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 125I-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.
McMahon
G A
GA
Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008, USA.
Dignam
J D
JD
Gentry
L E
LE
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002241:Carbohydrates / Q000032:analysis; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification; D008168:Lung / Q000166:cytology / Q000187:drug effects; D008360:Mannosephosphates / Q000032:analysis; D008907:Mink; D010446:Peptide Fragments; D011498:Protein Precursors; D017433:Protein Structure, Secondary; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism* / Q000502:physiology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D002241:Carbohydrates; D018076:DNA, Complementary; D008360:Mannosephosphates; D010446:Peptide Fragments; D011498:Protein Precursors; D011506:Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C027693:mannose-6-phosphate; D013762:Tetrahydrofolate Dehydrogenase