Yang Y, Dignam J D, Gentry L E
Department of Biochemistry and Molecular Biology, Paul Block, Jr., Health Science Building, 3035 Arlington Avenue, Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
Biochemistry. 1997 Sep 30;36(39):11923-32. doi: 10.1021/bi9710479.
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.
转化生长因子β1（TGF-β1）是一种强大的生长分化和形态发生因子。TGF-β1的氨基末端248个氨基酸的前体区域，即β1-潜伏相关肽（β1-LAP），在无活性复合物中与TGF-β1非共价结合。先前的研究表明，去糖基化的β1-LAP不能与TGF-β1形成这种潜伏复合物。为了研究β1-LAP的碳水化合物结构在其生物学功能中的作用，我们在大肠杆菌中表达了带有N端10个组氨酸残基标签的猴β1-LAP。该多肽用6 M盐酸胍从包涵体中溶解出来，并通过金属螯合亲和色谱法纯化。使用促溶剂介导的折叠程序将纯化的β1-LAP重折叠成其二聚体形式。二聚体β1-LAP与TGF-β1、TGF-β2和TGF-β3形成90 kDa的复合物，并逆转TGF-β1对Mv1Lu细胞的抑制活性。固相结合试验表明，重折叠的β1-LAP与肝素和血小板反应蛋白1结合。重折叠的β1-LAP促进的FET细胞粘附被RGD肽阻断。在中国仓鼠卵巢细胞中产生的纯化β1-LAP，用N-糖苷酶F去糖基化后，与成熟的TGF-β1形成80-90 kDa的复合物。β1-LAP的碳水化合物结构对于与配体的结合或其生物学活性不是必需的。
