Abstract
Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.
Highlights
In the studies described here, we investigated the additional possibility that analyses of microfibrils obtained from enzymatic digestion of tissues could lead to difficulties in interpretation, if fibrillin molecules are degraded during the extraction process
Fibrillin-1 monoclonal antibodies 26, 201, 15, and 69 have been characterized previously [3, 4, 6, 28, 29]. mAb 78 was produced from a fusion of splenocytes immunized with a pepsin fragment called PF3 [28] and has been characterized as an antibody specific for fibrillin-1.5 Polyclonal antibody 9543 has been demonstrated to be highly specific for fibrillin-1 [30]
The results presented in this study show that fibrillin-1 is cleaved during the crude collagenase digestions commonly used to extract microfibrils
Summary
Reagents—All chemicals, including collagenase (type 1A), were purchased from Sigma. Fibrillin-1 monoclonal antibodies (mAbs) 26, 201, 15, and 69 have been characterized previously [3, 4, 6, 28, 29]. mAb 78 was produced (as we have described for other mAbs) from a fusion of splenocytes immunized with a pepsin fragment called PF3 [28] and has been characterized as an antibody specific for fibrillin-1.5 Polyclonal antibody 9543 has been demonstrated to be highly specific for fibrillin-1 [30]. Recombinant fibrillin-1 peptides, rF11, rF23, rF20, rF30, rF31, and rF6, and recombinant fibrillin-2 peptide, rF32, were produced and purified as described elsewhere [3, 29, 32] These recombinant peptides were treated with 0.2 mg/ml Sigma collagenase (type 1A) in 50 mM Tris-HCl, pH 7.4, containing 0.01 M CaCl2, 2 mM phenylmethylsulfonyl fluoride, and 10 mM N-ethylmaleimide for 48 h at 4 °C. The product of the PCR amplification was cloned into an NdeI/BamHI-digested pET11a vector so that the final construct contained the entire human BMP-7 prodomain coding sequence starting from Asp, after the predicted endogenous signal peptide cleavage site, followed by a C-terminal His tag and a stop codon This vector was transformed into competent cells of E. coli DH5␣, and the insert structure was verified by restriction analysis and DNA sequencing. Fractions were analyzed by SDS-PAGE for their purity and molecular mass, and imidazole in these fractions was removed by dialysis against buffer A
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