Biomedical subjects

Douglas S Annis

Publications and source records attributed to Douglas S Annis.

At least 19 recordsLinked to original sources

PubMed2006

ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2.

Matrix metalloproteases regulate both physiological and pathological events by processing matrix proteins and growth factors. ADAMTS1 in particular is required for normal ovulation and renal function and has been shown to modulate angiogenesis. Here we report that TSP1 and 2 are substrates of ADAMTS1. Using a combination of mass spectrometry and Edman degradation, we mapped the cleavage sites and characterized the biological relevance of these processing events. ADAMTS1 cleavage mediates the release of polypeptides from the trimeric structure of both TSP1 and 2 generating a pool of antiangiogenic fragments from matrix-bound thrombospondin. Using neo-epitope antibodies we confirmed that processing occurs during wound healing of wild-type mice. However, TSP1 proteolysis is decreased or absent in ADAMTS1 null mice; this is associated with delayed wound closure and increased angiogenic response. Finally, TSP1-/- endothelial cells revealed that the antiangiogenic response mediated by ADAMTS1 is greatly dependent on TSP1. These findings have unraveled a mechanistic explanation for the angiostatic functions attributed to ADAMTS1 and demonstrated in vivo processing of TSP1 under situations of tissue repair.

ADAM Proteins↗

PubMed2006

Differential engagement of modules 1 and 4 of vascular cell adhesion molecule-1 (CD106) by integrins alpha4beta1 (CD49d/29) and alphaMbeta2 (CD11b/18) of eosinophils.

We have studied adhesion of eosinophils to various forms of vascular cell adhesion molecule 1 (VCAM-1, CD106), an integrin counter-receptor implicated in eosinophil recruitment to the airway in asthma. Full-length 7d-VCAM-1, with seven immunoglobulin-like modules, contains integrin-binding sites in modules 1 and 4. The alternatively spliced six-module protein, 6d-VCAM-1, lacks module 4. In static assays, unactivated purified human blood eosinophils adhered similarly to recombinant soluble human 6d-VCAM-1 and 7d-VCAM-1 coated onto polystyrene microtiter wells. Further experiments, however, revealed differences in recognition of modules 1 and 4. Antibody blocking indicated that eosinophil adhesion to 6d-VCAM-1 or a VCAM-1 construct containing only modules 1-3, 1-3VCAM-1, is mediated by alpha4beta1 (CD49d/29), whereas adhesion to a construct containing modules 4-7, 4-7VCAM-1, is mediated by bothalpha4beta1 andalphaMbeta2 (CD11b/18). Inhibitors of phosphoinositide 3-kinase, which block adhesion of eosinophils mediated by alphaMbeta2, blocked adhesion to 4-7VCAM-1 but had no effect on adhesion to 6d-VCAM-1. Consistent with the antibody and pharmacological blocking experiments, eosinophilic leukemic cell lines lacking alphaMbeta2 did not adhere to 4-7VCAM-1 but did adhere to 6d-VCAM-1 or 1-3VCAM-1. Activation of eosinophils by interleukin (IL)-5 enhanced static adhesion to 6d-VCAM-1, 7d-VCAM-1, or 4-7VCAM-1; IL-5-enhanced adhesion to all 3 constructs was blocked by anti-alphaMbeta2. Adhesion of unstimulated eosinophils to 7d-VCAM-1 under flow conditions was inhibited by anti-alpha4 or anti-alphaM. IL-5 treatment decreased eosinophil adhesion to 7d-VCAM-1 under flow, and anti-alphaM had the paradoxical effect of increasing adhesion. These results demonstrate that alphaMbeta2 modulatesalpha4beta1-mediated eosinophil adhesion to VCAM-1 under both static and flow conditions.

Binding Sites↗

PubMed2006

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin.

Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5 min following ligand addition at concentrations as low as 5 nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with alpha6 and beta1 integrin subunits at the base of the extensions. alpha5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectin-fibronectin interactions during progression of assembly.

