HEPARIN-BINDING EGF--STRUCTURE AND FUNCTION

HB-EGF是EGF家族的一个新的肝素结合成员， 最近被纯化至同质，测序并克隆（Higashiyama 等人，Science，1991，251 936-939）。该提案的总体目标是 深入分析HB-EGF的结构和生物学特性。 初步的结构研究表明i）虽然HB-EGF是20- 30， 22 kDa，它只含有85-90个氨基酸，这表明它是 （2）经过广泛修改后，（2）它最初是 表达为208个氨基酸的跨膜前体并被加工， 和iii）其含有调节其肝素结合结构域， 与低亲和力硫酸乙酰肝素相互作用的促有丝分裂活性 蛋白聚糖（HSPG）受体。初步生物 研究表明，HB-EGF i）由巨噬细胞合成，光滑 肌肉细胞（SMC）和一些癌细胞，ii）是有效的SMC 丝裂原（比EGF强40倍，与PDGF同样有效）， 和SMC趋化因子，iii）是上皮细胞的有效有丝分裂原， 细胞（例如角质形成细胞和间皮细胞），和iv）是 伤口液体的成分。HB-EGF的这些生物活性可能 在体内具有生理和病理后果。EGF作为一种 巨噬细胞产物，以及上皮细胞和成纤维细胞的有丝分裂原， 可以参与炎症性伤口愈合过程， 肉芽组织形成和再上皮化。HB-EGF作为SMC 巨噬细胞和SMC自身产生的丝裂原可能部分地 负责与动脉粥样硬化相关的SMC增生。的 本提案的具体目标是：1. HB-EGF结构分析 包括获得成熟HB-EGF的完整一级序列， 多种形式的HB-EGF的分离，翻译后分析， HB-EGF的生物活性分析 前体，和多克隆抗HB-EGF抗体的生产; 2） 鉴定HB-EGF中的肝素结合结构域; 3）为了分析i）HB-EGF中的肝素结合结构域， 通过巨噬细胞和其他细胞调节HB-EGF表达，和ii） HB-EGF促有丝分裂活性分析及其在创伤愈合中的作用 对上皮细胞（例如角质形成细胞）的作用及其在 伤口流体; 4.分析HB-EGF对平滑肌细胞的促有丝分裂作用， 体外和体内SMC对HB-EGF的生物合成 表征低（硫酸乙酰肝素蛋白聚糖）和高亲和力HB-EGF 受体。

HB-EGF is a novel heparin-binding member of the EGF family, that has recently been purified to homogeneity, sequenced and cloned (Higashiyama et al. Science, 1991, 251 936-939). The overall goal of the proposal is to analyze in-depth the structural and biological properties of HB-EGF. Preliminary structural studies indicate i) that while HB-EGF is 20- 22kDa, it contains only 85-90 amino acids suggesting that it is extensively modified post-translationally, ii) that it is originally expressed as a 208 amino acid transmembrane precursor and is processed, and iii) that it contains a heparin-binding domain that modulates its mitogenic activity by interaction with low affinity heparan sulfate proteoglycan (HSPG) receptors on cell surfaces. Preliminary biological studies indicate that HB-EGF i) is synthesized by macrophages, smooth muscle cells (SMC) and by some carcinoma cells, ii) is a potent SMC mitogen (40 times more potent than EGF and equally as potent as PDGF), and a SMC chemotactic factor, iii) is a potent mitogen for epithelial cells (e.g. keratinocytes and mesothelial cells), and iv) is a constituent of wound fluid. These biological activities of HB-EGF might have physiological and pathological consequences in vivo. HB-EGF, as a macrophage product, and mitogen for epithelial cells and fibroblasts, could participate in inflammatory wound healing processes such as granulation tissue formation and re-epithelialization. HB-EGF as a SMC mitogen produced by macrophages and SMC themselves could be in part responsible for the SMC hyperplasia associated with atherosclerosis. The Specific Aims of this proposal are: 1. To analyze HB-EGF structure including obtaining the complete primary sequence of mature HB-EGF, isolation of multiple forms of HB-EGF, analysis of post-translational modifications, analysis of the biological activity of the HB-EGF precursor, and production of polyclonal anti-HB-EGF antibodies; 2) To identify a heparin-binding domain in HB-EGF; 3) To analyze i) the regulation of HB-EGF expression by macrophages and other cells, and ii) the involvement of HB-EGF in wound healing by analysis of its mitogenic effects on epithelial cells (e.g. keratinocytes) and its presence in wound fluid; 4. To analyze the mitogenic effects of HB-EGF on SMC and the biosynthesis of HB-EGF by SMC in vitro and in vivo; 5) To characterize low (heparan sulfate proteoglycan) and high affinity HB-EGF receptors.