Figure 5.

The Discoidin Domain of ACLP Binds to Fibrillar Collagens and Enhances Polymerization of Collagen I In Vitro

(A) Binding assays were performed as previously described.²¹ Different extracellular matrix proteins were diluted to 10 μg/mL in PBS and each sample was coated to individual wells of a 96-well Corning cell culture plate overnight at room temperature. Proteins used were bovine gelatin (Sigma), rat collagen type I, bovine collagen type II, mouse type III collagen (Fibrogen), mouse collagen type IV, human collagen type V, and human fibronectin (BD Biosciences). Wells were washed with TBST (50 mM Tris [pH 7.4], 150 mM NaCl, 1% Tween-20) and blocked with 1 mg/mL casein (Sigma) in TBST for 1 hr followed by additional washes in TBST. Recombinant anti-Xpress tagged Discoidin-Like-Domain (DLD) protein was purified from BL21 bacteria as previously described.¹⁵ Recombinant DLD binding assays were performed as above with coating of 10 μg/mL of extracellular matrix proteins to a 96-well Corning cell culture plate as described above. Wells were washed in TBST and blocked in 1 mg/mL casein in TBST for 1 hr followed by additional washes in TBST. DLD protein was diluted in TBST to 500 nM and was incubated with each extracellular matrix proteins for 2 hr at room temperature. Wells were washed 3× with TBST and anti-Xpress (Invitrogen) was diluted 1:1,000 in TBST then incubated with each sample for 1 hr. Additional TBST washes were performed and anti-mouse HRP was diluted 1:1,000 in TBST and incubated with each sample for 1 hr followed by washing. Signal was detected using the HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (eBiosciences) and reactions were stopped by the addition of 2 M H₂SO₄. Plates were read at OD 450 nm and results are presented as either fold control (plastic-only wells).

(B) Recombinant ACLP was generated as previously described.¹⁸ In order to explore the role of ACLP in collagen fibril formation, a collagen polymerization assay was used.^{26, 27} Collagen I (0.6 mg/mL) was diluted in PBS containing 20 μg/mL rACLP and allowed to polymerize at 37°C for 1 hr in a clear 96-well plate. Readings at 410 nm were taken every 30 s using a BioTek Synergy HT system (Biotek). PBS containing only ACLP was also analyzed as a control.