Abstract
Actinopterygii express two types of chitinase (acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2)) that are active at acidic pHs and involved in digestion in the stomach. We proposed the existence of a new fish chitinase that has a non-digestive function. In this study, we used Sebastiscus marmoratus, for which characteristics and cDNA cloning of chitinase isozymes (SmChi-1, SmChi-2) in the stomach have been reported. Initially, we examined the distribution of chitinase and β-N-acetylhexosaminidase (Hex) in the body and then we tried to clone novel chitinase cDNA from the kidney. Chitinase and Hex activities were measured using pNP-(GlcNAc)n, (n = 2, 3) and pNP-GlcNAc as substrates, respectively. Total RNA was extracted from the kidney. RT-PCR was performed to obtain chitinase cDNA fragments using reverse transcriptase with an oligo dT primer. The RACE method was used to obtain sequences of the upstream and downstream regions of cDNA. The full-length chitinase cDNA was determined using PrimeSTAR® Max DNA polymerase with proofreading activity. High chitinase activity was observed in the stomach, as previously reported. In addition, relatively high activity was observed in the liver, spleen, kidney, and heart. In contrast, Hex activity was detected in all organs. This result is consistent with the report that Hex is related to body-wide metabolism. Full-length cDNA (SmChi-3) of the novel chitinase was obtained from the kidney, which contained 1440 bp open reading frames. The domain structure of SmChi-3 was assumed to be similar to those of SmChi-1 and SmChi-2. SmChi-1 and SmChi-2 have a serine and glycine-rich linker region, which is characteristic of AMCase. In contrast, SmChi-3 contained no apparent sequence in the linker region. Phylogenetic analysis revealed the existence of a new chitinase group, which was named fish chitinase-3 (FCase-3) and differed from AFCase-1 and AFCase-2.
Highlights
Chitin, a β-1,4-linked aminopolysaccharide of N-acetyl-D-glucosamine (GlcNAc), is the second most abundant component of biomass in the world, after cellulose, and is found in the exoskeletons of arthropods, the cell walls of fungi, and the cuticles of nematodes [1] [2] [3]
Measurement of activity of chitinolytic enzymes in organs of S. marmoratus (Figure 1) revealed that the chitinase activity assayed using pNP-(GlcNAc)2 and pNP-(GlcNAc)3 as substrates agreed with results that were reported previously [32], in which glycol chitin was used as a substrate and had the highest value in the stomach
We thought that after ingested chitin was degraded to (GlcNAc)n, by chitinases in the stomach, S. marmoratus would degrade (GlcNAc)n into GlcNAc with chitinases and Hex, which would allow for the metabolism and use of (GlcNAc)n that had been absorbed in the intestines and transferred to the bloodstream
Summary
A β-1,4-linked aminopolysaccharide of N-acetyl-D-glucosamine (GlcNAc), is the second most abundant component of biomass in the world, after cellulose, and is found in the exoskeletons of arthropods, the cell walls of fungi, and the cuticles of nematodes [1] [2] [3]. Chitin oligosaccharides (GlcNAc)n, have been found to promote the growth of bifidobacteria, have immunostimulatory activity [4], and GlcNAc has been found to improve osteoarthritis [5] and dry skin [6]. Chitinolytic enzymes can be classified into the following two categories according to their degradation patterns: Endo-type chitinolytic enzymes, which degrade β-1,4-glycosidic bonds in a chitin polymer at random to produce chitin oligosaccharides (GlcNAc)n and are called chitinases (EC 3.2.1.14) [7] [8] [9]; and exo-type chitinolytic enzymes, which degrade a chitin polymer from the nonreducing end, one by one, to produce GlcNAc and are called β-N-acetylhexosaminidase (Hex) (EC 3.2.1.52) [8] [9] [10]. 19 chitinases, on the other hand, are found primarily in higher plants and are reported to have strong antibacterial properties [20] [23]
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