Publications (15)66.13 Total impact
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Article: Mapping structural landmarks, ligand binding sites, and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions, and variation in phenotypes in inherited diseases affecting basement membranes.
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ABSTRACT: Collagen IV is the major protein found in basement membranes. It comprises three heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity.We constructed linear maps of the collagen IV heterotrimers ("interactomes") that indicated major structural landmarks, known and predicted ligand-binding sites, and missense mutations, in order to identify functional and disease-associated domains, potential interactions between ligands, and genotype–phenotype relationships. The maps documented more than 30 known ligand-binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin, and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection, and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases, and heparin. The maps indicated that mutations affecting major ligand-binding sites, for example, for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms, and muscle Cramps [HANAC] syndrome, and early-onset Alport syndrome, respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets.Human Mutation 02/2011; 32(2):127-43. · 5.69 Impact Factor -
Article: Dinosaur peptides suggest mechanisms of protein survival.
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ABSTRACT: Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.PLoS ONE 01/2011; 6(6):e20381. · 4.09 Impact Factor -
Article: A key role for the integrin alpha2beta1 in experimental and developmental angiogenesis.
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ABSTRACT: The alpha2beta1 integrin receptor plays a key role in angiogenesis. Here we investigated the effects of small molecule inhibitors (SMIs) designed to disrupt integrin alpha2 I or beta1 I-like domain function on angiogenesis. In unchallenged endothelial cells, fibrillar collagen induced robust capillary morphogenesis. In contrast, tube formation was significantly reduced by SMI496, a beta1 I-like domain inhibitor and by function-blocking anti-alpha2beta1 but not -alpha1beta1 antibodies. Endothelial cells bound fluorescein-labeled collagen I fibrils, an interaction specifically inhibited by SMI496. Moreover, SMI496 caused cell retraction and cytoskeletal collapse of endothelial cells as well as delayed endothelial cell wound healing. SMI activities were examined in vivo by supplementing the growth medium of zebrafish embryos expressing green fluorescent protein under the control of the vascular endothelial growth factor receptor-2 promoter. SMI496, but not a control compound, interfered with angiogenesis in vivo by reversibly inhibiting sprouting from the axial vessels. We further characterized zebrafish alpha2 integrin and discovered that this integrin is highly conserved, especially the I domain. Notably, a similar vascular phenotype was induced by morpholino-mediated knockdown of the integrin alpha2 subunit. By live videomicroscopy, we confirmed that the vessels were largely nonfunctional in the absence of alpha2beta1 integrin. Collectively, our results provide strong biochemical and genetic evidence of a central role for alpha2beta1 integrin in experimental and developmental angiogenesis.American Journal Of Pathology 09/2009; 175(3):1338-47. · 4.89 Impact Factor -
Article: Heparan sulfate proteoglycans: a GAGgle of skeletal-hematopoietic regulators.
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ABSTRACT: This review summarizes our current understanding of the presence and function of heparan sulfate proteoglycans (HSPGs) in skeletal development and hematopoiesis. Although proteoglycans (PGs) comprise a large and diverse group of cell surface and matrix molecules, we chose to focus on HSPGs owing to their many proposed functions in skeletogenesis and hematopoiesis. Specifically, we discuss how HSPGs play predominant roles in establishing and regulating niches during skeleto-hematopoietic development by participating in distinct developmental processes such as patterning, compartmentalization, growth, differentiation, and maintenance of tissues. Special emphasis is placed on our novel hypothesis that mechanistically links endochondral skeletogenesis to the establishment of the hematopoietic stem cell (HSC) niche in the marrow. HSPGs may contribute to these developmental processes through their unique abilities to establish and mediate morphogen, growth factor, and cytokine gradients; facilitate signaling; provide structural stability to tissues; and act as molecular filters and barriers.Developmental Dynamics 10/2008; 237(10):2622-42. · 2.54 Impact Factor -
Article: Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates.
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ABSTRACT: Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The "cell interaction domain" is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The "matrix interaction domain" may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.Journal of Biological Chemistry 08/2008; 283(30):21187-97. · 4.77 Impact Factor -
Article: Non-enzymatic glycation of type I collagen diminishes collagen-proteoglycan binding and weakens cell adhesion.
