Molecular cloning and characterization of placental tissue protein 18 (PP18a)/human mitochondrial branched-chain aminotransferase (BCATm) and its novel alternatively spliced PP18b variant.
Five different insert-length cDNAs encoding for soluble placental tissue protein 18 (PP18) variants were isolated by screening a human placental cDNA library using monospecific anti-PP18 serum. Sequence analysis of the longest clone showed that the insert contains an open reading frame encoding for a 392 residue-long protein with a 27 amino acid mitochondrial targeting sequence. The mature protein-designated PP18a-is 41.264 kDa consisting of 365 residues and is identical to the previously isolated and characterized PP18 antigen described in 1985. We also found a new, alternatively spliced cDNA encoding for a 300 residue-long, 33.776 kDa protein, which was designated PP18b. Alignment search of the protein databank showed that PP18a is almost entirely identical to the human mitochondrial branched-chain aminotransferase, while PP18b is its newly discovered splicing variant. We detected the two PP18 variants in normal adult and fetal human tissues besides the mitochondrial (only PP18a) and cytosolic (only PP18b) fractions of term placenta with chemiluminescence Western blot analysis. The 41 kDa PP18a variant was expressed ubiquitously, while the 33 kDa PP18b variant was found in smaller amounts in nearly all tissues. Trace amounts of the variants were present in the sera of non-pregnant healthy controls, as well as in pregnant women, but there was no real change in serum levels during pregnancy. In conclusion, PP18 variants are not specific for the placenta. Aminotransferase activity of placental origin PP18 antigens was verified by structural analysis and by a coupled branched-chain aminotransferase/glutamate dehydrogenase assay.
Than
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Institute of Biochemistry, Medical School, University of Pécs, Edesanyák 17, H-7624 Pécs, Hungary. gthan@apacs.pote.hu
Sümegi
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B
Than
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GN
Bellyei
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Bohn
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H
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D014644:Genetic Variation; D006801:Humans; D008163:Luminescent Measurements; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry*; D011247:Pregnancy; D011257:Pregnancy Proteins; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D000637:Transaminases
D018076:DNA, Complementary; D015778:Minor Histocompatibility Antigens; D011257:Pregnancy Proteins; D011506:Proteins; D000637:Transaminases; C424463:BCAT2 protein, human