Biophysical Characterization of the Signature Domains of Thrombospondin-4 and Thrombospondin-2

Misenheimer, Tina M.; Mosher, Deane F.

doi:10.1074/jbc.m504696200

Cited by 19 publications

(23 citation statements)

References 27 publications

(39 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Lowering the calcium concentration to 200 M, therefore, lowers the temperatures of the reversible transitions and, as in 2 mM calcium, the Ser 700 protein was more sensitive to heating than the Asn 700 protein. The four peaks seen in the THBS-1 proteins contrasts with DSC studies on the signature domain of THBS-2, which melts with two peaks (24,25) (Fig. 2C).…”

Section: The Polymorphism Leads To Altered Thermal Stability Of the Ementioning

confidence: 56%

Influences of the N700S Thrombospondin-1 Polymorphism on Protein Structure and Stability

Carlson¹,

Liu

Keck

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Thrombospondins (THBSs)3 are secreted metalloglycoproteins found in organisms as diverse as Drosophila, Ciona, and tetrapods, in which there are 5 THBSs (1, 2). THBSs are organized into two groups, based on their modular structure. Group A THBSs (THBS-1 and THBS-2) form trimers and are composed of an N-terminal module, an oligomerization domain, a von Willebrand Factor type C homology module, three THBS type 1 or properdin modules, and a signature domain comprising three epidermal growth factor-like modules (EGF1, EGF2, and EGF3), a calcium-binding wire, and a C-terminal lectin-like module (Fig. 1A). Group B THBSs (THBSs 3-5) form pentamers, lack von Willebrand factor type C and properdin modules, and have an additional EGF-like module, EGF2Ј (3). The most C-terminal EGF-like module, the wire, and lectin-like module have the highest identity, 53-82% identity, when comparing Drosophila to vertebrate THBSs (3).Crystal structures of the signature domains of THBS-1 and THBS-2 revealed that the wire is composed of 13 calcium-binding repeats: eight C-type and five N-type (4, 5). N-type repeats are 13 or 15 residues long, whereas C-type repeats are 23 residues long. There is an insert in repeat 1C of the wire that contains an Asn-Ala-Thr glycosylation sequence in THBS-1 and THBS-2 (Fig. 1, B and C). The THBS-2 signature domain binds 30 calcium ions, with one calcium ion present in EGF2 at the EGF1-EGF2 interface, 26 sites in the wire, and three sites in the lectin-like module. The structure of the THBS-2 signature domain revealed extensive interactions among EGF2, EGF3, the wire, and the lectin-like module (Fig. 1B). Most strikingly, the wire wraps around the lectin-like module, making contact at repeats 1C and 9C. EGF2 and EGF3 also interact with the wire-lectin complex (5).THBSs have diverse roles in angiogenesis, cell motility, apoptosis, cytoskeletal organization, and extracellular matrix organization (3). Various studies have demonstrated the ability of recombinant THBS-1 proteins made in insect cells to recapitulate functions of naturally derived proteins (6 -9). There is additional interest in these proteins because of the large number of disease-associated genetic alterations occurring within the signature domain. Heterozygosity or homozygosity for the Ser 700 allele of the A8831G non-synonymous single nucleotide polymorphism in the THBS-1 gene is associated with the occurrence of premature (age Ͻ 45) coronary heart disease (7, 10). Over 100 identified mutations in THBS-5, also known as cartilage oligomeric matrix protein (COMP) (the gene is designated COMP), lead to skeletal dysplasias when present as a single copy (11). When considered in the context of the crystal struc-* This work was supported, in whole or in part, by National Institutes of Health Grant HL54462 (to D. F. M.). This work was also supported by a Shaw Foundation for Medical Research grant (to J. L. K.). Differential scanning calorimetry data were obtained at the University of Wisconsin-Madison Biophysics Instrumentation Facility, which was est...

show abstract

Section: The Polymorphism Leads To Altered Thermal Stability Of the Ementioning

confidence: 56%

Influences of the N700S Thrombospondin-1 Polymorphism on Protein Structure and Stability

Carlson¹,

Liu

Keck

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recent advances in understanding the structures of TSPs have revealed that there are extensive interactions between the EGF domains, type 3 repeats and L-lectin-like domain, such that the whole multidomain region should be considered a single structural unit (Kvansakul et al, 2004;Misenheimer and Mosher, 2005;Carlson et al, 2005). These findings are corroborated by immunochemical analyses (Annis et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Extracellular matrix retention of thrombospondin 1 is controlled by its conserved C-terminal region

