In order to characterize a specific extracellular 21-kDa calmodulin-binding protein (named: ECBP21) from Angelica dahurica L. suspension-cultured cells, the cDNA coding for the protein has been cloned. Here, Southern blot analysis shows that there are at least two copies of ECBP21 gene in Angelica genome. Using truncated versions of ECBP21 and synthetic peptide in CaM binding assays, we mapped the calmodulin-binding domain to a 16-amino acid stretch (residues 200-215) at the C-terminal region. The ECBP21 was localized in the cell wall area by the immunogold electron microscopy and by GFP labeling method. These results define ECBP21 as a kind of an extracellular calmodulin-binding protein (CaMBP). Furthermore, using Northern blot analysis, we examined the expression dynamics of ecbp21 during the incubation of Angelica suspension-cultured cells and the treatments with some growth regulators. The above studies further provide the molecular evidence for the existence of the gene coding for extracellular CaMBPs and imply a possible role for ECBP21.
Lipid transfer proteins in plants are believed to be involved in many processes of cell physiology and development. In this work, a full-length cDNA encoding a novel lipid transfer protein, designated BcLTP was isolated from Brassica chinensis. At least two copies of BcLTP are present in whole genome of B. chinensis, and its transcripts preferably accumulate in second-year organs, implying its role in reproductive growth stage. The 118 amino acid sequence deduced from a 354 bp open reading frame (ORF) shares common features with other members of plants LTPs family. A putative signal peptide at the N terminus was tested for secretion function by the yeast signal sequence trap (YSST) system, and further confirmed by vesicular and extracellular localization of YFP fusion protein. A highly conserved CaM binding site at C terminus was found and the binding properties with two representative CaM isoforms, one is convergent AtCaM2, one is divergent SCaM5, were determined by gel overlay. We found that convergent AtCaM2 prefer high concentration of Ca(2+) for binding BcLTP, while SCaM5 does not depend on Ca(2+ )concentration too much for binding BcLTP. The lipid binding feature of BcLTP was demonstrated using florescence-marked 1-pyrenedodecanoic acid, which can be enhanced by AtCaM2 in Ca(2+ )dependent manner and by SCaM5 in either presence or absence of Ca(2+). The collected data suggest that BcLTP may secrete and combine extracellular CaM isoforms, which in turn, facilitate lipid binding of BcLTP via Ca(2+) mediated signaling.
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