Animals↗

PubMed2006

Cleavage of human 7-domain VCAM-1 (CD106) by thrombin.

Vascular cell adhesion molecule 1 (VCAM-1, CD106) is expressed as a type I transmembrane integrin counter-receptor on activated endothelium and mediates white blood cell attachment. The alternatively spliced 7-domain (7d) form of VCAM-1 contains a potential thrombin cleavage site. Thrombin proteolysis of 7d-VCAM-1 may help regulate adhesive activity ofVCAM-1. We determined whether 7d-VCAM-1 is proteolyzed and rendered inactive by thrombin. Recombinant extracellular domain of 7d-VCAM-1 was cleaved by thrombin to generate 33- and 44-kDa products. Cleavage was in the sequence PGPR/IAAQIG near the N-terminal border of the alternatively spliced fourth immunoglobulin (Ig)-like module. There was no cleavage of 6d-VCAM-1 lacking the fourth module. Expression of full-length 7d-VCAM-1 presented on Chinese hamster ovary (CHO) monolayers, as detected by flow cytometry with an antibody directed to Ig-like modules 1-3, was reduced by thrombin treatment whereas there was no reduction in the expression of full-length 6d-VCAM-1. Adhesion of blood eosinophils to full-length 7d-VCAM-1 was reduced after treatment of CHO cells with thrombin, whereas adhesion to full-length 6d-VCAM-1 was not affected. We conclude that cleavage of 7d-VCAM-1 by thrombin is a potential mechanism for differential regulation of VCAM-1 splice forms in white blood cell adhesion and trafficking.

Amino Acid Sequence↗

PubMed2005

Low-density lipoprotein receptor-related protein contributes to the antiangiogenic activity of thrombospondin-2 in a murine glioma model.

Host antiangiogenesis factors defend against tumor growth. The matricellular protein, thrombospondin-2 (TSP-2), has been shown to act as an antiangiogenesis factor in a carcinogen-induced model of skin cancer. Here, using an in vivo malignant glioma model in which the characteristics of the tumors formed after intracerebral implantation of GL261 mouse glioma cells are assessed, we found that tumor growth and microvessel density were significantly enhanced in tumors propagated in TSP-2(-/-) mice. Mechanistically, matrix metalloproteinase (MMP)-2 has been associated with neoangiogenesis and it has been proposed that the levels of available MMP-2 may be down-regulated by formation of a complex with TSP-2 that is internalized by low-density lipoprotein receptor-related protein 1 (LRP1). We found elevated expression of MMP-2 and MMP-9 in tumors propagated in TSP-2(-/-) mice, with a preferential localization in the microvasculature. In wild-type mice, MMP-2 was coexpressed with TSP-2 in the tumor microvasculature. In vitro, addition of recombinant (rec) TSP-2 to mouse brain microvessel endothelial cells reduced MMP-2 levels and invasion through mechanisms that could be inhibited by a competitive inhibitor of ligand binding to LRP1 or by siLRP1. Thus, the antiangiogenic activity of TSP-2 is capable of inhibiting the growth of gliomas in part by reducing the levels of MMP-2 in the tumor microvasculature. This mechanism is mediated by LRP1.

Animals↗

PubMed2005

Structure of the calcium-rich signature domain of human thrombospondin-2.

Thrombospondins (THBSs) are secreted glycoproteins that have key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6-A-resolution crystal structure of the glycosylated signature domain of human THBS2, which includes three epidermal growth factor-like modules, 13 aspartate-rich repeats and a lectin-like module. These elements interact extensively to form three structural regions termed the stalk, wire and globe. The THBS2 signature domain is stabilized by these interactions and by a network of 30 bound Ca(2+) ions and 18 disulfide bonds. The structure suggests how genetic alterations of THBSs result in disease.

