STRUCTURAL AND FUNCTIONAL STUDIES OF HEPARIN-BINDING EGF

那个女人这项工作的长期目标是阐明 肝素结合EGF样生长因子（HB-EGF）的生理作用， 一种最近描述的表皮生长因子（BGF）家族成员。 HB-EGF是一种阳离子型肝素结合剂，分子量为20，000 - 22，000-Mr，热-和酸- 成纤维细胞、上皮细胞和平滑肌的稳定促分裂原 细胞，最初被认为是培养的分泌产物， 人类巨噬细胞。尽管HB-EGF以约86%的蛋白质形式分泌 氨基酸，它最初是作为208个氨基酸的前体合成的。 酸被预测为膜锚定。的具体目标 本研究的主要目的是：（1）分析HB-EGF的功能意义 研究HB-EGF的生物学活性 前体;和（3）鉴定HB-IgM的肝素结合结构域， 确定其功能意义。假设（1）： 糖基化影响生物或物理化学性质， （2）HB-EGF前体可以存在于一种新的表皮生长因子中， （3）在细胞分裂之前， HB-cAg分子内的区域解释了其结合 肝素和这种结合改变HB-EGF之间的相互作用 EGF受体（EGF R）本研究的设计和方法是：（1） 比较生物活性、稳定性、半衰期、亲和常数和 非糖基化的降解和活化的受体后途径 HB-EGF在E. coil原核表达系统 在牛痘病毒真核表达中产生的糖基化HB-BGF 系统这些参数也将评估HB-EGF， 进行定点诱变以防止糖基化，以及 对于在糖基化缺陷细胞系中产生的HB-EGF;（2） 为了在大肠杆菌中表达HB-EGF前体，这可能无法表达HB-EGF前体。 将其切割成其成熟形式，并使用定点诱变， 牛痘病毒表达系统，以防止其裂解;和（3）使用 缺失诱变，以去除推定的肝素结合结构域， HBEGF，以测试天然和突变体HB-EGF与野生型和 硫酸肝素缺陷型CHO细胞，其已经用 EGF-R嵌合融合蛋白的特性研究 含有EGF和HB-EGF的肝素结合结构域。健康 这个项目的相关性是，HB-EGF可能发挥作用， 发育和分化，修复过程，如伤口愈合 和子宫重塑，以及诸如动脉粥样硬化和 肿瘤发生

The broad. long-term objectives of this work are to elucidate the physiological roles of heparin-binding EGF-like growth factor (HB-EOF), a recently described member Of the epidermal growth factor (BGF) family. HB-EGF is a cationic, heparin-binding, 20,000-22,000-Mr, heat- and acid- stable mitogen for fibroblasts, epithelial cells, and smooth muscle cells, that was initially recognized as a secreted product of cultured human macrophage. Although HB-EGF is secreted as a protein of about 86 amino acids, it is initially synthesized as a precursor of 208 amino acids that is predicted to be membrane-anchored. The specific aims of this proposal are (l) To analyze the functional significance of HB-EGF glycosylation; (2) To study the biological activity of the HB-EGF precursor; and (3) To identify heparin-binding domains of HB-EOF and to establish their functional significance. The hypotheses are (l) That glycosylation influences biological or physico-chemical properties of HB-EGF; (2) That the HB-EGF precursor can be presented in a juxtacrine fashion to neighboring cells prior to cleavage; and (3) That specific regions within the HB-EOF molecule account for its ability to bind to heparin and that this binding modifies the interaction between HB-EGF and the EGF receptor (EGF-R). The research design and methods are (l) To compare the bioactivity, stability, half-life, affinity constants and post receptor pathways of degradation and activation of non-glycosylated HB-EGF produced in an E. coil procaryotic expression system with that of glycosylated HB-BGF produced in a vaccinia virus eucaryotic expression system. These parameters will also be assessed for HB-EGF that has undergone site-directed mutagenesis to prevent glycosylation, as well as for HB-EGF that is produced in glycosylation-deficient cell lines; (2) To express the HB-EGF precursor in E.coli, which may not be able to cleave it to its mature form, and to use site-directed mutagenesis in a vaccinia virus expression system to prevent its cleavage; and (3) To use deletion mutagenesis to remove putative heparin-binding domain(s) from HBEGF, to test the binding of native and mutant HB-EGF to wild type and heparin sulfate-deficient CHO cells that have been transfected with the EGF-R, and to study the properties of a chimeric fusion protein containing EGF and a heparin-binding domain of HB-EGF. The health relatedness of this project is that HB-EGF may play a role in development and differentiation, repair processes such as wound healing and uterine remodeling, and pathologies such as atherosclerosis and oncogenesis.