Structural characterization of the latent complex between transforming growth factor β1 and β1-latency-associated peptide

The formation of a non-covalent complex between mature transforming growth factor β1 (TGF-β1) and its pro region, the β1-latency-associated peptide (β1-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian β1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line which secretes β1-LAP into the culture medium at > 1 mg/l, thus enabling structural studies of complex formation between β1-LAP and TGF-β1. The simian β1-LAP expressed in CHO cells reversed the growth inhibitory effect of exogenous TGF-β1 on Mv1Lu (mink lung epithelial) cells and was able to form a cross-linked complex with ¹²⁵I-TGF-β1. Simian β1-LAP was purified to homogeneity by a combination of ammonium sulphate precipitation, gel filtration, dye ligand chromatography and anion-exchange chromatography, with a yield of 15%. The purified protein had an apparent molecular mass of 114 kDa as determined by SDS/PAGE, which is greater than that determined for the transient expression of simian β1-LAP in COS-1 and for the simian precursor of TGF-β1 (pro-TGF-β1) in CHO cells, this major difference being due to more extensive glycosylation of β1-LAP expressed by this CHO clone. Far-UV CD spectroscopy of simian β1-LAP indicates a mostly β-sheet structure, with extensive structural rearrangements occurring upon formation of the latent complex between TGF-β1 and β1-LAP.