We developed a method using a single set of degenerate oligonucleotide primers for amplification of the conserved active site of transglutaminases by reverse transcription-polymerase chain reaction (RT-PCR) and identification of the PCR products by cleavage with diagnostic restriction enzymes. We demonstrate amplification of tissue transglutaminase (TG C ), keratinocyte transglutaminase (TG K ), prostate transglutaminase (TG P ), the a-subunit of factor XIII, and band 4.2 protein from different human cells or tissues. Analysis of normal human keratinocytes revealed expression of a transglutaminase different from the expected and characterized transglutaminase gene products. A full-length cDNA for the novel transglutaminase (TG X ) was obtained by anchored PCR. The deduced amino acid sequence encoded a protein with 720 amino acids and a molecular mass of ϳ81 kDa. A comparison of TG X to the other members of the gene family revealed that the domain structure and the residues required for enzymatic activity and Ca 2؉ binding are conserved and showed an overall sequence identity of about 35%. Two transcripts with an apparent size of 2.2 and 2.8 kilobases were detected with a specific probe for TG X on Northern blots of human foreskin keratinocyte mRNA, indicating the presence of alternatively spliced mRNAs. cDNA sequencing revealed a shorter TG X transcript lacking the sequence homologous to that encoded by exon III of other transglutaminase genes. TG X expression increased severalfold when keratinocyte cultures were induced to differentiate by suspension or growth to postconfluency, suggesting that TG X contributes to the formation of the cornified envelope.
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