A Major Urinary Protein of the Domestic Cat Regulates the Production of Felinine, a Putative Pheromone Precursor
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CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Chemistry & Biology 13, 1071–1079, October 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.chembiol.2006.08.013 A Major Urinary Protein of the Domestic Cat Regulates the Production of Felinine, a Putative Pheromone Precursor Masao Miyazaki,1 Tetsuro Yamashita,2 Introduction Yusuke Suzuki,1 Yoshihiro Saito,3 Satoshi Soeta,4 Hideharu Taira,2 and Akemi Suzuki1,* Most mammals have a highly developed olfactory 1 Sphingolipid Expression Laboratory sense, and urinary odorants and pheromones are impor- RIKEN Frontier Research System tant molecules for chemical communication used in 2-1 Hirosawa reproduction, territoriality, and conspecific recognition. Wako-shi It is thought that the biosynthetic and excretion mecha- Saitama 351-0198 nisms of the molecules have evolved independently in Japan each animal species, although evidence is limited to 2 Department of Agro-Bioscience a small number of species, which include rodents and Faculty of Agriculture elephants [1]. Iwate University The domestic cat (Felis catus) is a popular pet, and it is 3-18-8 Ueda commonly known that cat urine has a characteristic Morioka-shi ‘‘catty odor.’’ Male cats frequently spray urine with the Iwate 020-8550 odor onto vertical objects, such as walls, in order to Japan mark their territory. In cat urine, a sulfur-containing 3 Analytical Applications Department amino acid, 2-amino-7-hydroxy-5,5-dimethyl-4-thia- Tokyo Customer Support Center heptanoic acid, known as felinine (Figure 1), is excreted Shimadzu Corporation [2, 3]. Felinine excretion is regulated by testosterone [4], 380-1 Horiyamashita and male cats excrete a large amount of felinine (122 Hadano-shi mmol/kg body weight/day) compared to castrated males Kanagawa 259-1304 and mature females (41 and 36 mmol/kg body weight/ Japan day, respectively) [5]. Chemically synthesized felinine 4 Department of Veterinary Anatomy and native felinine purified from urine develop an odor Nippon Veterinary and Animal Science University during storage that is characteristic of cat urine [6]. 1-7-1 Kyonan-cho Based on these findings, it has been postulated that Musashino-shi felinine is a biologically important molecule in the cat, Tokyo 180-0023 and that it is used as a territorial marker for intraspecies Japan communication [7, 8] and is a putative precursor of a pheromone that plays a role in attracting females [4]. A felinine precursor is present in cat blood as 3-meth- Summary ylbutanol-glutathione (3-MBG) and is formed via a gluta- thione S-conjugation reaction between glutathione and Domestic cats spray urine with species-specific odor isopentenyl pyrophosphate as an intermediate of cho- for territorial marking. Felinine (2-amino-7-hydroxy- lesterol biosynthesis [9]. In general, glutathione S-con- 5,5-dimethyl-4-thiaheptanoic acid), a putative phero- jugates such as 3-MBG are converted to cysteinylgly- mone precursor, is excreted in cat urine. Here, we re- cine S-conjugates by g-glutamyl transferase (Enzyme port that cauxin, a carboxylesterase excreted as a Commission [EC] numbers 2.3.2.2) in the kidney, and major urinary component, regulates felinine produc- the cysteinylglycine S-conjugates are hydrolyzed by tion. In vitro enzyme assays indicated that cauxin renal dipeptidase (EC 3.4.13.11). The resulting cysteine hydrolyzed the felinine precursor 3-methylbutanol- S-conjugates, including felinine, are N-acetylated by cysteinylglycine to felinine and glycine. Cauxin and microsomal N-acetyltransferase (EC 2.3.1.5) and are felinine were excreted age dependently after 3 months ultimately excreted in the urine as N-acetyl cysteine of age. The age-dependent increases in cauxin and S-conjugates, known as mercapturic acids [10, 11]. felinine excretion were significantly correlated. In ma- The metabolic pathway by which 3-MBG is hydrolyzed ture cats, cauxin and felinine levels were sex-depen- to felinine, however, remains unknown. dently correlated and were higher in males than in Here, we report that cauxin, a major urinary protein, is females. In headspace gas of cat urine, 3-mercapto- involved in the production of felinine in the domestic cat. 3-methyl-1-butanol, 3-mercapto-3-methylbutyl for- We previously found that urine of normal male cats con- mate, 3-methyl-3-methylthio-1-butanol, and 3-methyl- tains a high concentration of proteins (about 0.5–1.0 mg/ 3-(2-methyldisulfanyl)-1-butanol were identified as ml), and we found that more than 90% of these proteins candidates for felinine derivatives. These findings are a 70 kDa component named cauxin [12]. Cauxin is demonstrate that cauxin-dependent felinine produc- a member of the carboxylesterases (EC 3.1.1.1), which tion is a cat-specific metabolic pathway, and they hydrolyze a wide variety of aromatic and aliphatic sub- provide information for the biosynthetic mechanisms strates containing ester, thioester, and amide bonds of species-specific molecules in mammals. [13, 14]. Cauxin is expressed only in the kidney and is secreted from the kidney-proximal straight tubular cells into the urine. Recently, we found that cauxin is excreted in a species-, sex-, and age-dependent manner [15]. *Correspondence: [email protected] Although the cauxin gene is conserved in several Chemistry & Biology 1072 Figure 1. Chemical Structures of Felinine-Related Molecules Discussed in This Paper (A) 3-Methylbutanol-glutathione (3-MBG). (B) 3-Methylbutanol-cysteinylglycine (3-MBCG). (C) 2-Amino-7-hydroxy-5,5-dimethyl-4-thiahepanoic acid (felinine). (D) 3-Methylbutanol-N-acetyl cysteine (N-acetyl felinine). mammals, including humans, mice, and dogs, a large time-of-flight mass spectrometry (MALDI-TOF MS) indi- amount of cauxin is found only in the domestic cat, bob- cated that product a purified by HPLC with an octade- cat (Lynx rufus), and lynx (Lynx lynx), all of which are rel- cylsilica (ODS) column exhibited ions at m/z 208.1 for atively close members in the Felidae lineage [16]. Urinary [M+H]+, m/z 230.1 for [M+Na]+, and m/z 246.1 for cauxin is detected in cats older than w3 months of age [M+K]+, confirming that product a was felinine (molecu- and is significantly higher in intact males than in cas- lar weight: 207.29). When 3-MBG was used as the sub- trated males or mature females. Based on these find- strate, no product was detected by HPTLC (Figure 2A, ings, we reasoned that products of the cauxin enzyme lane 5). These results indicate that cauxin hydrolyzed reaction should vary with species, sex, and age in the the peptide bond of 3-MBCG and produced felinine same way as cauxin. We therefore focused our attention and glycine, but did not hydrolyze 3-MBG. The Km and on felinine, whose excretion is species, sex, and age de- Vmax values of cauxin for hydrolysis of 3-MBCG, which pendent [8], and we hypothesized that cauxin hydro- were calculated from a Lineweaver-Burk plot, were 1.8 lyzes the felinine precursors, 3-methylbutanol-cysteinyl- mM and 3.3 nmol/mg/min, respectively (Figure 2D). glycine (3-MBCG) and/or 3-MBG. In this study, we found Cauxin had hydrolytic activity toward 3-MBCG in the that cauxin hydrolyzes the peptide bond of 3-MBCG pH range of 5.5–7.5, as shown in Figure 2E, and cat urine and produces felinine. Furthermore, in the analysis of falls within this range (males 6.37 6 0.07; females 5.97 6 headspace gas of cat urine, cat-specific odorants 0.10) [17]. 3-mercapto-3-methyl-1-butanol, 3-mercapto-3-methyl- butyl formate, 3-methyl-3-methylthio-1-butanol, and Temporal Changes in Excretion of Cauxin, Felinine, 3-methyl-3-(2-methyldisulfanyl)-1-butanol were identi- and 3-MBCG in Immature Cats fied as candidates for degradative products of felinine. Figure 3 shows the excretion levels of cauxin, felinine, To our knowledge, these findings provide new infor- and 3-MBCG in male (Figure 3A) and female (Figure 3B) mation for the biosynthetic mechanisms of species- littermate cats from birth to 4.8 months of age. Cauxin specific urinary chemicals in mammals. concentrations (g/l) were normalized to urinary creati- nine levels (g/l), and felinine and 3-MBCG concentra- Results tions (nmol/ml) were normalized to urinary creatinine concentrations (mmol/ml). Cauxin excretion was de- Cauxin Enzyme Activity toward Felinine Precursors tected in males older than 2.8 months of age and in Using in vitro enzyme assays, we examined the pepti- females older than 3.1 months of age, and levels were dase activity of cauxin toward the felinine precursors, found to increase with age. Felinine was detected in 3-MBCG and 3-MBG. The reaction products ana- males older than 2.5 months of age and in females older lyzed by high-performance thin-layer chromatography than 3 months of age, and its excretion level increased (HPTLC) are shown in Figure 2A. When 3-MBCG was with age in a very similar manner to that of cauxin for used as the substrate (lane 3), two products, a and b, cats older than 3 months of age. Notably, the felinine pre- comigrated with the authentic compounds of felinine cursor 3-MBCG was present for cats younger than 3 (lane 1) and glycine (lane 2), respectively. As shown in months of age, and its concentration did not vary with Figure 2B, the reaction products were also analyzed age. During this time, cauxin was below the detection by an amino acid analyzer, and peaks a and b showed limit of western blotting with anti-cauxin antibody, and the same elution times as those of the authentic com- very little felinine was excreted in the urine. pounds of felinine and glycine, respectively.