Marquette K A, Pittman D D, Kaufman R J
Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, USA.
J Biol Chem. 1995 Apr 28;270(17):10297-303. doi: 10.1074/jbc.270.17.10297.
Factor VIII is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Factor VIII is homologous to blood coagulation factor V, both having a domain structure of A1-A2-B-A3-C1-C2. Previous transfection studies demonstrated that factor VIII is 10-fold less efficiently expressed than the homologous coagulation factor, factor V. The inefficient expression correlated with interaction of the factor VIII primary translation product with the protein chaperonin BiP in the lumen of the endoplasmic reticulum. In contrast, factor V was not detected in association with BiP and was secreted efficiently. To determine whether specific amino acid sequences within factor VIII inhibit secretion, we have studied the secretion of factor VIII deletion and factor VIII/factor V chimeric proteins upon transient transfection of COS-1 monkey cells. A chimeric factor VIII protein that contained the A1- and A2-domains of factor V was secreted with a similar efficiency as wild-type factor V, whereas the complementary chimera having the A1- and A2-domains of factor VIII was secreted with low efficiency, similar to wild-type factor VIII. These results suggested that sequences within the A1- and A2-domains were responsible for the low secretion efficiency of factor VIII. Secretion of A1-domain-deleted factor VIII was increased approximately 10-fold compared to wild-type factor VIII or A2-domain-deleted factor VIII. Expression of the factor VIII A1-domain alone did not yield secreted protein, whereas expression of the factor VIII A2-domain alone or the factor V A1-domain or A2-domain alone directed synthesis of secreted protein. Secretion of a hybrid in which the carboxyl-terminal 110 amino acids of the A1-domain were replaced by homologous sequences from the factor V A1-domain was also increased 10-fold compared to wild-type factor VIII, however, the secreted protein was not functional and the heavy and light chains were not associated. These results localize a 110-amino acid region within the A1-domain that inhibits factor VIII secretion. This region is clustered with multiple short peptide sequences that have potential to bind BiP.
凝血因子VIII是一种在X染色体连锁的出血性疾病血友病A中缺乏的凝血因子。凝血因子VIII与凝血因子V同源，二者都具有A1-A2-B-A3-C1-C2的结构域结构。先前的转染研究表明，凝血因子VIII的表达效率比同源凝血因子V低10倍。这种低效表达与凝血因子VIII初级翻译产物在内质网腔中与蛋白伴侣BiP的相互作用有关。相比之下，未检测到凝血因子V与BiP结合，并且其分泌效率很高。为了确定凝血因子VIII内的特定氨基酸序列是否抑制分泌，我们研究了在COS-1猴细胞瞬时转染后凝血因子VIII缺失蛋白和凝血因子VIII/凝血因子V嵌合蛋白的分泌情况。一种包含凝血因子V的A1和A2结构域的嵌合凝血因子VIII蛋白的分泌效率与野生型凝血因子V相似，而具有凝血因子VIII的A1和A2结构域的互补嵌合体的分泌效率很低，与野生型凝血因子VIII相似。这些结果表明，A1和A2结构域内的序列是凝血因子VIII分泌效率低的原因。与野生型凝血因子VIII或缺失A2结构域的凝血因子VIII相比，缺失A1结构域的凝血因子VIII的分泌增加了约10倍。单独表达凝血因子VIII的A1结构域不会产生分泌蛋白，而单独表达凝血因子VIII的A2结构域或凝血因子V的A1结构域或A2结构域则指导分泌蛋白的合成。与野生型凝血因子VIII相比，一种将A1结构域的羧基末端110个氨基酸替换为凝血因子V A1结构域的同源序列的杂合体的分泌也增加了10倍，然而，分泌的蛋白没有功能，重链和轻链也没有关联。这些结果将抑制凝血因子VIII分泌的区域定位在A1结构域内的一个110个氨基酸的区域。该区域聚集了多个有可能与BiP结合的短肽序列。
