CHARACTERIZATION OF LATENT FORMS OF TRANSFORMING GROWTH FACTOR-BETA

转化生长因子-β 的潜在形式的表征

• 批准号: 3874661
• 负责人: L M WAKEFIELD
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3874661
• 关键词: biotransformation; carcinogenesis inhibitor; cell differentiation; cell growth regulation; cell type; genetic manipulation; laboratory rat; neoplastic transformation; peptides; platelets; posttranslational modifications; protein biosynthesis; protein sequence; protein structure function; secretion; steroid hormone; tissue /cell culture; transforming growth factors

Under normal physiological conditions, TGF-Beta1 is found predominantly in latent forms, which show no biological activity unless they are first activated in vitro by denaturants or extremes of pH. We have identified and characterized a naturally occurring latent TGF-Beta1 complex. Cells in culture and degranulating human platelets secrete TGF-Beta1 in a latent complex that consists of mature TGF-Beta1, non-covalently associated with the remainder of the TGF-Beta1 precursor, "pro" sequence, and a third unidentified protein of 135 KDa. We have further shown that TGF-Beta1 made by recombinant expression systems using the entire coding sequence of the human TGF-Beta1 gene is also biologically latent, and that this complex is identical to the natural cellular complex, except that it lacks the 135 KDa component. This demonstrates that the TGF-Beta1 precursor "pro" region alone is sufficient to confer latency on TGF-Beta1. This region is now referred to as the TGF-beta1 latency-associated peptide (LAP). We have purified the TGF-Beta1 LAP from recombinant sources, complexed it with (125)I-TGF-Beta1 and analyzed the pharmacokinetics of the resultant latent complex in rats. We have shown that, providing the LAP is correctly sialylated by the expression system, the plasma half-life of TGF-Beta1 in the latent complex is 109 min., compared with just 2.7 min. for active, uncomplexed TGF-Beta. This suggests that whereas endogenous active TGF-Beta probably has very local autocrine or paracrine action, endogenous latent TGF-Beta may have a more endocrine mode of action. The more favorable pharmacokinetcs of the latent TGF-Beta1 also suggest that this may be the form of choice for human clinical use.

在正常生理条件下，TGF-β 1主要存在于 潜在形式，除非它们是第一个，否则不显示生物活性 在体外被变性剂或极端pH激活。我们已经鉴定并 其特征在于天然存在的潜在TGF-β 1复合物。细胞 培养和脱粒人血小板分泌TGF-β 1， 由成熟的TGF-β 1组成的复合物，与 TGF-β 1前体的剩余部分，即“原”序列，和第三个 未鉴定的蛋白质135 KDa。我们进一步证明，TGF-β 1使 通过重组表达系统，使用 人TGF-β 1基因也是生物学上潜伏的， 与天然细胞复合物相同，除了它缺乏135 KDa的 成分这表明TGF-β 1前体“亲”区域 单独足以赋予TGF-β 1潜伏期。该地区目前 被称为TGF-β 1潜伏相关肽（TGF-β 1 latency-associated peptide，简称TGF-LTP）。我们有 从重组来源中纯化TGF-β 1 β，将其与 （125）I-TGF-β 1，并分析了所得潜伏期的药代动力学。 复杂的老鼠我们已经证明，只要LAP正确， 通过表达系统进行唾液酸化， 潜伏络合物为109分钟，而活性组只有2.7分钟， 未复合的TGF-β。这表明，虽然内源性活性TGF-β 可能具有非常局部的自分泌或旁分泌作用，内源性潜伏 TGF-β可能具有更多的内分泌作用模式。越有利 潜在的TGF-β 1的药物动力学也表明，这可能是 人类临床使用的选择形式。