Recombinant latent transforming growth factor β-binding protein 2 assembles to fibroblast extracellular matrix and is susceptible to proteolytic processing and release

Latent transforming growth factor P-binding protein 2 (LTBP-2) belongs to the fibrillin-LTBP gene family and is a component of 10-nm microfibrils. LTBP-2 consists mainly of domains of 8-cysteine and EGF-Like repeats linked by proline-rich regions. To characterize the biochemical properties of LTBP-2, its assembly to the extracellular matrix, and its proteolytic release from the matrix, LTBP-2 was expressed recombinantly in Chinese hamster ovary cells and purified to homogeneity under nondenaturing conditions. Purified LTBP-2 bound calcium and was glycosylated at the central domain of EGF-like repeats. Antibodies made against the recombinant LTBP-2 decorated fibrillar structures in fibroblast extracellular matrix. Treatment of matrices with plasmin or elastase released a soluble similar to 160-kDa LTBP-2 fragment. Processing of LTBP-2 was studied by treating purified LTBP-2 with plasmin or porcine pancreatic elastase. LTBP-2 was processed with these proteases initially to a similar to 160-kDa fragment, and with higher concentrations to a protease-resistant similar to 120-kDa fragment. Processing sites were localized by amino acid sequencing to proline-rich regions at the N-terminal part of LTBP-2, suggesting that the matrix binding sites locate to the N-terminal similar to 500 amino acids of LTBP-2. Purified and biotinylated LTBP-2 could be assembled to fibrillar structures in fibroblast extracellular matrix during cell cultivation, indicating that LTBP-2 assembly to the matrix is not strictly linked to cells that make it and suggesting that microfibril assembly may involve soluble intermediates.