Recombinant Expression and Characterization of a Novel Fibronectin Isoform Expressed in Cartilaginous Tissues

Tomohiro Kozaki,Yoshito Matsui,Jianguo Gu,Ryoko Nishiuchi,Nobuo Sugiura,Koji Kimata,Keiichi Ozono,Hideki Yoshikawa,Kiyotoshi Sekiguchi

doi:10.1074/jbc.m307432200

Abstract

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.

Highlights

A novel fibronectin (FN) isoform lacking the segment from IIICS through the I-10 module is expressed predominantly in normal cartilaginous tissues
It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin ␣5␤1, it was more active than plasma-type FN in binding to chondroitin sulfate E
Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing

Summary

EXPERIMENTAL PROCEDURES

CDNA Construction—The cDNA expression vector pAIFNC, which encodes recombinant plasma-type FN (designated rFN/C), was described previously [13]. The flow-through fractions were applied to an immunoaffinity column containing mAb 119A to capture recombinant FNs. Proteins bound to the columns were eluted with 0.1 M triethylamine (pH 11.5), immediately neutralized with 2 M NaH2PO4 (pH 3.6), and dialyzed against phosphate-buffered saline (PBS). Cells were washed three times with PBS, fixed with 3.7% paraformaldehyde, and double-stained with mouse antihuman FN mAb 136H and rabbit anti-FN polyclonal antibody at room temperature for 1 h. For cell adhesion inhibition assays, cells were preincubated with function-blocking mAbs against different types of integrins or synthetic peptides in serum-free medium at a density of 3 ϫ 105 cells/ml at 4 °C for 30 min before plating. Purified recombinant FNs were applied to a HiTrap heparin column (1-ml volume), which had been equilibrated with 10 mM phosphate buffer containing 0.14 M NaCl, and eluted with a gradient of 0.14 –1.0 M NaCl (flow rate of 0.1 ml/min). The amounts of FN bound to the PE-GAGs were quantified by enzymelinked immunosorbent assay using anti-human FN mAb 136H and horseradish peroxidase-conjugated anti-mouse IgG

RESULTS

Functional Characterization of Cartilage Fibronectin

DISCUSSION