Amino Acid Sequence↗

PubMed2004

Actions of the functional upstream domain of protein F1 of Streptococcus pyogenes on the conformation of fibronectin.

Fibronectin (Fn), discovered by Harvard's Plasma Protein Program as plasma "cold-insoluble globulin" in the 1940s, has attracted much interest over the past three decades. One of the most interesting features of Fn is its ability to change shape in response to various environmental conditions and interactions with other substances found in the extra-cellular space. Here we examine the potential of the functional upstream domain (FUD) of Streptococcus pyogenes protein F1 to bring about changes in structure of Fn. In particular, we investigate the accessibility of Fn's 10th type III module that contains the integrin binding RGD motif. By use of monoclonal antibodies in a competitive ELISA assay, we found that FUD interacts with the amino-terminal type I modules of Fn to unveil the cell-binding region of Fn. This conformational change was achieved at sub-equimolar ratios of FUD/Fn monomer. We discuss the functional relevance of the interaction for both Fn and S. pyogenes and correlate the results with a conformational model of Fn that arose out of a collaboration between our laboratory and that of John Ferry.

Adhesins, Bacterial↗

PubMed2004

Identification of novel beta1 integrin binding sites in the type 1 and type 2 repeats of thrombospondin-1.

In addition to the three known beta(1) integrin recognition sites in the N-module of thrombospondin-1 (TSP1), we found that beta(1) integrins mediate cell adhesion to the type 1 and type 2 repeats. The type 1 repeats of TSP1 differ from typical integrin ligands in that recognition is pan-beta(1)-specific. Adhesion of cells that express one dominant beta(1) integrin on immobilized type 1 repeats is specifically inhibited by antagonists of that integrin, whereas adhesion of cells that express several beta(1) integrins is partially inhibited by each alpha-subunit-specific antagonist and completely inhibited by combining the antagonists. beta(1) integrins recognize both the second and third type 1 repeats, and each type 1 repeat shows pan-beta(1) specificity and divalent cation dependence for promoting cell adhesion. Adhesion to the type 2 repeats is less sensitive to alpha-subunit antagonists, but a beta(1) blocking antibody and two disintegrins inhibit adhesion to immobilized type 2 repeats. beta(1) integrin expression is necessary for cell adhesion to the type 1 or type 2 repeats, and beta(1) integrins bind in a divalent cation-dependent manner to a type 1 repeat affinity column. The widely used TSP1 function blocking antibody A4.1 binds to a site in the third type 2 repeat. A4.1 proximally inhibits beta(1) integrin-dependent adhesion to the type 2 repeats and indirectly inhibits integrin-dependent adhesion mediated by the TSP1 type 1 repeats. Although antibody A4.1 is also an antagonist of CD36 binding to TSP1, these data suggest that some biological activities of A4.1 result from antagonism of these novel beta(1) integrin binding sites.

Antibodies, Monoclonal↗

PubMed2004

Eosinophils adhere to vascular cell adhesion molecule-1 via podosomes.

Vascular cell adhesion molecule (VCAM)-1 supports specific eosinophil adhesion via alpha4beta1 integrin. We tested the hypothesis that adhesive contacts formed by eosinophils on VCAM-1 are different from focal adhesions formed by adherent fibroblasts. Eosinophils adherent on VCAM-1 formed punctate adhesions that fit the criteria for podosomes, highly dynamic structures found in adherent transformed fibroblasts, osteoclasts, and macrophages. The structures contained beta1 integrin subunit, phosphotyrosine-containing proteins, punctate filamentous actin, and gelsolin, a podosome marker. In contrast, nontransformed fibroblasts on VCAM-1 formed peripheral focal adhesions that were positive for alpha4, beta1, phosphotyrosine, vinculin, talin, and paxillin; negative for gelsolin; and associated with microfilaments. Phorbol myristate acetate or tumor necrosis factor-alpha and interleukin-5 stimulated podosome formation in adherent eosinophils. Because podosomes in tumor cells are associated with extracellular matrix degradation, we analyzed the VCAM-1 layer. VCAM-1 was lost under adherent eosinophils but not under adherent fibroblasts. This loss was inhibited by the metalloproteinase inhibitor ortho-phenanthroline and correlated with expression and podosome localization of a membrane-tethered metalloproteinase, a disintegrin and metalloproteinase domain 8. Podosome-mediated VCAM-1 clearance may be a mechanism to regulate eosinophil arrest and extravasation in allergic conditions such as asthma.

