Recombinant Latent Transforming Growth Factor β-binding Protein 2 Assembles to Fibroblast Extracellular Matrix and Is Susceptible to Proteolytic Processing and Release*
- From the ‡Department of Virology, The Haartman Institute, P. O. Box 21, Haartmaninkatu 3, FIN-00014, University of Helsinki, Finland, and the §Ludwig Institute for Cancer Research, Box 595, S-751 24, Uppsala, Sweden
Abstract
Latent transforming growth factor β-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 ∼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 ∼160-kDa fragment, and with higher concentrations to a protease-resistant ∼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 ∼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.
Footnotes
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↵* This work was supported by the Academy of Finland, the University of Helsinki, Helsinki University Hospital, the Sigrid Juselius Foundation, the Finnish Cancer Organizations, and the Novo Nordisk Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed. Tel.: 358-9-191-26476; Fax: 358-9-191-26475; E-mail:Jorma.Keski-Oja{at}Helsinki.fi.
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↵2 M. Hyytiäinen, unpublished observations.
- Abbreviations:
- ECM
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extracellular matrix
- LAP
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latency-associated protein
- LTBP
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latent TGF-β-binding protein
- TGF
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transforming growth factor
- CHO
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Chinese hamster ovary
- Ab
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antibody
- PAGE
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polyacrylamide gel electrophoresis.
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- Received March 9, 1998.
- Revision received June 5, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
