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Cloning, Expression and Functional Analysis of Cathepsin D in Amphioxus Branchiostoma Japonicus

Author: ChenRong
Tutor: JiGuangDong
School: Ocean University of China
Course: Developmental Biology
Keywords: amphioxus cathepsin D gene cloning expression histone H2A
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 38
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Abstract


Cathepsin D (CTSD) (EC 3.4.23.5) is a lysosomal endoproteolytic aspartic proteinase which also has been found in endosomes of macrophage. It is thought to play key roles in the developmental and physiological process of animals. It have been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates, the amphioxus Branchiostoma japonicus. Amphioxus or lancelet, a cephalochordate, has long been regarded as the closest relative of vertebrates, and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. In this paper, we report the cloning, characterization, expression, phylogenetic analysis and functional characterization of amphioxus Cathepsin D gene (AmphiCTSD).The full-length cDNA of AmphiCTSD was 1772 bp long, containing an open reading frame (ORF) of 1188 bp which encoded a protein of 395 amino acids with a predicted molecular mass of approximately 42.92 kDa. The deduced protein had a signal peptide (16 amino acids) and had a single Asp superfamily domain with the two conserved Asp (Asp-Thr -Gly) motif, which are both characteristic of cathepsin D. The phylogenetic tree constructed using the sequences of representative cathepsin Ds including that of B. japonicus demonstrated that AmphiCTSD formed an independent group together with invertebrate cluster, suggesting that AmphiCTSD gene may be the archetype of vertebrate cathepsin D genes.Tissue in situ hybridization histochemistry demonstrated that AmphiCTSD transcript was most abundant in the ovary, epidermis, hind-gut and hepatic caecum, but little signal was observed in the muscle, neural tube and notochord. Embryo in situ hybridization demonstrated that AmphiCTSD transcript was expressed during every different developing stage, and was most abundant in neurula of 12hr and one-day larvae. Challenge with LPS and LTA soon (12 and 24 h) resulted in a down-regulation of AmphiCTSD expression, suggesting that AmphiCTSD may be involved in the host immune defense.The purified AmphiCTSD are able to be converted into active protein of～37.8 kDa via a N-terminal autocleavage at acid pH3.5 to efficiently degrade the cathepsin D-specific substrate haemoglobin. Both autocatalytic activity and the enzymatic activity are inhibited potently by pepstain A, but not by leupeptin, PMSF, EDTA and aprotinin. AmphiCTSD can also digest histone H2A, the cell wall of E. coli and S. aureus.In summary, this study reports the first characterization of cathepsin D in lower chordates, and suggests a function for this enzyme associated with the host immune defense.

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