Actin Cytoskeleton↗

PubMed2003

Alpha4beta1 integrin mediates selective endothelial cell responses to thrombospondins 1 and 2 in vitro and modulates angiogenesis in vivo.

We examined the function of alpha4beta1 integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. Alpha4beta1 supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by alpha4beta1, whereas antagonism of fibroblast growth factor-2-stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an alpha4beta1-dependent manner. Soluble alpha4beta1 antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1-dependent and not observed in explants from thrombospondin-1-/- mice. Antagonizing alpha4beta1 may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the alpha4beta1 binding sequence stimulate angiogenesis in vivo. Therefore, alpha4beta1 is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.

Animals↗

PubMed2003

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin.

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.

Amino Acid Sequence↗

PubMed2003

Interactions among the three structural motifs of the C-terminal region of human thrombospondin-2.

The C-terminal regions of thrombospondins (TSPs) contain three elements, EGF-like modules (E), a series of Ca(2+)-binding repeats (Ca), and a C-terminal sequence (G). We have looked for interactions among these elements in four recombinant proteins based on human TSP-2: E3CaG-2, CaG-2, E3Ca-2, and Ca-2. When bound Ca(2+) was assayed by atomic absorption spectroscopy or an equilibrium dialysis protocol in which Ca(2+) was removed from the proteins prior to equilibrium dialysis, E3CaG-2 bound 22-27 Ca(2+), CaG-2 bound 17-20 Ca(2+), and E3Ca-2 and Ca-2 bound 14-20 Ca(2+). Approximately 10 of the bound Ca(2+) in E3CaG-2 were exchangeable. The far UV circular dichroism (CD) spectrum of Ca(2+)-replete E3CaG-2 contained a strong negative band at 203 nm attributable to Ca and a less intense negative band at 218 nm attributable to Ca and G. Chelation of Ca(2+) with EDTA shifted the 203 nm band of all four proteins and the 218 nm band of E3CaG-2 and CaG-2 to less negative positions. The apparent EC50 for the far UV CD transition was 0.22 mM Ca(2+) for all proteins, indicating that Ca(2+) binding to Ca is primarily responsible for the CD change. Near UV CD and intrinsic fluorescence revealed that the tryptophan residues in G are sensitive to changes in Ca(2+). Differential scanning calorimetry of the proteins in 2 mM Ca(2+) showed that E3CaG-2 melts with two transitions, 44-51 degrees C and 75-83 degrees C. The lower transition required G, while the higher transition required Ca. Both transitions were stabilized in constructs containing E3. These results indicate that E3, Ca, and G function as a complex structural unit, and that the structures of both Ca and G are influenced by the presence or absence of Ca(2+).

Amino Acid Sequence↗

PubMed2003

A polymorphism in thrombospondin-1 associated with familial premature coronary heart disease causes a local change in conformation of the Ca2+-binding repeats.

