Wang Jianghua, Cawley Niamh X, Voutetakis Antonis, Rodriguez Yazmin M, Goldsmith Corinne M, Nieman Lynnette K, Hoque A T M Shamsul, Frank Stuart J, Snell Chris R, Loh Y Peng, Baum Bruce J
Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
Hum Gene Ther. 2005 May;16(5):571-83. doi: 10.1089/hum.2005.16.571.
Regulated secretory pathway proteins, when delivered as transgenes to salivary glands, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, for example, human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. The C terminus of hGH is highly conserved and contains a domain similar to the regulated pathway sorting domain of pro-opiomelanocortin (POMC). We hypothesized that, similar to POMC, mutation of this domain would divert hGH secretion from the regulated to the constitutive pathway, which in salivary glands leads to the bloodstream. Several mutations were made in the C terminus of the hGH cDNA and tested in vitro. One biologically active mutant containing E174A and E186A substitutions, and with an included C-terminal extension, was studied in greater detail. Compared with wild-type hGH, we found that this mutant hGH accumulated in the Golgi/trans-Golgi network and showed increased basal secretion in AtT20 cells, a model endocrine cell line. Importantly, in vivo, the mutant hGH displayed a relative increase in the proportion of constitutive pathway secretion seen from rat salivary glands, with a significantly lower saliva-versus-serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by reengineering its sorting determinants.
受调控的分泌途径蛋白作为转基因导入唾液腺后，主要分泌到唾液中。这对于那些需要全身性治疗功能的蛋白质（例如人生长激素（hGH））来说并无用处。提高hGH分泌到血液中的效率的一种策略涉及对现有的分选信号进行操控。hGH的C末端高度保守，并且包含一个与促黑素细胞皮质激素原（POMC）的受调控途径分选结构域相似的结构域。我们推测，与POMC类似，该结构域的突变会使hGH的分泌从受调控途径转向组成型途径，而在唾液腺中，组成型途径会通向血液。在hGH cDNA的C末端进行了多处突变并在体外进行了测试。对一种含有E174A和E186A替换且带有C末端延伸的生物活性突变体进行了更详细的研究。与野生型hGH相比，我们发现这种突变体hGH在高尔基体/反式高尔基体网络中积累，并在模型内分泌细胞系AtT20细胞中显示出基础分泌增加。重要的是，在体内，突变体hGH在大鼠唾液腺中组成型途径分泌的比例相对增加，唾液与血清的分泌比显著降低（p = 0.03）。尽管这种突变体不太可能具有治疗益处，但这些结果表明，转基因分泌蛋白的最终去向可能通过重新设计其分选决定因素来控制。
