Isolation and characterization of a lectin-like chitinase from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii

A lectin was isolated from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii by affinity chromatography using mucin-sepharose matrix. The purity of the isolated lectin was confirmed in native gradient PAGE that showed a single protein band of ∼37.9 kDa. In SDS-PAGE also one band of ∼...

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Published in:Biochimie 2024-06, Vol.221, p.125-136
Main Authors: Sahoo, Sonalina, Badhe, Mohan R., Paul, Anirban, Sahoo, Pramoda Kumar, Suryawanshi, Amol R., Panda, Debabrata, Pillai, Bindu R., Baliarsingh, Snigdha, Patnaik, Bharat Bhusan, Mohanty, Jyotirmaya
Format: Article
Language:English
Subjects:
Animals
Chitinase
Chitinases - chemistry
Chitinases - isolation & purification
Chitinases - metabolism
Chitinases - pharmacology
Electrophoresis, Polyacrylamide Gel
ELISA
Hepatopancreas
Hepatopancreas - enzymology
Lectin
Lectins - chemistry
Lectins - isolation & purification
Lectins - pharmacology
Macrobrachium rosenbergii
Palaemonidae - enzymology
Rabbits
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Summary:A lectin was isolated from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii by affinity chromatography using mucin-sepharose matrix. The purity of the isolated lectin was confirmed in native gradient PAGE that showed a single protein band of ∼37.9 kDa. In SDS-PAGE also one band of ∼43.3 kDa molecular weight was observed that indicated the protein to be a monomer. The band from the SDS-PAGE gel was identified through mass spectrometry as chitinase 1. The purified chitinase (50 μg/ml) hemagglutinated rabbit RBCs and, mucin and glucose inhibited hemagglutination with minimum concentrations of 0.1 mg/ml and 100 mM, respectively. Bacterial agglutination with Gram –ve Vibrio harveyi, Aeromonas sobria and Escherichia coli was also observed by this protein. Thus, chitinase 1 showed lectin-like properties besides its chitin hydrolytic activity. In western blot with hepatopancreas sample, rabbit antiserum against chitinase 1 cross-reacted to two additional proteins namely, chitinase 1C and obstructor E (a chitin-binding protein, CBP), besides its specific reactivity. An indirect ELISA was developed with the antiserum to quantify chitinases/CBP in hepatopancreas and serum samples of M. rosenbergii. The assay was used in samples from juvenile prawns following V. harveyi challenge. At 72 h post-challenge, significantly higher levels of chitinases/CBP were quantified in the hepatopancreas of the challenged group (1.8 ± 0.2 mg/g tissue) compared to the control (1.2 ± 0.1 mg/g tissue). This study suggests that the chitinase 1 protein with lectin-like properties is possibly induced at the protein level and can be putatively involved in the innate immune response of M. rosenbergii. •A ∼43.3 kDa Chitinase 1 was purified from the hepatopancreas of M. rosenbegii.•It possesses chitinolytic, hemagglutinating and bacterial agglutinating properties.•Antiserum to chitinase 1 cross-reacted to chitinase 1C and obstructor E proteins.•ELISA was developed to quantify chitinases/CBP in hepatopancreas and serum samples.•Vibrio harveyi challenge revealed chitinases/CBP to be inducible in nature.
ISSN:0300-9084
1638-6183
DOI:10.1016/j.biochi.2023.09.025