ROLE OF CARBOHYDRATE STRUCTURES IN THE BINDING OF BETA-1-LATENCY-ASSOCIATED PEPTIDE TO LIGANDS

Transforming growth factor beta 1 (TGF-beta 1) is a potent growth diff erentiation and morphogenesis factor. The amino-terminal 248 amino aci d pro region of TGF-beta 1, the beta 1-latency-associated peptide (bet a 1-LAP), is noncovalently associated with TGF-beta 1 in an inactive c omplex, Previous studies suggested that deglycosylated beta 1-LAP can not form this latent complex with TGF-beta 1. To study the role of the carbohydrate structures of beta 1-LAP in its biological functions, we expressed simian beta 1-LAP in Escherichia coli with a 10 histidine r esidue tag on the N-terminus. This polypeptide was solubilized from in clusion bodies with 6 M guanidine hydrochloride and purified by metal chelate affinity chromatography, Purified beta 1-LAP was refolded to i ts dimeric form using a chaotrope-mediated folding procedure, The dime ric beta 1-LAP forms 90 kDa complexes with TGF-beta 1, TGF-beta 2, and TGF-beta 3, and reverses the inhibitory activity of TGF-beta 1 on Mvl Lu cells. Solid phase binding assays demonstrate that refolded beta 1- LAP binds to heparin and thrombospondin 1. FET cell adhesion promoted by refolded beta 1-LAP was blocked by an RGD peptide. Purified beta 1- LAP produced in Chinese hamster ovary cells, deglycosylated with N-gly cosidase F, forms a 80-90 kDa complex with mature TGF-beta 1. The carb ohydrate structures of beta 1-LAP are not required for binding to liga nds or for its biological activity.