Role of Carbohydrate Structures in the Binding of β1-Latency-Associated Peptide to Ligands

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Abstract
Transforming growth factor β1 (TGF-β1) is a potent growth differentiation and morphogenesis factor. The amino-terminal 248 amino acid pro region of TGF-β1, the β1-latency-associated peptide (β1-LAP), is noncovalently associated with TGF-β1 in an inactive complex. Previous studies suggested that deglycosylated β1-LAP can not form this latent complex with TGF-β1. To study the role of the carbohydrate structures of β1-LAP in its biological functions, we expressed simian β1-LAP in Escherichia coli with a 10 histidine residue tag on the N-terminus. This polypeptide was solubilized from inclusion bodies with 6 M guanidine hydrochloride and purified by metal chelate affinity chromatography. Purified β1-LAP was refolded to its dimeric form using a chaotrope-mediated folding procedure. The dimeric β1-LAP forms 90 kDa complexes with TGF-β1, TGF-β2, and TGF-β3, and reverses the inhibitory activity of TGF-β1 on Mv1Lu cells. Solid phase binding assays demonstrate that refolded β1-LAP binds to heparin and thrombospondin 1. FET cell adhesion promoted by refolded β1-LAP was blocked by an RGD peptide. Purified β1-LAP produced in Chinese hamster ovary cells, deglycosylated with N-glycosidase F, forms a 80−90 kDa complex with mature TGF-β1. The carbohydrate structures of β1-LAP are not required for binding to ligands or for its biological activity.