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 ␤1 (TGF-␤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-␤. 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.

Molecular Interactions of Biglycan and Decorin with Elastic Fiber Components

“…67-kDa cell surface elastin-binding protein (35); the elastinmicrofibril interface protein emilin (36); the fibrillin-like latent transforming growth factor B-1 binding proteins (37,38); and the small dermatan sulfate proteoglycans decorin and biglycan (13,14). Recently Trask et al (13) demonstrated the importance of sulfation for the assembly of elastin, fibrillin-1, and MAGP-1 into extracellular matrix, and this finding suggested that a proteoglycan(s) is involved in elastic fiber assembly.…”

Analysis of the expression pattern of the latent transforming growth factor β binding protein isoforms in normal and diseased human liver reveals a new splice variant missing the proteinase-sensitive hinge region

“…10 LTBP, initially identified as part of the large latent TGF-␤ complex, 11 is a component of the ECM of various cell types and tissues. [12][13][14] Although LTBP does not directly mediate the latency of TGF-␤, 15 it is known to be important for the assembly and secretion of the complex, 6 and additionally for the storage of TGF-␤ in the ECM. The release of latent TGF-␤ from ECM by proteolytic cleavage of LTBP is discussed to be an initial step in the activation of TGF-␤.…”

Expression and Matrix Deposition of Latent Transforming Growth Factor β Binding Proteins in Normal and Fibrotic Rat Liver and Transdifferentiating Hepatic Stellate Cells in Culture

“…Thus, LTBPs are supposed to be important constituents of the microfibrillar structure of connective tissue. [23][24][25] The presence of LTBPs in parenchymal and nonparenchymal liver cells and its proposed biological functions prompted us to investigate in some detail the expression of LTBP isoforms in experimental fibrosis and transdifferentiating cultured rat HSC. In addition, the time-dependent binding of TGF-␤ components (LTBP, LAP, TGF-␤) to the extracellular matrix and possible biological functions of LTBP in cultured transdifferentiating HSC were studied.…”