Cloning, sequencing, structural and molecular biological characterization of placental protein 20 (PP20)/human thiamin pyrophosphokinase (hTPK).
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.
Bellyei
Sz
S
Department of Biochemistry and Medical Chemistry, University of Pecs, Pecs, Hungary.
Szigeti
A
A
Boronkai
A
A
Szabo
Z
Z
Bene
J
J
Janaky
T
T
Barna
L
L
Sipos
K
K
Minik
O
O
Kravjak
A
A
Ohmacht
R
R
Melegh
B
B
Zavodszky
P
P
Than
G N
GN
Sumegi
B
B
Bohn
H
H
Than
N G
NG
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002277:Carcinoma / Q000097:blood / Q000737:chemistry; D003001:Cloning, Molecular; D005260:Female; D015723:Gene Library; D005865:Gestational Age; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000097:blood / Q000737:chemistry; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry* / Q000235:genetics; D020539:Sequence Analysis, Protein; D013836:Thiamin Pyrophosphokinase / Q000737:chemistry* / Q000235:genetics; D014327:Trophoblasts / Q000737:chemistry
D011257:Pregnancy Proteins; D013836:Thiamin Pyrophosphokinase