Limits...
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.

Show MeSH
The full-length cDNA sequences and deduced amino acid sequences of BmCDA7. The predicted signal peptide is underlined with a broken line. A potential polyadenylation signal sequence is boxed. The chitin deacetylase domain is underlined. Cysteines in the mature protein sequence are in bold type and circled. The N-glycosylation sites and O-glycosylation sites are showed with bold type and grey background.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3958831&req=5

f2-ijms-15-01946: The full-length cDNA sequences and deduced amino acid sequences of BmCDA7. The predicted signal peptide is underlined with a broken line. A potential polyadenylation signal sequence is boxed. The chitin deacetylase domain is underlined. Cysteines in the mature protein sequence are in bold type and circled. The N-glycosylation sites and O-glycosylation sites are showed with bold type and grey background.

Mentions: The protein sequence of CDA (BGIBMGA013757) was used to search the silkworm genome database. Another seven genes showing significant homology with CDA were identified (Table S2). We named the silkworm CDA in accord with Dixit et al. [28]. BGIBMGA013757, which was identified from PM, was named BmCDA7. We cloned and sequenced the BmCDA7 gene. The cDNA and predicted protein sequences of the BmCDA7 are shown in Figure 2. Rapid amplification of cDNA ends (RACE) experiments were used to obtain the 5′ and 3′ ends of BmCDA7. Finally, the full length of the BmCDA7 cDNA is 1357 bp followed by an A+T-rich region with two typical polyadenylation signal sequences (AATAAA). The full length of BmCDA7 cDNA contains an open reading frame of 1140 bp, a 27 bp upstream-untranslated region and a 190 bp downstream-untranslated region. The putative BmCDA7 cDNA encoded for a 379 amino acid residue protein consisting of a 16 amino acid residue signal peptide according to SignalP software [29] and a mature polypeptide of 363 amino acid residues. After removal of the signal peptide, the deduced protein was predicted by the ExPASy server to have a molecular mass of 41.26 kDa and a theoretical pI of 5.12 [30]. Prediction of potential glycosylation sites using the NetNglyc 1.0 [31] and NetOglyc 3.1 [32] server showed that the protein contains a putative N-glycosylation site at Asn168, and putative O-glycosylation sites at Thr209 and Thr215. These sequence features suggest that the BmCDA7 protein might be both N- and O-glycosylated. BmCDA7 has a putative polysaccharide deacetylase-like domain (residues 46–182) and 15 cysteine residues present in three clusters of five situated at residues 24–83, 183–243 and 332–365 as determined by SMART analysis [33]. The polysaccharide deacetylase-like domain showed sequence similarities to the CDA domain sequences from fungi and a bacterium according to Guo et al. and the cysteine-rich regions are common to PM proteins. These sequence characteristics are similar to TnPM-P42 of T. ni, which is different from the peritrophin-type PM proteins but, instead, has a chitin deacetylase-like domain and uses a CDA-like domain for chitin binding [15]. Therefore, our results indicate BmCDA7 does not resemble any of the peritrophin domains from types I or II PMs [2], but belongs to Class 3 of PM proteins. The distribution patterns of the cysteine residues in BmCDA7 are dissimilar compared to the known peritrophin-type PM proteins. Such arrangements of the cysteine residues could constitute a new type of chitin-binding domain, and provide an important mechanism for the protein-chitin association in PM formation. Wang and Granados suggested the conserved cysteine residues form intra-domain disulfide bonds, which confer the stability of PM proteins in the protease-rich gut environment [34]. They might be a basic module that combines with other protein sequences to generate a new function or modify an existing function.


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)

The full-length cDNA sequences and deduced amino acid sequences of BmCDA7. The predicted signal peptide is underlined with a broken line. A potential polyadenylation signal sequence is boxed. The chitin deacetylase domain is underlined. Cysteines in the mature protein sequence are in bold type and circled. The N-glycosylation sites and O-glycosylation sites are showed with bold type and grey background.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ijms-15-01946: The full-length cDNA sequences and deduced amino acid sequences of BmCDA7. The predicted signal peptide is underlined with a broken line. A potential polyadenylation signal sequence is boxed. The chitin deacetylase domain is underlined. Cysteines in the mature protein sequence are in bold type and circled. The N-glycosylation sites and O-glycosylation sites are showed with bold type and grey background.
Mentions: The protein sequence of CDA (BGIBMGA013757) was used to search the silkworm genome database. Another seven genes showing significant homology with CDA were identified (Table S2). We named the silkworm CDA in accord with Dixit et al. [28]. BGIBMGA013757, which was identified from PM, was named BmCDA7. We cloned and sequenced the BmCDA7 gene. The cDNA and predicted protein sequences of the BmCDA7 are shown in Figure 2. Rapid amplification of cDNA ends (RACE) experiments were used to obtain the 5′ and 3′ ends of BmCDA7. Finally, the full length of the BmCDA7 cDNA is 1357 bp followed by an A+T-rich region with two typical polyadenylation signal sequences (AATAAA). The full length of BmCDA7 cDNA contains an open reading frame of 1140 bp, a 27 bp upstream-untranslated region and a 190 bp downstream-untranslated region. The putative BmCDA7 cDNA encoded for a 379 amino acid residue protein consisting of a 16 amino acid residue signal peptide according to SignalP software [29] and a mature polypeptide of 363 amino acid residues. After removal of the signal peptide, the deduced protein was predicted by the ExPASy server to have a molecular mass of 41.26 kDa and a theoretical pI of 5.12 [30]. Prediction of potential glycosylation sites using the NetNglyc 1.0 [31] and NetOglyc 3.1 [32] server showed that the protein contains a putative N-glycosylation site at Asn168, and putative O-glycosylation sites at Thr209 and Thr215. These sequence features suggest that the BmCDA7 protein might be both N- and O-glycosylated. BmCDA7 has a putative polysaccharide deacetylase-like domain (residues 46–182) and 15 cysteine residues present in three clusters of five situated at residues 24–83, 183–243 and 332–365 as determined by SMART analysis [33]. The polysaccharide deacetylase-like domain showed sequence similarities to the CDA domain sequences from fungi and a bacterium according to Guo et al. and the cysteine-rich regions are common to PM proteins. These sequence characteristics are similar to TnPM-P42 of T. ni, which is different from the peritrophin-type PM proteins but, instead, has a chitin deacetylase-like domain and uses a CDA-like domain for chitin binding [15]. Therefore, our results indicate BmCDA7 does not resemble any of the peritrophin domains from types I or II PMs [2], but belongs to Class 3 of PM proteins. The distribution patterns of the cysteine residues in BmCDA7 are dissimilar compared to the known peritrophin-type PM proteins. Such arrangements of the cysteine residues could constitute a new type of chitin-binding domain, and provide an important mechanism for the protein-chitin association in PM formation. Wang and Granados suggested the conserved cysteine residues form intra-domain disulfide bonds, which confer the stability of PM proteins in the protease-rich gut environment [34]. They might be a basic module that combines with other protein sequences to generate a new function or modify an existing function.

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