Differential alternative splicing of human transglutaminase 4 in benign prostate hyperplasia and prostate cancer

Abstract

Transglutaminase 4 is a member of enzyme family that catalyzes calcium-dependent posttranslational modification of proteins. Although transglutaminase 4 has been shown to have prostate-restricted expression pattern, little is known about the biological function of transglutaminase 4 in human. To gain insight into its role in prostate, we analyzed the expression status of human transglutaminase 4 in benign prostate hyperplasia (BPH) and prostate cancer (PCa). Unexpectedly, RT-PCR and nucleotide sequence analysis showed four alternative splicing variants of transglutaminase 4: transglutaminase 4-L, -M (-M1 and -M2) and -S. The difference between transglutaminase 4-M1 and -M2 is attributed to splicing sites, but not nucleotide size. The deduced amino acid sequences showed that transglutaminase 4-L, -M1 and -M2 have correct open reading frames, whereas transglutaminase 4-S has a truncated reading frame. RT-PCR analysis of clinical samples revealed that transglutaminase 4-M and -S were detected in all tested prostate tissue (80 BPH and 48 PCa). Interestingly, transglutaminase 4-L was found in 56% of BPH (45 out of 80) and only in 15% of PCa (7 out of 48). However, transglutaminase 4-L expression did not correlate with serum prostate-specific antigen (PSA) level, prostate volumes or PSA densities. These results will provide a clue to future investigation aiming at delineating physiological and pathological roles of human transglutaminase 4.

Figure 1

Identification of human transglutaminase 4 spliced variants in BPH tissues. RACE was performed on cDNA synthesized from BPH samples and resulted in three different-sized PCR products, 350 bp, 450 bp and 600 bp.

Figure 2

Comparison of nucleotide and amino acid sequences of human transglutaminase (TG) 4 isoforms. (A) Transglutaminase 4-M2 has four more nucleotide (GTGA, underlined) compare to transglutaminase 4-M1, which results in frameshift. Among three consecutive initiation codons (ATGATGGATG), transglutaminase 4-M1 is translated from first and transglutaminase 4-M2 is from third initiation codon. (B) Deduced amino acid sequence of transglutaminase 4-L. The underlined 45 amino acids are found only in transglutaminase 4-L, not in transglutaminase 4-M or -S. (C) Deduced amino acid sequence of transglutaminase 4-S. Transglutaminase 4-S shows 107 nucleotide deletion compared to transglutaminase 4-M1 and encodes truncated protein comprising 91 amino acids.

Figure 3

Structure and alternative splicing of human transglutaminase (TG) 4 gene. (A) Human transglutaminase 4 gene has 15 exons and 14 introns. Exon 2, containing 135 nuclotides, is found only in transglutaminase 4-L and Exon 4, comprising 107 nucleotides, is deleted in transglutaminase 4-S. (B) Nucleotide sequences of the exon-intron junction between exon 1 and intron 1. The splicing site of transglutaminase 4-M2 is four-nucleotide apart from the site of transglutaminase 4-L and -M. The dots above the nucleotide sequences indicate initiation codons.

Figure 4

Schematic representation of alternatively spliced transcripts for human transglutaminase (TG) 4.

Figure 5

Characterization of transglutaminase 4 isoforms expressed in yeast and HeLa cell lysate. (A) Yeast INVSc1 cells were transformed with pYes2.0 vector containing cDNA for hemagglutinin-tagged transglutaminase 4 isoform. Yeast lysates were analyzed by Western blot analysis using anti-hemagglutinin antibody. (B) transglutaminase activities of yeast lysates were estimated by measuring incorporation of [¹⁴C]-putrescine to N,N'-dimethylcasein. Fold increase of transglutaminase activity is expressed as a relative value to that of vector transfected cells, which was normalized by protein level of transglutaminase 4 isoforms. (C) HeLa cells were transfected with cDNA for hemagglutinin-tagged transglutaminase 4 isoforms. Cell lysates and lyophilized culture supernatants were analyzed by Western blot analysis using anti-hemagglutinin antibody. L, transglutaminase 4-L; M1, transglutaminase 4-M1; M2, transglutaminase 4-M2.

Figure 6

Expression of human transglutaminase (TG) 4 isoforms in BPH. (A) Localization of primers used to amplify each spliced variants of human transglutaminase 4. (B) Representative of RT-PCR analysis for transglutaminase 4 isoforms. cDNA synthesized from BPH tissues are amplified with TP-5/TP-4 primers to detect transglutaminase 4-L and -M isoforms.