A single nucleotide polymorphism that substitutes a serine for an asparagine at residue 700 in the Ca2+-binding repeats of thrombospondin-1 is associated with familial premature coronary heart disease. We expressed the Ca2+-binding repeats alone (Ca) or with the third epidermal growth factor-like module (E3Ca), without (Asn-700) or with (Ser-700) the disease-associated polymorphism. The intrinsic fluorescence of a single tryptophan (Trp-698) adjacent to the polymorphic residue was quenched cooperatively by adding Ca2+. The third epidermal growth factor-like repeat dramatically altered the Ca2+-dependent fluorescence transition for the Asn-700 constructs; the half-effective concentration (EC50) of Ca Asn-700 was 390 microM, and the EC50 of E3Ca Asn-700 was 70 microM. The Ser-700 polymorphism shifted the EC50 to higher Ca2+ concentrations (Ca Ser-700 EC50 of 950 microM and E3Ca Ser-700 EC50 of 110 microM). This destabilizing effect is due to local conformational changes, as the Ser-700 polymorphism did not influence the secondary structure of E3Ca or Ca as assessed by far UV circular dichroism. At 200 microM Ca2+, in which both E3Ca Asn-700 and Ser-700 are in the Ca2+-replete conformation at 37 degrees C, the fluorescence of E3Ca Ser-700 reverted to the Ca2+-depleted spectrum at 50 degrees C compared with 65 degrees C for E3Ca Asn-700. These findings indicate that the Ser-700 polymorphism subtly but significantly sensitizes the calcium-binding repeats to removal of Ca2+ and thermal denaturation.

Calcium↗

PubMed2002

Biophysical characterization, including disulfide bond assignments, of the anti-angiogenic type 1 domains of human thrombospondin-1.

Thrombospondin-1 (TSP1), a modular secreted glycoprotein, possesses anti-angiogenic activity both in vitro and in vivo. This activity has been localized to the thrombospondin type 1 repeats/domains (TSR). A TSP1 monomer contains three TSRs, each with a hydrophobic cluster with three conserved tryptophans (WxxWxxW), a basic cluster with two conserved arginines (RxR), and six conserved cysteines. Using the baculovirus system, we expressed TSRs of human TSP1 as either the three domains in tandem (P123) or the third domain alone (P3) and demonstrated that both P123 and P3 at nanomolar concentrations inhibit either basic fibroblast-growth-factor or sphingosine-1-phosphate induced endothelial cell migration. Far-UV circular dichroism (CD) indicated that P123 and P3 have a common global fold that is very similar to properdin, a protein with six TSRs. Near-UV CD and fluorescence quenching studies indicated the conserved tryptophans are in a structured, partially solvent-accessible, positively charged environment. N-terminal sequence and mass spectrometry analysis of trypsin-digested TSRs indicated that the RFK linker sequence between P1 and P2 is readily proteolyzed and the conserved arginines are solvent accessible. By a combination of proteolysis and mass spectrometry, the recombinant TSRs were determined to be fully disulfide bonded with a connectivity of 1-5, 2-6, and 3-4 (cysteines are numbered sequentially from N- to C-terminus). TSRs are found in numerous extracellular proteins. These TSRs share the hydrophobic and basic clusters of the TSP TSRs but some have quite different placement of cysteine residues. We propose a sorting of TSRs into six groups that reconciles our results with information about other TSRs.

Amino Acid Sequence↗

PubMed2002

The von Willebrand factor-reducing activity of thrombospondin-1 is located in the calcium-binding/C-terminal sequence and requires a free thiol at position 974.

Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury; however, only the very large VWF multimers are effective in promoting platelet adhesion in flowing blood. The multimeric size of VWF can be controlled by the glycoprotein, thrombospondin-1 (TSP-1), which facilitates reduction of the disulfide bonds that hold VWF multimers together. The TSP family of extracellular glycoproteins consists of 5 members in vertebrates, TSP-1 through TSP-4 and TSP-5/COMP. TSP-1 and TSP-2 are structurally similar trimeric proteins composed of disulfide-linked 150-kDa monomers. Recombinant pieces of TSP-1 and TSP-2 incorporating combinations of domains that span the entire subunit were produced in insect cells and examined for VWF reductase activity. VWF reductase activity was present in the Ca(++)-binding repeats and C-terminal sequence of TSP-1, but not of TSP-2. Alkylation of Cys974 in the C-terminal TSP-1 construct, which is a serine in TSP-2, ablated VWF reductase activity. These results imply that the reductase function of TSP-1 centers around Cys974 in the C-terminal sequence.

