Bellyei Sz, Szigeti A, Boronkai A, Szabo Z, Bene J, Janaky T, Barna L, Sipos K, Minik O, Kravjak A, Ohmacht R, Melegh B, Zavodszky P, Than G N, Sumegi B, Bohn H, Than N G
Department of Biochemistry and Medical Chemistry, University of Pecs, Pecs, Hungary.
Placenta. 2005 Jan;26(1):34-46. doi: 10.1016/j.placenta.2004.03.008.
Full-length cDNAs of placental protein 20 (PP20) were cloned by screening a human placental cDNA library, which encode a 243 amino acid protein, identical to human thiamin pyrophosphokinase (hTPK) as confirmed by protein sequence analysis. Genomic alignment showed that the PP20/hTPK gene contains 9 exons. It is abundantly expressed in placenta, as numerous EST clones were identified. As thiamine metabolism deficiencies have been seen in placental infarcts previously, these indicate that PP20/hTPK may have a role in placental diseases. Analysis of the 1kb promoter region showed numerous putative transcription factor binding sites, which might be responsible for the ubiquitous PP20/hTPK expression. This may also be in accordance with the presence of the protein in tissues responsible for the regulation of the exquisite balance between cell division, differentiation and survival. TPK activity of the purified and recombinant protein was proved by mass spectrometry with electrospray ionization. By Western blot, PP20/hTPK was found in all human normal and tumorous adult and fetal tissues in nearly equal amounts, but not in sera. By immunohistochemical and immunofluorescent confocal imaging methods, diffuse labelling in the cytoplasm of the syncytiotrophoblasts and weak staining of the trophoblasts were observed, and the amount of PP20/hTPK decreased from the first trimester to the end of gestation. A 3D model of PP20/hTPK was computed (PDB No.: 1OLY) by homology modelling. A high degree of structural homology showed that the thiamin binding site was highly similar to that of the mouse enzyme, but highly different from the bacterial ones. Comparison of the catalytic centre sequences revealed differences, raising the possibility of designing new drugs which specifically inhibit bacterial and fungal enzymes without affecting PP20/hTPK and offering the possibility for safe antimicrobial therapy during pregnancy.
通过筛选人胎盘cDNA文库克隆了胎盘蛋白20（PP20）的全长cDNA，其编码一种243个氨基酸的蛋白质，经蛋白质序列分析证实与人硫胺素焦磷酸激酶（hTPK）相同。基因组比对显示PP20/hTPK基因包含9个外显子。由于在胎盘梗死中先前已观察到硫胺素代谢缺陷，这些表明PP20/hTPK可能在胎盘疾病中起作用。对1kb启动子区域的分析显示有许多假定的转录因子结合位点，这可能是PP20/hTPK普遍表达的原因。这也可能与该蛋白在负责调节细胞分裂、分化和存活之间精确平衡的组织中的存在情况一致。通过电喷雾电离质谱法证明了纯化的重组蛋白的TPK活性。通过蛋白质印迹法，发现PP20/hTPK在所有人类正常和肿瘤性成人及胎儿组织中的含量几乎相等，但在血清中未检测到。通过免疫组织化学和免疫荧光共聚焦成像方法，观察到合体滋养层细胞质中的弥漫性标记和滋养层的弱阳性染色，并且PP20/hTPK的量从妊娠早期到妊娠末期减少。通过同源建模计算出PP20/hTPK的三维模型（PDB编号：1OLY）。高度的结构同源性表明硫胺素结合位点与小鼠酶的高度相似，但与细菌的差异很大。催化中心序列的比较揭示了差异，这增加了设计新药物的可能性，这些药物可以特异性抑制细菌和真菌酶而不影响PP20/hTPK，并为孕期安全的抗菌治疗提供了可能性。
