Fig. 6.

LTBP-1 interactions with fibrillin-1. (A) BIAcore analysis of the binding of immobilized full-length LTBP-1 to PF1, the unprocessed N-terminal fibrillin-1 fragment (Cain et al., 2008). The sensorgram (inset) shows analyte concentrations ranging from 0-2000 nM, with duplicate concentrations in every run. One representative experiment is shown. Response difference is the difference between experimental and control flow cells, in response units. Time is shown in seconds. Evaluation of the kinetics of the interactions was performed according to a 1:1 Langmuir binding model. PF1 bound only weakly to full-length LTBP-1 (K_D=1270±186 nM). Data are mean ± s.e.m. of at least two separate experiments. Similarly, PF1^Ex1-11 and PF1^Ex5-7 showed only weak binding to full-length LTBP-1 (not shown). (B) BIAcore analysis of the binding of C-terminal LTBP-1 (C-LTBP-1), immobilized on a CM5 chip, to PF1 (i), PF1^Ex5-7 (ii) or PF1^Ex6+7 (iii). Sensorgrams show analyte concentrations ranging from 0-3000 nM, with duplicate concentrations in every run. One representative experiment is shown in each case. Response difference is the difference between experimental and control flow cells, in response units. Time is shown in seconds. Evaluation of the kinetics of the interactions was performed according to a 1:1 Langmuir binding model. Data are mean ± s.e.m. of at least two separate experiments. PF1 bound with moderate affinity to C-LTBP-1 (K_D=134.4±36.9 nM), whereas PF1^Ex5-7 and PF1^Ex6+7 bound strongly to C-LTBP-1 (K_D values=9.3±3.3 nM and 31.2±0 nM, respectively). (C) Solid-phase binding assays showing soluble biotinylated C-terminal LTBP-1 (C-LTBP-1) binding to immobilized wild-type PF1 or to eight N-terminal Marfan syndrome mutants (shown on the left). PF1 mutants were immobilized onto Immulon-4 HBX microtitre plates at 100 nM, and soluble biotinylated C-LTBP-1 was plated at 100 nM and detected at 405 nm. GraphPad Prism 4.0 was used to calculate significance using unpaired Student's t-tests. One representative experiment is shown. Data are shown with the negative (biotinylated LTBP-1 only) control subtracted. Results are shown as the mean ± s.e.m. of triplicate values. Soluble biotinylated LTBP-1 bound strongly to PF1 and seven of the mutants, but binding to mutant C166S was significantly reduced (***P<0.0001 by Student's t-test in comparison with wild-type PF1). (D) Human dermal fibroblasts were cultured for up to 7 days. Medium and cell-layer extracts were immunoprecipitated with anti-LTBP-1 mAb388, and analyzed by SDS-PAGE and immunoblotting using anti-fibrillin-1 proline-region antibody. Fibrillin-1 was detected mainly in cell-layer immunoprecipitates, as a 350 kDa band.