A quantitative study on splice variants of N-acylethanolamine acid amidase in human prostate cancer cells and other cells

Highlights

• mRNA of human N-acylethanolamine-hydrolyzing acid amidase (NAAA) has splice variants.

• We determined sequences of four major NAAA variants in LNCaP prostate cancer cells.

• We developed quantitative PCR for each variant in prostate cancer cells and others.

• Ratios of the four variants were different by cell type.

• Two variants encoded catalytically inactive truncated proteins.

Abstract

N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme, hydrolyzing various bioactive N-acylethanolamines with a preference for palmitoylethanolamide. Human NAAA mRNA was previously reported to consist of multiple 3′-end splice variants. However, their tissue distributions and roles have not been examined yet. In the present study, we first identified four major splice variants (tentatively referred to as a1, a2, b2, and c2) in a human prostate cancer cell line LNCaP, which were composed of exons 1–11, exons 1–10 and 12, exons 1–9 and 12, and exons 1–8 and 12, respectively. We next developed quantitative polymerase chain reaction methods to individually quantify these NAAA variants as well as collectively measure all the variants. Among various human prostate cancer cells, the total levels of NAAA mRNAs in androgen-sensitive cells like LNCaP were higher than those in androgen-insensitive cells. In all of these prostate cells and other human cells, variants a1 and b2 showed the highest and lowest expression levels, respectively, among the four variants. Interestingly, ratios of the four variants were different by cell type. Variants a1 and a2 encoded the same full-length NAAA protein, which was catalytically active, while b2 and c2 were translated to C-terminally truncated proteins. As expressed in HEK293 cells these truncated forms were detected as catalytically inactive precursor proteins, but not as mature forms. These results revealed wide distribution of multiple variants of NAAA mRNA in various human cells and suggested that the proteins from some variants are catalytically inactive.

Introduction

N-Acylethanolamines are ethanolamides of long-chain fatty acids and ubiquitously exist in animal tissues [1], [2]. N-Acylethanolamines are well known as a class of endogenous lipid mediators, showing different biological activities depending on acyl species. Arachidonoylethanolamide (anandamide) has been most extensively studied as an endogenous ligand of cannabinoid receptor CB1, which is thus defined as an endocannabinoid [3], as well as an endogenous ligand of transient receptor potential vanilloid 1 (an endovanilloid) [4]. In addition to marijuana-like pharmacological activities, endocannabinoids, including anandamide, may be involved in the growth and transformation of tumor cells [5], [6], [7]. On the other hand, cannabinoid receptor-insensitive palmitoylethanolamide and oleoylethanolamide bind to other receptors such as peroxisome proliferator-activated receptor-α (PPAR-α) [8], [9] and show anti-inflammatory, analgesic, appetite-suppressing, and anti-obesity effects [10], [11].

Fatty acid amide hydrolase (FAAH) [12] and N-acylethanolamine acid amidase (N-acylethanolamine-hydrolyzing acid amidase, NAAA) [13], [14], [15] are two major enzymes responsible for the degradation of N-acylethanolamines in animal tissues. FAAH is a membrane-associated enzyme with an optimal pH value of 8.5–10, while NAAA is a lysosomal enzyme acting at acidic pH [16], [17]. NAAA hydrolyzes various N-acylethanolamines with a preference for palmitoylethanolamide [14], and its specific inhibitors increase endogenous levels of palmitoylethanolamide and other N-acylethanolamines [18], [19], [20]. Specific NAAA inhibitors are thus expected as anti-inflammatory and analgesic drugs. NAAA is distributed in various tissues with predominant expression in macrophages [21], [22] and prostate [23]. Recently, Liu et al. reported that NAAA is abundantly expressed in non-aggressive prostate cancer and is potentially useful as a tissue biomarker related to cancer aggressiveness [24].

Hong et al. showed that human NAAA gene is composed of 11 exons and that the full length mRNA can be translated to a protein composed of 359 amino acid residues (GenBank™ accession number NM_014435.3) [25]. However, Goodchild et al. had earlier reported that human placenta expresses multiple splice variants of NAAA and that one of them contains exon 12 instead of exon 11 [26]. In agreement with this report, many splice variants are found for human NAAA on NCBI database. All the splice variants contain exons 1–8 in common, and alternative splicing occurs in exons 9–12. However, the tissue distribution of each variant and the catalytic activity of the proteins translated have not been investigated. Considering a potential usefulness of NAAA variants as tissue biomarkers, it was also interesting to elucidate how the percentage composition of NAAA variants is different among various cells, which differ in characteristics and origins.

In the present study, we first focused on six splice variants (NM_014435.3, XM_005262923.2, XM_005262924.2, XM_005262920.2, XM_006714180.2, and NM_001042402.1) to which we tentatively refer as a1, b1, c1, a2, b2, and c2, respectively (Fig. 1). a1, b1, and c1 form a group of variants containing exon 11, while a2, b2, and c2 contain exon 12. Since a1, a2, b2, and c2 were found to be major NAAA variants in the prostate cancer cell line LNCaP, we investigated how these four splice variants of NAAA are differently expressed in various cells originated from human prostate and other tissues. We also expressed recombinant NAAA protein isoforms corresponding to these variants as well as several related mutant proteins in mammalian cells and examined their enzyme activities.