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ABSTRACT: Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans(1) (PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content-effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, and had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.Journal of Cellular Biochemistry 04/2008; 104(5):1684-98. · 2.87 Impact Factor -
Article: Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans.
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ABSTRACT: Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proalpha1(I) and proalpha2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype-phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in alpha1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691-823 and 910-964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril-matrix interactions. Recurrences at the same site in alpha2(I) are generally concordant for outcome, unlike alpha1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In alpha2(I), lethal exon skipping events are located in the carboxyl half of the chain. Our data on genotype-phenotype relationships indicate that the two collagen chains play very different roles in matrix integrity and that phenotype depends on intracellular and extracellular events.Human Mutation 04/2007; 28(3):209-21. · 5.69 Impact Factor -
Article: Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo.
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ABSTRACT: Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)(n) tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [K(d)], approximately 50 nM), similar potencies in neutralizing anti-Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)(3)VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti-Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.Blood 03/2004; 103(4):1356-63. · 9.90 Impact Factor -
Article: Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly.
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ABSTRACT: Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.Journal of Biological Chemistry 09/2003; 278(33):30516-24. · 4.77 Impact Factor -
Article: Angiogenesis in Collagen I Requires α2β1 Ligation of a GFP*GER Sequence and Possibly p38 MAPK Activation and Focal Adhesion Disassembly
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ABSTRACT: Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6–8 h and capillary-like lumens by 24 h. Blocking of α2β1, but not α1 or αvβ3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative α2β1 binding site: α1(I)496–507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: α1(I)127–138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial α2β1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.Journal of Biological Chemistry 08/2003; 278(33):30516-30524. · 4.77 Impact Factor -
Article: Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan.
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ABSTRACT: Perlecan, a ubiquitous basement membrane heparan sulfate proteoglycan, plays key roles in blood vessel growth and structural integrity. We discovered that the C terminus of perlecan potently inhibited four aspects of angiogenesis: endothelial cell migration, collagen-induced endothelial tube morphogenesis, and blood vessel growth in the chorioallantoic membrane and in Matrigel plug assays. The C terminus of perlecan was active at nanomolar concentrations and blocked endothelial cell adhesion to fibronectin and type I collagen, without directly binding to either protein; henceforth we have named it "endorepellin." We also found that endothelial cells possess a significant number of high affinity (K(d) of 11 nm) binding sites for endorepellin and that endorepellin binds endostatin and counteracts its anti-angiogenic effects. Thus, endorepellin represents a novel anti-angiogenic product, which may retard tumor neovascularization and hence tumor growth in vivo.Journal of Biological Chemistry 03/2003; 278(6):4238-49. · 4.77 Impact Factor -
Article: Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen.
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ABSTRACT: Type I collagen is the most abundant protein in humans, and it helps to maintain the integrity of many tissues via its interactions with cell surfaces, other extracellular matrix molecules, and growth and differentiation factors. Nearly 50 molecules have been found to interact with type I collagen, and for about half of them, binding sites on this collagen have been elucidated. In addition, over 300 mutations in type I collagen associated with human connective tissue disorders have been described. However, the spatial relationships between the known ligand-binding sites and mutation positions have not been examined. To this end, here we have created a map of type I collagen that includes all of its ligand-binding sites and mutations. The map reveals the existence of several hot spots for ligand interactions on type I collagen and that most of the binding sites locate to its C-terminal half. Moreover, on the collagen fibril some potentially relevant relationships between binding sites were observed including the following: fibronectin- and certain integrin-binding regions are near neighbors, which may mechanistically relate to fibronectin-dependent cell-collagen attachment; proteoglycan binding may potentially impact upon collagen fibrillogenesis, cell-collagen attachment, and collagen glycation seen in diabetes and aging; and mutations associated with osteogenesis imperfecta and other disorders show apparently nonrandom distribution patterns within both the monomer and fibril, implying that mutation positions correlate with disease phenotype. These and other observations presented here may provide novel insights into evaluating type I collagen functions and the relationships between its binding partners and mutations.Journal of Biological Chemistry 03/2002; 277(6):4223-31. · 4.77 Impact Factor -