Adams

Bentley

Kvansakul

et al. 2008

Journal of Cell Science

View full text Add to dashboard Cite

domain or type 1 repeats. Using a novel mRFP-tagged TSP1 C-terminal trimer, we demonstrate that ECM retention involves the RGD site and a novel site in the L-lectin domain with structural similarity to the ligand-binding site of cargo transport proteins. CD47 and ␤1 integrins are dispensable for ECM retention, but ␤1 integrins enhance activity. These novel data advance concepts of the molecular processes that lead to ECM retention of TSP1. Journal of Cell Science Matrix retention of thrombospondinsTSP2 is present in cartilage and various connective tissues (Bornstein et al., 2004). TSP4 is present in the ECM of the nervous system, at neuromuscular junctions, in the developing retina, adult eye and tendon (Arber and Caroni, 1995; Hauser et al., 1995;Stenina et al., 2003; Dunkle et al., 2007). TSP5 or COMP (cartilage oligomeric matrix protein) is a component of ECM in cartilage, synovium and tendon, which associates with collagen fibrils and binds collagens II and IX, matrilin-3 and fibronectin in vitro (Hedbom et al., 1992; DiCesare et al., 1995; DiCesare et al., 1997; DiCesare et al., 2002;Sodersten et al., 2005). In vivo models and human diseases provide evidence that TSPs contribute functionally to ECM organisation and can modulate the adhesive or structural properties of ECM. For example, TSP2-null mice have aberrant assembly of collagen fibrils because of reduced cell surface tissue transglutaminase, resulting in lax tendons and decreased tensile strength of the skin (Kyriakides et al., 1998; Agah et al., 2005). Interaction of Drosophila TSP (D-TSP) with ␣PS2 integrin on muscle cells is needed for stable tendon and muscle attachment during development (Chanana et al., 2007;Subramanian et al., 2007). Polymorphisms in the coding sequences of human THBS1 and THBS4, which predispose to cardiovascular disease, enhance platelet aggregation in the case of TSP1 and affect endothelial cell adhesion in the case of TSP4 (Stenina et al., 2007).In vitro binding assays with purified proteins have demonstrated that TSPs can bind to multiple structural ECM components and to various growth factors that may themselves bind ECM (reviewed by Adams, 2001). These findings indicate targets for binding by extracellular TSPs, but do not address the molecular processes by which TSPs come to be retained within ECM. Knowledge of these processes is important with regard to the clear biological significance of TSPs in connective ECM, tumour biology, immune response, synaptogenesis and vascular function. Prospective therapeutic modulations or construction of synthetic ECM would also benefit from this knowledge. Until recently, the ability to analyse these mechanisms was hampered by a lack of understanding of the complex structure of TSP polypeptides, particularly the large and highly conserved C-terminal region. In recent years, structures for all the major domains of TSP1 and TSP2 have been solved at the atomic level (Tan et al., 2002;Tan et al., 2006;Kvansakul et al., 2004; Carlson et al., 2005). We demonstrate here that ECM retention...

show abstract

“…These elements are three or more contiguous EGF-like modules, the Ca 2ϩ -binding wire, and the lectin-like module (4). Atomic absorption spectroscopy estimated that 28 Ca 2ϩ bind to the TSP-2 signature domain (E123CaG-2) in solution (5). This number agrees with the crystal structure, which revealed 26 Ca 2ϩ ions bound to the wire, 3 to the lectin-like module, and 1 to EGF2, for a total of 30 bound Ca 2ϩ ions (4).…”

Section: Thrombospondins (Tsp)mentioning

confidence: 99%

Immunochemical Analysis of the Structure of the Signature Domains of Thrombospondin-1 and Thrombospondin-2 in Low Calcium Concentrations

Annis

Gunderson

Mosher

2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Thrombospondins (TSPs) undergo conformational changes upon removal of calcium. The eight C-type and five N-type calcium-binding repeats of TSP-2 form a circuitous wire that, in 2 mM calcium, interacts at its ends with more N-terminal epidermal growth factor (EGF)-like modules, EGF2 and EGF3, and the C-terminal lectin-like module. These components, along with the other EGF-like module(s), form the signature domain of TSPs. Characterization of conformation-sensitive epitopes of monoclonal antibodies to human TSP-2 and its TSP-1 homolog have given insights into the structure of the signature domain in the absence of calcium. The epitope for 4B6.13 anti-TSP-2 was localized to His-722 and Leu-703 in repeat 1C of the wire; recognition only occurred in constructs that included EGF3, the rest of the wire, and the lectin-like module and in the presence of calcium. The epitope for C6.7 anti-TSP-1 was localized to Glu-609 in the EGF2 module. The C6.7 epitope was preferentially recognized when EGF2 was expressed in the context of EGF1, EGF3, the wire, and the lectin-like module. Preferential recognition of the C6.7 epitope did not require calcium. Rotary shadowing electron microscopy of TSP-1 has shown elongation of the stalk and diminution of the C-terminal globule. We propose a model whereby at low calcium concentrations the lectin-like module drops away from EGF3 concomitant with changes in conformation of the wire and loss of the 4B6.13 epitope. A critical feature of the model is interaction of repeat 12N of the wire with EGF2 in both the presence and absence of calcium.

show abstract

Biophysical Characterization of the Signature Domains of Thrombospondin-4 and Thrombospondin-2

Cited by 19 publications

References 27 publications

Influences of the N700S Thrombospondin-1 Polymorphism on Protein Structure and Stability

Influences of the N700S Thrombospondin-1 Polymorphism on Protein Structure and Stability

Extracellular matrix retention of thrombospondin 1 is controlled by its conserved C-terminal region

Immunochemical Analysis of the Structure of the Signature Domains of Thrombospondin-1 and Thrombospondin-2 in Low Calcium Concentrations

Contact Info

Product

Resources

About