THE IDENTIFICATION AND QUANTITATIVE ANALYSIS OF mRNA SPLICE VARIANTS OF PEPTIDYLGLYCINE α-AMIDATING MONOOXYGENASE (PAM)

Abstract

Many biologically active peptides and peptide hormones are produced by specific endoproteolytic cleavage of higher molecular weight, inactive precursor proteins. Post-translational processing of these precursors is required to generate the mature and biologically active form of the peptide. This processing involves enzymes that perform such modifications as: N-terminal acetylation, sulfation of tyrosine residues, glycosylation and C-terminal amidation. One of these processing enzymes, PAM (peptidylglycine α-amidating monooxygenase), catalyzes the post-translational carboxy-terminal amidation of many bioactive peptides in a sequential two-step process carried out by two separate catalytic domains present in the PAM precursor protein, designated PHM (peptidylglycine a - hydroxylating monooxygenase) and PAL (peptidyl α- hydroxyglyc me a -amidating lyase). The PAM proenzyme is itself subject to tissue specific post-translational processing and several alternatively spliced forms have been identified. It has been demonstrated that peptide hormones can function as autocrine growth factors in lung cancer cells and their role in the promotional stages of lung cancer is being extensively studied. Since amidation is a necessary step in the synthesis of approximately 50% of all known biologically active peptides, the enzymes involved in this process may become potential targets for early detection and targets for new intervention strategies for certain types of cancers. Moreover, understanding the biochemistry of amidating enzymes is essential for the development of chemopreventive agents that can allay the effects of peptides and peptide growth factors that play a role in the early stages of carcinogenesis. In the rat, it has recently been demonstrated that alternative mRNA splicing of exons in the 3' region of the PAM precursor and in the linker region joining the PHM and PAL domains generates tissue specific forms of the enzyme with marked biochemical differences. In contrast to rat PAM enzymes, the biochemical nature of human PAM enzymes that can arise via alternative mRNA splicing at these same sites and the identification of their respective mRNA splice forms has not been well characterized. The study presented here focuses on the identification of alternatively spliced forms of human PAM mRNA in a number of human tumor cell lines and normal tissue. Using reverse transcription coupled to the polymerase chain reaction (RT-PCR) and DNA sequencing four alternatively spliced human PAM mRNA transcripts that differ in the region joining the PHM and PAL domains have been identified. Two of the mRNAs represent previously reported splice variants that are differentiated by the presence (hPA M-A) or absence (hPA M-B) of a 321- nucleotide segment (optional exon A) within the coding region of the PAM gene. The remaining mRNAs represent two new splice variants, hPA M- C and hPA M-D, that show the presence of an exon not previously reported for PAM in any other species. This new exon, designated exon C, is 54 nucleotides in length, encodes an 18 amino acid peptide conserving a dibasic amino acid endoproteolytic motif and is located 3' of optional exon A in human genomic DNA. The structural features of the exon C linker suggest it will result in PAM proteins with significant biochemical and biological differences to either hPAM- A or hPA M-B. Semi-quantitative RT-PCR was used to determine the range of expression of the different hPA M linker mRNA forms in human tissues and human lung tumor cell lines. Both semiquantitative as well as quantitative PCR analysis revealed that hPAM-A is expressed at a higher level in neuroendocrine lung cancer cell types (carcinoid, small cell) than either hPA M-B or the newly identified hPAM-C or hPAM-D.