Dunstan S L, Sala-Newby G B, Fajardo A B, Taylor K M, Campbell A K
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom.
J Biol Chem. 2000 Mar 31;275(13):9403-9. doi: 10.1074/jbc.275.13.9403.
Pholasin is the photoprotein responsible for luminescence in the bivalve Pholas dactylus and consists of a luciferin tightly bound to a glycosylated protein. It is a sensitive indicator of reactive oxygen species. A full-length clone encoding apopholasin was isolated from a P. dactylus light organ cDNA library. The unprocessed apoprotein contained 225 amino acids, starting with a signal peptide of 20 amino acids, 3 predicted N-linked glycosylation sites, 1 O-linked site, no histidines, and 7 cysteines. The recombinant apoprotein was expressed in cell extracts and insect cells. The size of the apoprotein expressed in cell extracts and the cytosol of insect cells was 26 kDa but that of the fully processed protein was 34 kDa, as was native pholasin. Both the processed and unprocessed recombinant apoproteins were recognized by a polyclonal antibody raised against native pholasin. Acid methanol extracts from Pholas added to recombinant apoprotein resulted in chemiluminescence triggered by sodium hypochlorite but not photoprotein formation. These results have important implications in understanding the molecular evolution of bioluminescence and will allow the development of recombinant pholasin as an intracellular indicator of reactive oxygen species.
磷光蛋白是双壳贝类斧蛤发光的光蛋白，由紧密结合在糖基化蛋白上的荧光素组成。它是活性氧的敏感指示剂。从斧蛤发光器官cDNA文库中分离出编码脱辅基磷光蛋白的全长克隆。未加工的脱辅基蛋白含有225个氨基酸，起始为20个氨基酸的信号肽，3个预测的N-糖基化位点，1个O-糖基化位点，无组氨酸，7个半胱氨酸。重组脱辅基蛋白在细胞提取物和昆虫细胞中表达。在细胞提取物和昆虫细胞胞质溶胶中表达的脱辅基蛋白大小为26 kDa，但完全加工后的蛋白大小为34 kDa，与天然磷光蛋白相同。加工和未加工的重组脱辅基蛋白都能被针对天然磷光蛋白产生的多克隆抗体识别。添加到重组脱辅基蛋白中的斧蛤酸甲醇提取物会导致由次氯酸钠引发的化学发光，但不会形成光蛋白。这些结果对理解生物发光的分子进化具有重要意义，并将有助于开发重组磷光蛋白作为活性氧的细胞内指示剂。
