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 gr owth factor beta(1) (TGF-beta(1)) and its pro region, the beta(1)-late ncy-associated peptide (beta(1)-LAP), is important in regulating the a ctivity of this multipotent growth factor. We have overexpressed simia n beta 1-LAP in Chinese hamster ovary (CHO) cells to produce a cell li ne which secretes beta(1)-LAP into the culture medium at > 1 mg/l, thu s 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 l ung 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 li gand chromatography and anion-exchange chromatography, with a yield of 15%. The purified protein had an apparent molecular mass of 114 kDa a s determined by SDS/PAGE, which is greater than that determined for th e transient expression of simian beta 1-LAP in COS-1 and for the simia n precursor of TGF-beta 1 (pro-TGF-beta 1) in CHO cells, this major di fference being due to more extensive glycosylation of beta 1-LAP expre ssed by this CHO clone. Far-UV CD spectroscopy of simian beta 1-LAP in dicates a mostly beta-sheet structure, with extensive structural rearr angements occurring upon formation of the latent complex between TGF-b eta 1 and beta 1-LAP.