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Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane.

Zhong XW, Wang XH, Tan X, Xia QY, Xiang ZH, Zhao P - Int J Mol Sci (2014)

Bottom Line: Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels.The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars.The functions and regulatory mechanism of BmCDA7, however, need further investigation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. zxw_strive@163.com.

ABSTRACT
The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. PM proteins are important determinants for PM structure and formation. In this study, the silkworm Bombyx mori midgut PM protein BmCDA7 was identified by proteomic tools. The full-length BmCDA7 cDNA is 1357 bp; the deduced protein is composed of 379 amino acid residues and includes a 16 amino acid residue signal peptide, a putative polysaccharide deacetylase-like domain and 15 cysteine residues present in three clusters. The heterologously expressed proteins of the BmCDA7 gene in yeast displayed chitin deacetylase activity. Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels. The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars. The functions and regulatory mechanism of BmCDA7, however, need further investigation.

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Two-dimensional gel electrophoresis of proteins associated with the peritrophic membrane of B. mori larvae. One hundred and fifty micrograms of protein was applied to the IPG strip (18 cm, pH 3–10, L) and 15% SDS-PAGE was carried out for separation in the second dimension. The major expressed protein spots on the gel are numbered.
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f1-ijms-15-01946: Two-dimensional gel electrophoresis of proteins associated with the peritrophic membrane of B. mori larvae. One hundred and fifty micrograms of protein was applied to the IPG strip (18 cm, pH 3–10, L) and 15% SDS-PAGE was carried out for separation in the second dimension. The major expressed protein spots on the gel are numbered.

Mentions: Until now, the mode of action and catalytic mechanism of CDAs were not well understood. The function of fungal and bacterial CDAs has been demonstrated, including modification of the insect cuticular chitin to aid mycelial penetration and evasion of lysozyme action [8,14]. The role of insect CDAs, however, has had no more detailed investigation. Recently, several CDA genes were identified in the PM of various insects. In this study, we characterized silkworm CDA associated with the PM using a coupled proteomics and genomics approach. To study the protein profiles of silkworm PM, the total proteins were extracted from the silkworm larvae PM on day three of the fifth instar, separated by 2-D polyacrylamide electrophoresis and >60 protein spots were observed (Figure 1). We detected more spots in silkworm PM compared to other insects [20,22–24]. Most of the resolved protein spots had pI values between 5 and 9 and a molecular mass of 10–66 kDa. A total of 30 protein spots from PM were excised from the gel and investigated further by matrix-assisted lased desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, 12 proteins were identified (Table 1) and the detailed MS analysis for the identified silkworm PM proteins is given in Table S1. This investigation revealed these proteins were components of the PM. There were two structural peritrophic matrix proteins, chitin deacetylase (Spot 4) and PM chitin-binding protein 2 (Spot 17). They were also characterized by shotgun proteome technology in recent studies [25,26], which suggested that they have significant functional roles in silkworm PM.


Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane.

Zhong XW, Wang XH, Tan X, Xia QY, Xiang ZH, Zhao P - Int J Mol Sci (2014)

Two-dimensional gel electrophoresis of proteins associated with the peritrophic membrane of B. mori larvae. One hundred and fifty micrograms of protein was applied to the IPG strip (18 cm, pH 3–10, L) and 15% SDS-PAGE was carried out for separation in the second dimension. The major expressed protein spots on the gel are numbered.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3958831&req=5

f1-ijms-15-01946: Two-dimensional gel electrophoresis of proteins associated with the peritrophic membrane of B. mori larvae. One hundred and fifty micrograms of protein was applied to the IPG strip (18 cm, pH 3–10, L) and 15% SDS-PAGE was carried out for separation in the second dimension. The major expressed protein spots on the gel are numbered.
Mentions: Until now, the mode of action and catalytic mechanism of CDAs were not well understood. The function of fungal and bacterial CDAs has been demonstrated, including modification of the insect cuticular chitin to aid mycelial penetration and evasion of lysozyme action [8,14]. The role of insect CDAs, however, has had no more detailed investigation. Recently, several CDA genes were identified in the PM of various insects. In this study, we characterized silkworm CDA associated with the PM using a coupled proteomics and genomics approach. To study the protein profiles of silkworm PM, the total proteins were extracted from the silkworm larvae PM on day three of the fifth instar, separated by 2-D polyacrylamide electrophoresis and >60 protein spots were observed (Figure 1). We detected more spots in silkworm PM compared to other insects [20,22–24]. Most of the resolved protein spots had pI values between 5 and 9 and a molecular mass of 10–66 kDa. A total of 30 protein spots from PM were excised from the gel and investigated further by matrix-assisted lased desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, 12 proteins were identified (Table 1) and the detailed MS analysis for the identified silkworm PM proteins is given in Table S1. This investigation revealed these proteins were components of the PM. There were two structural peritrophic matrix proteins, chitin deacetylase (Spot 4) and PM chitin-binding protein 2 (Spot 17). They were also characterized by shotgun proteome technology in recent studies [25,26], which suggested that they have significant functional roles in silkworm PM.

Bottom Line: Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels.The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars.The functions and regulatory mechanism of BmCDA7, however, need further investigation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. zxw_strive@163.com.

ABSTRACT
The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. PM proteins are important determinants for PM structure and formation. In this study, the silkworm Bombyx mori midgut PM protein BmCDA7 was identified by proteomic tools. The full-length BmCDA7 cDNA is 1357 bp; the deduced protein is composed of 379 amino acid residues and includes a 16 amino acid residue signal peptide, a putative polysaccharide deacetylase-like domain and 15 cysteine residues present in three clusters. The heterologously expressed proteins of the BmCDA7 gene in yeast displayed chitin deacetylase activity. Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels. The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars. The functions and regulatory mechanism of BmCDA7, however, need further investigation.

Show MeSH
Related in: MedlinePlus