinant substrate, substituted with keratan sulfate, suitable for the study of aggrecanolysis in vitro. Recombinant human G1-G2 was produced in primary bovine keratocytes using a vaccinia virus expression system. Following purification and digestion with specific hydrolases, fluorophore-assisted carbohydrate electrophoresis was used to confirm the presence of the monosulfated Gal-GlcNAc6S and GlcNAc6s-Gal disaccharides and the disulfated Gal6S-GlcNAc6S disaccharides of keratan sulfate. Negligible amounts of fucose or sialic acid were detected, and the level of unsulfated disaccharides was minimal. Treatment with keratanases reduced the size of the recombinant G1-G2 by approximately 5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by approximately 5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is N-linked. Cleavage of G1-G2 by aggrecanase was markedly reduced when keratan sulfate chains were removed by treatment with keratanase, keratanase II, endo-beta-galactosidase, or N-glycosidase F. These results indicate that modification of oligosaccharides in the aggrecan interglobular domain with keratan sulfate, most likely at asparagine residue 368, potentiates aggrecanase activity in this part of the core protein.

Aggrecans↗
PubMed2005

Decorin prevents metastatic spreading of breast cancer.

Metastases in breast cancer are a vital concern in treatment, with epidermal growth factor receptor and ErbB2 strongly implicated in mediating tumor invasion and spreading. In this study, we investigated the role of decorin in suppressing both primary breast carcinomas and pulmonary metastases. We show that decorin causes marked growth suppression both in vitro and in vivo using a metastatic breast cancer cell line and an orthotopic mammary carcinoma model. Treatment with decorin protein core reduced primary tumor growth by 70% and eliminated observed metastases. An adenoviral vector containing the decorin transgene caused primary tumor retardation of 70%, in addition to greatly reducing observed metastases. Moreover, we demonstrate that ErbB2 phosphorylation and total receptor protein levels are reduced in this model system upon de novo expression of decorin under the control of a doxycycline-inducible promoter. Primary tumor growth in vivo was reduced by up to 67% upon decorin induction, and pulmonary metastases were markedly hampered as well. These effects are likely occurring through decorin's long-term downregulation of the ErbB2 tyrosine kinase cascade. These results demonstrate a novel role for decorin in reduction or prevention of tumor metastases in this breast cancer model and could eventually lead to improved therapeutics for metastatic breast cancer.

Adenoviridae↗
PubMed2003

Biologically active decorin is a monomer in solution.

It has been reported that decorin and its protein core can have molecular masses nearly double the size of those previously published, suggesting a dimeric structure. In this study we tested whether biologically active decorin and its glycoprotein core would form dimers in solution. We used homo- and hetero-bifunctional chemical cross-linking reagents, BS3 and sulfo-SMPB, respectively, as well as glutaraldehyde and found no preferential dimer formation, whether chemical cross-linking was performed in the presence or absence of live cells. Under the same experimental conditions, we easily detected dimers of epidermal growth factor receptor and basic fibroblast growth factor, two glycoproteins known to dimerize. Only at very high cross-linker to decorin molar ratios (2000:1) were trimers and multimers observed, but performing the chemical cross-linking in the presence of a reducing agent abolished these. The elution of decorin protein core in Superose 6 gel chromatography gave masses compatible with monomeric proteins, both before and after denaturation with 2.5 M guanidine HCl. Matrix-assisted laser desorption ionization gave a mass of 44,077 Da for decorin protein core, without any evidence of dimers or oligomers. Extensive oligomerization of the decorin protein core was observed only after dialysis against water and freeze-drying. These oligomers were considered artifacts because they were independent of chemical cross-linking and were resistant to heat denaturation and disulfide-bond reduction. Oligomeric preparations showed markedly reduced biological activity in both phosphorylation and collagen fibrillogenesis assays. Thus, biologically active decorin is a monomer in solution and, as such, is a monovalent ligand for various extracellular matrix proteins, growth factors, and cell surface receptors.

Cell Line↗
PubMed2003

Decorin binds fibrinogen in a Zn2+-dependent interaction.

We have previously shown that decorin, a member of the small leucine-rich proteoglycan family of extracellular matrix proteoglycans/glycoproteins is a Zn(2+) metalloprotein at physiological Zn(2+) concentrations (Yang, V. W-C., LaBrenz, S. R., Rosenberg, L. C., McQuillan, D., and Höök, M. (1999) J. Biol. Chem. 274, 12454-12460). We now report that the decorin proteoglycan binds fibrinogen in the presence of Zn(2+). The fibrinogen-binding site is located in the N-terminal domain of the decorin core protein and a 45-amino acid peptide representing this domain binds to the fibrinogen D fragment with an apparent K(D) of 1.7 x 10(-6) m, as determined from fluorescence polarization data. Furthermore, we show that Zn(2+) promotes the self-association of decorin. The N-terminal domain of the core protein also mediates this activity. The results of solid-phase binding assays and gel filtration chromatography suggest that the N-terminal domain of decorin, when present at low micromolar concentrations, forms an oligomer in a Zn(2+)-dependent manner. Thus, Zn(2+) appears to play a pivotal role in the interactions and biological function of decorin.

Animals↗