Bovine latent transforming growth factor β1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils

Abstract

Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of the guanidine-solubilized LTBP-2 appeared to be monomeric, indicating that it was not involved in disulfide-bonded aggregation either with itself or with latent TGF-beta. Additional LTBP-2 was resistant to solubilization with 6 M guanidine but was readily extracted with a reductive saline solution. This treatment is relatively specific for solubilization of microfibrillar constituents including fibrillin 1 and microfibril-associated glycoprotein. Therefore, it can be inferred that some LTBP-2 is bound covalently to the microfibrils by reducible disulfide linkages. The evidence suggests that LTBP-2 has a direct role in elastic fiber structure and assembly which may be independent of its growth factor-binding properties. Thus, LTBP-2 appears to share functional characteristics with both LTBP-1 and fibrillins.