Structural characterization of the latent complex between transforming growth factor beta 1 and beta 1-latency-associated peptide

The formation of a non-covalent complex between mature transforming growth factor beta(1) (TGF-beta(1)) and its pro region, the beta(1)-latency-associated peptide (beta(1)-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian beta 1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line which secretes beta(1)-LAP into the culture medium at > 1 mg/l, thus enabling structural studies of complex formation between beta 1-LAP and TGF-beta 1. The simian beta 1-LAP expressed in CHO cells reversed the growth inhibitory effect of exogenous TGF-beta(1) on Mv1Lu (mink lung epithelial) cells and was able to form a cross-linked complex with I-125-TGF-beta 1. Simian beta 1-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 beta 1-LAP in COS-1 and for the simian precursor of TGF-beta 1 (pro-TGF-beta 1) in CHO cells, this major difference being due to more extensive glycosylation of beta 1-LAP expressed by this CHO clone. Far-UV CD spectroscopy of simian beta 1-LAP indicates a mostly beta-sheet structure, with extensive structural rearrangements occurring upon formation of the latent complex between TGF-beta 1 and beta 1-LAP.