Structure/function analysis of LTBP-1

Introduction

The latent TGF-β binding proteins are multidomain extracellular matrix glycoproteins, comprising repeating units of EGF-like, calcium-binding EGF-like, eight-cysteine, four-cysteine and RGD motifs (Sinha et al. 1998). Four LTBP isoforms have been isolated, sharing sequence identity and domain homology with the fibrillin family of extracellular matrix proteins. LTBP-1 was first characterized as a 125-kD component of the large latent TGF-β complex secreted from platelets (Wakefield et al. 1988; Kanzaki et al. 1990) in which LTBP-1 is linked by a disulphide bond to the TGF-β propeptide, which remains noncovalently associated with TGF-β until activated (Saharinen et al. 1996; Gleizes et al. 1996). Four alternatively spliced variants of LTBP-1 have been identified and have been designated LTBP-1S, LTBP-1L and LTBP-1Δ53. The LTBP-1S cDNA encodes 1394 amino acids (Kanzaki et al. 1990). LTBP-1L encodes 1740 amino acids, arising as a result of a splicing event at codons 145–146 of LTBP-1S and incorporating an amino terminal extension of 346 highly basic amino acids (Olofsson et al. 1995). The LTBP-1Δ53 isoforms lack 53 amino acids from the first eight-cysteine motif (Öklüet al. 1998) and a ‘hinge’ region (Gong et al. 1998). We are especially interested in the differential expression and deposition of the LTBP-1S and 1L isoforms in the extracellular matrix, their molecular composition, and the matrix molecules that they might target and bind to.

Materials and methods

The cDNA encoding full-length LTBP-1S was modified by PCR to incorporate a factor Xa proteolytic cleavage site and a Flag octapeptide antigenic epitope into the 3′ end of the sequence immediately prior to the stop codon. This modified cDNA was subcloned into the MCS of a plasmid vector derived from pSecTag (Invitrogen), downstream from the T7 and CMV promoters. The LTBP-1S cDNA was transcribed and the mRNA used in an in vitro translation system utilizing semipermeabilized HT1080 cells (Wilson et al. 1995), supplemented with [³⁵S]-cysteine (ICN). The recombinant protein was analysed by reductive SDS-PAGE and autoradiography. Additionally, HT1080 cells were transfected with the modified LTBP-1S cDNA using the Superfect reagent (Qiagen) and grown for 48 h. After this time the culture medium was removed, the cells were removed by trypsinisation and lysed with SDS sample buffer. In similar culture dishes the cells were lysed and the extracellular matrix components solubilized with NET buffer (0.4 m NaCl, 5 mm Tris HCl pH 7.5, 1% (v/v) NP-40). The NET-insoluble components were solubilized with SDS sample buffer. The media, cell lysates, and extracellular matrix components were analysed for expression of recombinant LTBP-1S by SDS-PAGE and Western blotting, using a monoclonal M2 anti Flag antibody (Sigma), anti mouse IgG conjugated to alkaline phosphatase and NBT/BCIP (Biorad).

Results

Autoradiography of [³⁵S]-cysteine labelled recombinant protein produced by the in vitro translation system indicated the expression of a 210-kD protein. This protein was observed only in samples to which the modified LTBP-1S mRNA was added, and not in control samples, lacking mRNA. Western blotting indicated the expression of recombinant protein only in cells that had been exposed to the LTBP-1S cDNA. A protein with a molecular weight of 210 kD was observed in the media from these cells. However, the components of the NET soluble fraction yielded a protein with a molecular weight of 205 kD.

Discussion

We have produced a modified recombinant LTBP-1S which is secreted from HT1080 cells into the media and deposited in the extracellular layer. The protein extracted from the extracellular layer is reduced in size, indicating the involvement of a proteolytic cleavage event in the binding to matrix. The effects of coexpression of LTBP-1S with TGF-β are currently under investigation and will be compared to the expression and deposition of LTBP-1L in these systems.