Bound cells were fixed with 100 l/well 5% glutaraldehyde solution in PBS for 20 min. 20C25, and 30C50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate. (6) demonstrated for fibulin-5 a compact dimer in the presence of calcium. Open in a separate window FIGURE 1. Overview and characterization of fibulin-3, -4, and -5. above the schematic indicate proteolytic cleavage sites corresponding to the protein bands in and protein bands in and were analyzed by N-terminal sequencing and are summarized R-BC154 in Table 1 and schematically in were analyzed by mass spectrometry. indicate bands correlating to full-length proteins, and indicate degradation products. Globular marker proteins are indicated in kDa, and the stacking-resolving interface (binding experiments and mouse and human data, fibulin-4 and -5 have recently been incorporated into a working model of elastogenesis. Fibulin-4 is hypothesized to bring tropoelastin and pro-lysyl oxidase in close proximity to facilitate initial cross-linking within the coacervate (28). Fibulin-5 is presumed to then bind the partially cross-linked tropoelastin coacervate and direct it to the microfibril for further cross-linking and incorporation into the mature elastic fiber (12, 13, 29). This study aimed to advance the understanding of the characteristics of the short fibulins with a focus on proteolysis and cell attachment. Human short fibulins were recombinantly expressed and purified, and SDS-PAGE analysis revealed that all three proteins were for 15 min at 4 C. 100 m phenylmethanesulfonyl fluoride (PMSF) was then added to prevent serine protease proteolysis of the collected material, and the collection was stored at ?20 C until protein purification. Treatment of stably transfected HEK293 cells with doxycycline (DOX; Sigma, product D9891) was attempted to inhibit MMPs to prevent proteolytic cleavage of the recombinant short fibulins. 25 m DOX supplementation commenced when the cells were transferred onto eight triple layer flasks and was repeated with each collection of serum-free medium every 48 h. As a control, the transfected HEK293 cells on eight R-BC154 triple layer flasks were cultured without DOX. Human skin fibroblasts were extracted from foreskins of healthy boys (2C5 years of age) following a standard circumcision procedure. This procedure was approved by the Montreal Children’s Hospital Research Ethics Committee (PED-06-054), and written consent of each patient’s parents was obtained. Cell lines of human fetal lung fibroblasts (ATCC, product CCL-75) and of human umbilical vein smooth muscle cells (ATCC, product CRL-2481) were commercially purchased. Human umbilical artery smooth muscle cells were a generous gift R-BC154 from REV7 Dr. Cynthia Goodyer. Cell culture conditions were identical to those used for HEK293 cells but without G418 supplementation. Cells in passages 4C10 were used for all experiments. Recombinant Protein Purification Recombinant hexahistidine-tagged short fibulins were purified to homogeneity by immobilized metal ion affinity chromatography with a Ni2+-loaded HisTrap HP 1-ml column (GE Healthcare, product 17-5247-01) using an ?ktaPurifier 10 low pressure liquid chromatography system (GE Healthcare) as follows. 2.5 liters of conditioned medium was concentrated to 50 ml in pressurized ultrafiltration cells (Amicon, products 5124 and 6028) through a 10-kDa cutoff membrane (Millipore, product PLGC07610) over 24 h at 4 C. The resulting 50 ml were dialyzed two times for 6 h against 2 liters of 20 mm HEPES, 500 mm NaCl, pH 7.4 (running buffer) at 4 C. Any remaining precipitate was centrifuged at 11,000 for 15 min at 4 C, and the supernatant was used to fill a 50-ml Superloop (GE Healthcare, product 19-7850-01). The concentrate was loaded at a flow rate of 0.5 ml/min followed by a 5-ml wash with running buffer. Bound protein was eluted from the column at 1 ml/min with a 0C500 mm linear imidazole gradient in 20 mm HEPES, 500 mm NaCl, pH 7.4. 1-ml eluate fractions were collected, and aliquots were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. Fractions containing purified short fibulins were pooled and dialyzed two times for 6 h against 1 liter of CaCl2-containing Tris-buffered saline (TBS/Ca; 50 mm Tris-HCl, 150 mm NaCl, 2 mm CaCl2, pH 7.4). The BCA protein assay.
