Regulation of HAM function by proteolytic processing

肝细胞生长因子激活因子抑制剂（HAI-1）是肝细胞生长因子激活因子（HGFA）的蛋白质类抑制剂，其通过蛋白水解激活肝细胞生长因子（HGF）。HGF是一种多功能细胞因子，参与损伤后的再生和组织修复。HAI-1作为I型膜蛋白合成，并通过蛋白水解加工作为40 kD HAI-1或58 kD HAI-1分泌。40 kD HAI-1含有一个Kunitz结构域（Kunitz 1），而58 kD HAI-1含有两个Kunitz结构域（Kunitz 1和2）。我们构建了Kunitz结构域的缺失突变体，发现Kunitz 1主要参与HGFA的抑制。虽然58 kD HAI-1含有两个Kunitz结构域，但其对HGFA的抑制活性很弱。我们发现两个Kunitz结构域相互掩盖，从而降低了整个蛋白的抑制活性。当切除三分之二的大鼠肝脏时，残留肝脏中的pro-HGF急剧增加，但活性HGF仅少量存在。然后，我们通过门静脉给予活性HGFA部分肝切除术后24小时，并检查对肝再生的影响。HGFA的施用引起残余肝脏中的pro-HGF活化并诱导c-Met（HGF受体）的酪氨酸磷酸化。此外，肝细胞标记指数增加，促进肝质量的恢复。然而，我们需要大量的HGFA来获得肝再生的显著增强，这表明组织来源的抑制剂如HAI-1在体内调节HGFA的活性中起重要作用。

HGF activator inhibitor (HAI-1) is a proteinaceous inhibitor of HGF activator (HGFA), which proteolytically activates hepatocyte growth factor (HGF). HGF is a multifunctional cytokine that is involved in regeneration and tissue repair after injury. HAI-1 is synthesized as a type I membrane protein, and secreted as a 40 kD HAI-1 or 58 kD HAI-1 by proteolytic processing. 40kD HAI-1 contains one Kunitz domain (Kunitz 1), whereas 58 kD HAI-1 contains two Kunitz domains (Kunitz 1 and 2). We generated deletion mutants of each Kunitz domains, and found that Kunitz 1is mainly involved in inhibition of HGFA. Although 58 kD HAI-1contains two Kunitz domains, its inhibitory activity for HGFA is very weak. We found that two Kunitz domains masked each other, thus lowering the inhibitory activity of the whole protein.When two-thirds of rat liver is resected, pro-HGF in the residual liver increases dramatically, but the active HGF is present in only a small amount. We then administered active HGFA via portal vein 24 h after partial hepatectomy, and examined the effect on liver regeneration. Administration of HGFA caused activation of pro-HGF in the residual liver and induced tyrosine phosphorylation of c-Met (HGF receptor). In addition, labeling index of hepatocytes was increased, and recovery of liver mass was promoted. We needed, however, a large amount of HGFA to obtain significant enhancement of liver regeneration, indicating that tissue-derived inhibitors such as HAI-1 play important roles in regulating activity of HGFA in vivo.