Alkylation↗

PubMed2002

Regulation of integrin function by CD47 ligands. Differential effects on alpha vbeta 3 and alpha 4beta1 integrin-mediated adhesion.

We examined the regulation of alpha4beta1 integrin function in melanoma cells and T cells by ligands of CD47. A CD47 antibody (B6H12) that inhibited alphavbeta3-mediated adhesion of melanoma cells induced by CD47-binding peptides from thrombospondin-1 directly stimulated alpha4beta1-mediated adhesion of the same cells to vascular cell adhesion molecule-1 and N-terminal regions of thrombospondin-1 or thrombospondin-2. B6H12 also stimulated alpha4beta1- as well as alpha2beta1- and alpha5beta1-mediated adhesion of CD47-expressing T cells but not of CD47-deficient T cells. alpha4beta1 and CD47 co-purified as a detergent-stable complex on a CD47 antibody affinity column. CD47-binding peptides based on C-terminal sequences of thrombospondin-1 also specifically enhanced adhesion of melanoma cells and T cells to alpha4beta1 ligands. Unexpectedly, activation of alpha4beta1 function by the thrombospondin-1 CD47-binding peptides also occurred in CD47-deficient T cells. CD47-independent activation of alpha4beta1 required the Val-Val-Met (VVM) motif of the peptides and was sensitive to inhibition by pertussis toxin. These results indicate that activation of alpha4beta1 by the CD47 antibody B6H12 and by VVM peptides occurs by different mechanisms. The antibody directly activates a CD47-alpha4beta1 complex, whereas VVM peptides may target an unidentified Gi-linked receptor that regulates alpha4beta1.

Amino Acid Sequence↗

PubMed2002

Trimeric assembly of the C-terminal region of thrombospondin-1 or thrombospondin-2 is necessary for cell spreading and fascin spike organisation.

Thrombospondin-1 (TSP-1) and the highly related protein thrombospondin-2 (TSP-2) are trimeric extracellular molecules that have complex roles in wound healing, angiogenesis and matrix organisation. At the cellular level, TSP-1 supports cell adhesion and migration by the organisation of fascin spike cytoskeletal structures. To define the molecular requirements for assembly of fascin spikes by thrombospondins, we developed a panel of recombinant protein units of TSP-1 and TSP-2; these were designed according to the domain boundaries and included matched monomeric and trimeric units. These proteins were tested for their effects on cell attachment and fascin spike organisation using C2C12 skeletal myoblasts and vascular smooth muscle cells. In monomeric units, cell attachment activity was localised to the type 1 repeats or type 3 repeats/C-terminal globule, and both regions need to be present in the same molecule for maximal activity. On a molar basis, cell-attachment activities with monomeric units were low compared with intact TSP-1, and no monomeric unit induced cell spreading. Trimeric versions of the type 1 repeats were more adhesive but did not induce cell spreading. Strikingly, trimers that contained the type 3 repeats/C-terminal globule of either TSP-1 or TSP-2 supported cell spreading and fascin spike organisation, producing a similar activity to intact TSP-1. We conclude that trimeric assembly of the highly conserved TSP C-terminal region is necessary for organisation of the fascin-based cytoskeletal structures that are needed for thrombospondin-induced cell motility.

Actin Cytoskeleton↗

PubMed2002

Interactions of thrombospondins with alpha4beta1 integrin and CD47 differentially modulate T cell behavior.

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin alpha4beta1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of alpha4beta1 integrin, and TSP1 inhibited interaction of activated alpha4beta1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The alpha4beta1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the alpha4beta1 integrin-dependent activities of TSP1. The beta1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.

Antigens, CD↗