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.
Department of Virology, The Haartman Institute, P. O. Box 21, Haartmaninkatu 3, FIN-00014, University of Helsinki, Finland.
M
M
Hyytiäinen
J
J
Taipale
C H
CH
Heldin
J
J
Keski-Oja
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