Sifers R N, Hardick C P, Woo S L
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1989 Feb 15;264(5):2997-3001.
Crystallographic studies have previously suggested that Lys290 forms a salt bridge with Glu342 in the serine protease inhibitor alpha 1-antitrypsin. Disruption of the formation of this structural feature by a Glu to Lys substitution at residue 342 in the PiZ variant has been implicated in causing the defective secretion of this mutant protein from hepatocytes (10-15% of normal). To test the validity of this hypothesis, mutant human alpha 1-antitrypsin cDNA constructs coding for specific amino acid substitutions at residues 290 and 342 were generated and the corresponding mutant proteins were expressed in mouse hepatoma cells. When the potential to form the salt bridge was reestablished by a Lys290 to Glu290 substitution in the PiZ variant, its secretion was increased to only 38% of normal. Furthermore, disruption of this structural feature by a Lys290 to Glu290 substitution in the normal inhibitor failed to reduce the secretion of alpha 1-antitrypsin to the extent observed for the PiZ variant (73% of normal). Finally, substitution of the neutral amino acid Gln at residue 342 only reduced the secretion of alpha 1-antitrypsin to 55% of normal. Of all mutant proteins tested, those bearing Lys at position 342 were secreted at the lowest levels. These findings demonstrate that although disruption of the 290-342 salt bridge does affect the secretion of alpha 1-antitrypsin, it is the substitution of Lys at residue 342 that causes the dramatic secretory defect of the PiZ variant.
晶体学研究先前表明，在丝氨酸蛋白酶抑制剂α1-抗胰蛋白酶中，赖氨酸290与谷氨酸342形成盐桥。在PiZ变体中，第342位残基处的谷氨酸被赖氨酸取代，破坏了这种结构特征的形成，这被认为是导致该突变蛋白从肝细胞分泌缺陷（为正常水平的10 - 15%）的原因。为了验证这一假设，构建了编码第290位和342位残基处特定氨基酸取代的突变型人α1-抗胰蛋白酶cDNA，并在小鼠肝癌细胞中表达相应的突变蛋白。当通过在PiZ变体中将赖氨酸290替换为谷氨酸290来重建形成盐桥的可能性时，其分泌量仅增加到正常水平的38%。此外，在正常抑制剂中通过将赖氨酸290替换为谷氨酸290来破坏这种结构特征，并没有将α1-抗胰蛋白酶的分泌减少到PiZ变体所观察到的程度（为正常水平的73%）。最后，在第342位残基处替换为中性氨基酸谷氨酰胺，仅将α1-抗胰蛋白酶的分泌减少到正常水平的55%。在所有测试的突变蛋白中，第342位带有赖氨酸的那些蛋白分泌水平最低。这些发现表明，虽然290 - 342盐桥的破坏确实会影响α1-抗胰蛋白酶的分泌，但正是第342位残基处赖氨酸的取代导致了PiZ变体显著的分泌缺陷。
