Recombinant latent transforming growth factor beta-binding protein 2 assembles to fibroblast extracellular matrix and is susceptible to proteolytic processing and release.
Latent transforming growth factor beta-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 approximately 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 approximately 160-kDa fragment, and with higher concentrations to a protease-resistant approximately 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 approximately 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.
Hyytiäinen
M
M
Department of Virology, The Haartman Institute, P. O. Box 21, Haartmaninkatu 3, FIN-00014, University of Helsinki, Finland.
Taipale
J
J
Heldin
C H
CH
Keski-Oja
J
J
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry*; D006224:Cricetinae; D005109:Extracellular Matrix / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D053779:Latent TGF-beta Binding Proteins; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D006023:Glycoproteins; C509035:LTBP2 protein, human; D053779:Latent TGF-beta Binding Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D010196:Pancreatic Elastase; D005341:Fibrinolysin; D002118:Calcium