Chapter: Glycosaminoglycans: Structure and Biological Functions
04/2001; , ISBN: 9780470015902 -
Article: Chondrogenic potential of chick embryonic calvaria: II. Matrix calcium may repress cartilage differentiation
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ABSTRACT: Chick embryos cultured in the absence of their eggshell are rendered severely calcium-deficient, and develop a cartilage-like phenotype in the calvarium, a normally osteogenic tissue. In the preceding paper (Jacenko and Tuan [1995] Dev. Dyn. 202:13–26), experiments using organ cultured calvaria from day-12 normal and shell-less embryos showed that depletion of calcium alone may be responsible in promoting chondrogenic differentiation in calvaria. Here these findings were extended using an in vivo calvarial grafting technique, such that the extent of calvarial matrix calcification was a function of the calcium status of both the graft and the host. In these calvarial grafts, undermineralized regions again were shown to support chondrogenesis. To identify possible mechanisms which promote chondrogenesis in the calvaria, cells were enzymatically dissociated from the calvaria and cultured in media with varied levels of soluble calcium, under conditions which should modulate cell-to-cell interactions, including monolayer, micromass, agarose gels, and suspension cultures. Soluble calcium had no effect on calvarial cell differentiation, whereas conditions which enhanced cell-cell interactions, e.g., suspension culture, elicited cartilage expression. Based on these findings, we propose that the calcified matrix of the calvarium is repressive to chondrogenesis during normal development, but that the lack of mineral in a calcium-deficient calvarium creates a microenvironment permissive for cell-to-cell interactions which lead to chondrogenic differentiation. © 1995 Wiley-Liss, Inc.Developmental Dynamics 12/1994; 202(1):27 - 41. · 2.54 Impact Factor -
Article: Polyionic regulation of cartilage development: Promotion of chondrogenesis in vitro by polylysine is associated with altered glycosaminoglycan biosynthesis and distribution
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ABSTRACT: The development of cartilage nodules in cultures of chick limb bud mesenchyme (Hamburger-Hamilton stages 23/24) is significantly promoted when the culture medium is supplemented with poly-l-lysine (PL) (Mr ⩾ 14K) (San Antonio and Tuan, 1986. Dev. Biol.115: 313). Here we present findings consistent with the hypothesis that PL may promote chondrogenesis by interacting electrostatically with sulfated glycosaminoglycans (GAGs): (1) poly-l-ornithine, poly-l-histidine, poly-d,l-lysine, and lysine-containing heteropolypeptides stimulate chondrogenesis in proportion to their contents of cationic residues; (2) the effects of PL are diminished when limb mesenchyme cultures are supplemented with exogenous GAGs, including heparin, dermatan sulfate, and chondroitin sulfate; (3) in high density cultures of limb bud mesenchyme, the release of sulfated macromolecules, but not of proteins in general, into the culture medium was significantly inhibited by PL (398K Mr) treatment, and a net increase in total GAG content of the PL-treated cultures was observed; and (4) in monolayer cultures of cells derived from other chick embryonic tissues, including liver, skeletal muscle, and calvaria, PL treatment promoted the cell layer-associated retention of sulfated GAG. These effects were not observed using the nonstimulatory, low Mr PL (4K). Based on the above findings and those from previous studies, it is proposed that PL may promote chondrogenesis by interacting electrostatically with cartilage GAGs, thus trapping the extracellular matrix around the newly emerging cartilage nodules and thereby stabilizing their growth and differentiation.Developmental Biology 09/1992; · 4.07 Impact Factor
Top co-authors
Top Journals
- Journal of Biological Chemistry (5)
- Human Mutation (2)
- Blood (1)
- Developmental Biology (1)
- Developmental Dynamics (1)
Institutions
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1992–2009
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Thomas Jefferson University
- • Department of Pathology, Anatomy & Cell Biology
- • Division of Hospital Medicine
Philadelphia, PA, USA
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2008
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University of Pennsylvania
- Cardiovascular Institute
Philadelphia, PA, USA
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1994
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Harvard University
Boston, MA, USA
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