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

Narayanapanicker Anilkumar¹, Douglas S. Annis², Deane F. Mosher² and Josephine C. Adams^1,*,

¹ MRC Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London, WC1E 6BT, UK
² Department of Medicine, University of Wisconsin, Madison, WI 53706, USA
Present address: Department of Cell Biology, NC1-110, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA

View larger version (33K): [in a new window]   Fig. 1. Schematic representations of the domains of a subgroup A thrombospondin subunit and the recombinant protein units of TSP-1 and TSP-2. Nomenclature: NTD or N, N-terminal domain; o, oligomerisation sequence; C, procollagen module, P, type 1 (properdin) repeat; E, type 2 (EGF-like) repeat; Ca, type 3 repeat/C-terminal globule; CTD, C-terminal globule; Del, deleted. The smaller protein units are designated by the domains they contain. To avoid lengthy terminology, the largest multidomain units are designated by the portions deleted. In the text, each unit is also designated (1) or (2) to indicate whether the protein unit is derived from TSP-1 or TSP-2, respectively.

View larger version (21K): [in a new window]   Fig. 2. Preparation of recombinant proteins. (A) The recombinant protein units DelN(1) (lane 1), DelNo(1) (lane 2), NoC(1) (lane 3), CP123(1) (lane 4), E123(1) (lane 5), E3Ca(1) (lane 6), DelN(2) (lane 7), DelNo(2) (lane 8), NoC(2) (lane 9), E3Ca(2) (lane 10) and oCP123(2) (lanes 11 and 12) were resolved by SDS-PAGE under non-reducing (lane 11) or reducing conditions (all other lanes) and stained with Coomassie blue. The asterisk marks the migration position of DelN proteins. (B) Western blots of trimeric DelN(1) (lane 1) and DelN(2) (lane 3), or monomeric DelNo(1) (lane 2) and DelNo(2), (lane 4), resolved under non-reducing conditions, transferred to nitrocellulose and probed with an antibody to TSP-1 (lanes 1,2) or an antibody to TSP-2 (lanes 3, 4). Molecular weight markers are indicated in kDa.

View larger version (25K): [in a new window]   Fig. 3. Cell attachment to TSP recombinant protein units. (A) 5x104 C2C12 cells (white bars) or VSM cells (shaded bars) were seeded on microtitre plates coated with 50 nM intact TSP-1, FN or 1 µM of the TSP units, and cell attachment quantified after 1 hour of incubation at 37°C. Attachment to TSP-1 or the modules is expressed as percentage of cell attachment to FN. The bars show the mean±s.e.m. of three independent experiments. (B) VSM cells use multiple adhesion systems to attach to the DelNo(1) unit. Cells were incubated in the presence of 1 mM GRGDS peptide, antibodies to integrin ß1, ß3, 2 or 4 subunits (all at 5 µg/ml), 100 µg/ml heparin, 300 µg/ml chondroitin sulphate A or 1 mM of CD47-binding peptide, KRFYVVMWKQVTQK, either alone or in combination. Cell attachment was quantified after 1 hour of incubation and is expressed as the percentage of cell attachment relative to untreated controls. Bars show mean values ± s.e.m. from three experiments.

View larger version (41K): [in a new window]   Fig. 4. Cell attachment properties of trimerised TSP recombinant protein units. (A) Attachment activity of trimeric TSP recombinant protein units. VSM cell attachment was quantified on surfaces coated with 100 nM monomeric or trimeric proteins as indicated and compared with the level of cell attachment to 100 nM TSP-1. Bars show mean values ± s.e.m. from three experiments. (B) Mechanisms of VSM cell attachment to the trimeric proteins, DelN(1), DelN(2) and oCP123(1). Cells were incubated in the presence of 1 mM GRGDS peptide, antibodies to integrin ß1, ß3, 2 or 4 subunits (all at 5 µg/ml), 100 µg/ml heparin, 300 µg/ml chondroitin sulphate A or 1 mM of CD47-binding peptide, KRFYVVMWKQVTQK, either alone or in combination. Cell attachment was quantified after 1 hour of incubation and is expressed as the percentage of cell attachment relative to untreated controls. Bars show mean values ± s.e.m. from three experiments.

View larger version (61K): [in a new window]   Fig. 5. The trimeric DelN(1) and DelN(2) units support cell spreading and fascin cytoskeletal organisation. C2C12 cells (left) or VSM cells (right) were seeded onto coverslips coated with 50 nM FN, TSP-1, 1 µM of the DelNo(1) or DelNo(2) units or 50 nM of the DelN(1), DelN(2) or oCP123(2) units, then fixed and stained for F-actin or for fascin. Bars, 10 µm.