The calpains are cytoplasmic, calcium-activated neutral cysteine-proteases. They function in Ca²⁺ signaling by modulating biological activities of their substrates through limited proteolysis. Several structural types of calpains are known, many of these being tissue-specific. Calpain 3 (CAPN3) is specifically expressed in muscle [1] , and mutations of the CAPN3 gene were reported in limb girdle muscular dystrophy type 2A [2] . A splice variant of calpain 3, Lp82, is expressed in the eye lens [3] . Calpain 8, predominantly expressed in stomach, shows two alternative splice products, one with and one without a calcium binding domain [4] . Calpain 9 (CAPN9) is predominantly expressed in the digestive tract and plays a role in gastric mucosal defense in collaboration with calpain 8 [5] [6] . Down-regulation of calpain 9 has been observed in stomach cancer [7] and hypertension [8] . The physiological function of calpain 9 in taste cells is still unclear.

All (classical) calpains are heterodimeric. Of the two subunits that comprise the calpain heterodimer, the larger one can be divided into four domains: domain I has a regulatory function; domain II is the cysteine protease domain; domain III is C2-like β-sandwich and domain IV is responsible for Ca²⁺-binding with five EF-hand motifs [9] . X-ray crystallographic analysis of rat calpain 4 revealed that several amino acid pairs in EF-hand motifs specified calcium-binding properties [10] [11] . Recently, the Km values (125 mM) for Ca²⁺ ions for human recombinant CAPN9 have been reported [12] . The physiological substrates of calpain 9 remain to be elucidated.

Knowing that calpains are involved in intracellular calcium-mediated events, we reasoned that calpains may play a role in taste transduction, a process regulated by calcium signaling. In the visual system, calpains were thought to play roles in adaptation processes through arrestin degradation [13] . The calpains may also play limited roles in cleavage of the IP₃ receptor [14] , PLC β₃ [15] , G proteins [16] , and other members of the calcium signaling cascades [17] .

Chemical stimulation of taste receptor cells generally results in changes in activity of intracellular calcium, leading to either release of neurotransmitter from the stimulated cell or communication with a specialized synaptic cell [18] . This taste-mediated increase in calcium may be sufficient to activate calpains that stimulate the degradation of signaling molecules.

Here we report on the identification of calpain 9 from catfish (designated Ip-CAPN9a) and on its expression in taste tissue. We first reported the molecular cloning of calpains from taste tissue in abstract form in 1997 [19] .

In this report, we described the isolation of a tissue-specific calpain cDNA out of a catfish barbell epithelium cDNA library. Calpains have been reported in other non-mammalian species including CAPN1 & CAPN2 in carp muscle [25] , those in trout muscle [26] and gill-specific calpain of rainbow trout [27] . The calpain reported on here encodes a 687 amino acid protein with a predicted molecular weight of 78,345 Da. From amino acid sequence comparison, this catfish calpain (Ip-CAPN9a) is most closely related to the CAPN9 of rainbow trout, zebrafish, human, mouse, rat and Xenopus (Figure 1(a)).

In a maximum-likelihood analysis with the whole protein sequence, the value of alpha was 0.926. Figure 2(a)

shows that Ip-CAPN9a clustered within the same node next to the CAPN3 subfamily, while rainbow trout capn1 and capn2 grouped with human capn1 and capn2. The same result was observed in the phylogenetic analysis with the Penta-EF hand motifs (α = 1.692) in Figure 2(b). These observations would suggest that CAPN3 and CAPN9 diverged from CAPN1 and CAPN2 prior to teleost evolution [28] .

A feature worth further study is the unique amino acid residues at the calcium binding site of Ip-CAPN9a, especially at the Y site (Figure 1(b)) [10] [29] . Considering their position within the calcium binding sites, it is likely that these unique amino acids participate in the calcium modulation of this enzyme.

Northern blot analysis suggests that Ip-CAPN9a is predominantly expressed in barbell, slightly in olfactory rosettes and intestine, but not in brain (Figure 3(a)). Even though we were unable to detect Ip-CAPN9a mRNA expression in brain by northern analysis, we could not rule out the possibility that some specific cells within the brain may express Ip-CAPN9a as shown in Drosophila CalpA [30] . Salem et al. showed that rainbow trout gill-specific calpain, closely related to calpain 9, was not expressed in the small intestine. Different detecting probes used in the northern blot analysis may explain this difference [27] .

The immuno-histochemical studies demonstrated that polyclonal antibodies against a deduced peptide from the N-terminal segment of Ip-CANP9a recognized the taste pore region (Figure 3(b)) and taste bud cells (Figure 3(c)). Taken together with the northern analysis, these data suggest that Ip-CAPN9a is a chemosensory and digestive tract specific calpain in fish. The barbell of the catfish detects taste stimuli as does the tongue in mammals. According to the current views of the taste system, where the sense of taste is considered a “metabolic sense” acting both at the perceptual and the metabolic levels, it may not be surprising to find a digestive tract calpain within cells of the taste bud.

In spite of much effort, we could not detect a band on SDS-PAGE-western blot that would correspond in molecular weight (78 kDa) to Ip-CAPN9a (data not shown). Several explanations can be considered including 1) the likely high turnover rate for tissue-specific calpains [1] and 2) high calcium concentration around taste cells leading to calpain activation and autolysis. A similar anomaly is seen with human CAPN3. This calpain is difficult to detect by western blotting, most probably due to its high turnover rate and its lability after dissociation from connectin, to which CAPN3 binds through its IS2 region [31] .

Human CAPN9 and Ip-CAPN9a might be more fragile than human calpain 3. M- and μ-calpains cleave their N-terminal segments upon activation (autolysis). The antibody against Ip-CAPN9a was raised against its N- terminal segment (3 to 16 amino acids). The presumptive cleavage site on autolysis localizes near the end of this segment. We thus assume that the antibody recognition segment would be mostly released from Ip-CAPN9a upon its activation. Na⁺, Ca²⁺, Mg²⁺ ions are rich in catfish barbell mucin layer. This high calcium concentration could activate calpains during sample preparation.

The calpains have been implicated in degradation of the IP₃ receptor [14] and of the enzyme, PLC beta-3 [15] . In a preliminary experiment [19] , we noted that the calpain inhibitor, the tripeptide, LLL, reduced the degradation of the IP₃ receptor in catfish barbell upon stimulation of the preparation by the potent taste stimulus, L-ar- ginine. As there are no subtype specific calpain inhibitors, we cannot determine which calpain subtypes are responsible for the degradation of taste-related proteins.

Our discovery of Ip-CAPN9a should lead to the characterization of other teleost-related calpain subtypes. Studies of other tissue-specific type fish calpains will clarify how these calcium-dependent calpains are activated and inhibited under physiological conditions.

T.O thanks to the laboratory members in the Monell Chemical Senses Center and NFRI.