The
cDNA encoding the 182
amino acid long precursor
stellacyanin from Cucumis sativus was isolated and characterized. The
protein precursor consists of four sequence domains: I, a 23
amino acid hydrophobic N-terminal
signal peptide with features characteristic of secretory
proteins; II, a 109
amino acid copper-binding domain; III, a 26
amino acid hydroxyproline- and
serine-rich
peptide characteristic of motifs found in the extension family, extracellular structural
glycoproteins found in plant cell walls; and IV, a 22
amino acid hydrophobic extension. Maturation of the
protein involves posttranslational processing of domains I and IV. The
copper-binding domain (domain II), which shares high sequence identity with other
stellacyanins, has been expressed without its
carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of
stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized
stellacyanin, indicating that this domain folds correctly, even in the absence of its
carbohydrate moiety. The presence of a
hydroxyproline- and
serine-rich domain III suggests that
stellacyanin may have a function other than that of a diffusible electron transfer
protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or
infection of the plant.