Role of carbohydrate structures in the binding of beta1-latency-associated peptide to ligands.
Transforming growth factor beta1 (TGF-beta1) is a potent growth differentiation and morphogenesis factor. The amino-terminal 248 amino acid pro region of TGF-beta1, the beta1-latency-associated peptide (beta1-LAP), is noncovalently associated with TGF-beta1 in an inactive complex. Previous studies suggested that deglycosylated beta1-LAP can not form this latent complex with TGF-beta1. To study the role of the carbohydrate structures of beta1-LAP in its biological functions, we expressed simian beta1-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 beta1-LAP was refolded to its dimeric form using a chaotrope-mediated folding procedure. The dimeric beta1-LAP forms 90 kDa complexes with TGF-beta1, TGF-beta2, and TGF-beta3, and reverses the inhibitory activity of TGF-beta1 on Mv1Lu cells. Solid phase binding assays demonstrate that refolded beta1-LAP binds to heparin and thrombospondin 1. FET cell adhesion promoted by refolded beta1-LAP was blocked by an RGD peptide. Purified beta1-LAP produced in Chinese hamster ovary cells, deglycosylated with N-glycosidase F, forms a 80-90 kDa complex with mature TGF-beta1. The carbohydrate structures of beta1-LAP are not required for binding to ligands or for its biological activity.
Yang
Y
Y
Department of Biochemistry and Molecular Biology, Paul Block, Jr., Health Science Building, 3035 Arlington Avenue, Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
Dignam
J D
JD
Gentry
L E
LE
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000737:chemistry*; D002448:Cell Adhesion; D006224:Cricetinae; D005109:Extracellular Matrix / Q000378:metabolism; D006031:Glycosylation; D008024:Ligands; D010446:Peptide Fragments; D017510:Protein Folding; D011498:Protein Precursors; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
D002241:Carbohydrates; D008024:Ligands; D010446:Peptide Fragments; D011498:Protein Precursors; D011506:Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1