TITLE:
Distribution of Chitinolytic Enzyme in the Organs and Molecular Cloning of a Novel Chitinase Gene from the Kidney of Marbled Rockfish Sebastiscus marmoratus
AUTHORS:
Miku Watanabe, Hiromi Kakizaki, Taro Tsukamoto, Miku Fujiwara, Hideto Fukushima, Mitsuhiro Ueda, Masahiro Matsumiya
KEYWORDS:
Chitinolytic Enzyme, Chitinase, Sebastiscus marmoratus, Distribution, cDNA Cloning
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.9 No.1,
January
30,
2018
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.