Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form.
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.
Nersissian
A M
AM
Department of Chemistry and Biochemistry, UCLA 90095, USA. jsv@chem.ucla.edu
Mehrabian
Z B
ZB
Nalbandyan
R M
RM
Hart
P J
PJ
Fraczkiewicz
G
G
Czernuszewicz
R S
RS
Bender
C J
CJ
Peisach
J
J
Herrmann
R G
RG
Valentine
J S
JS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D018553:Cucumis sativus / Q000737:chemistry*; D018076:DNA, Complementary; D006031:Glycosylation; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D008667:Metalloproteins; D010940:Plant Proteins; C003343:stellacyanin protein, plant; D003300:Copper