Characterization of cucurbita maxima phloem serpin-1 (CmPS-1). A developmentally regulated elastase inhibitor.
We report on the molecular, biochemical, and functional characterization of Cucurbita maxima phloem serpin-1 (CmPS-1), a novel 42-kDa serine proteinase inhibitor that is developmentally regulated and has anti-elastase properties. CmPS-1 was purified to near homogeneity from C. maxima (pumpkin) phloem exudate and, based on microsequence analysis, the cDNA encoding CmPS-1 was cloned. The association rate constant (k(a)) of phloem-purified and recombinant His(6)-tagged CmPS-1 for elastase was 3.5 +/- 1.6 x 10(5) and 2.7 +/- 0.4 x 10(5) m(-)(1) s(-)(1), respectively. The fraction of complex-forming CmPS-1, X(inh), was estimated at 79%. CmPS-1 displayed no detectable inhibitory properties against chymotrypsin, trypsin, or thrombin. The elastase cleavage sites within the reactive center loop of CmPS-1 were determined to be Val(347)-Gly(348) and Val(350)-Ser(351) with a 3:2 molar ratio. In vivo feeding assays conducted with the piercing-sucking aphid, Myzus persicae, established a close correlation between the developmentally regulated increase in CmPS-1 within the phloem sap and the reduced ability of these insects to survive and reproduce on C. maxima. However, in vitro feeding experiments, using purified phloem CmPS-1, failed to demonstrate a direct effect on aphid survival. Likely roles of this novel phloem serpin in defense against insects/pathogens are discussed.
Yoo
B C
BC
Department of Entomolgy, College of Agriculture and Environmental Sciences, University of California, Davis, California 95616, USA.
Aoki
K
K
Xiang
Y
Y
Campbell
L R
LR
Hull
R J
RJ
Xoconostle-Cázares
B
B
Monzer
J
J
Lee
J Y
JY
Ullman
D E
DE
Lucas
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids; D001665:Binding Sites; D015153:Blotting, Western; D002918:Chymotrypsin / Q000494:pharmacology; D003001:Cloning, Molecular; D019662:Cucurbitaceae / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014633:Valine / Q000737:chemistry
D018076:DNA, Complementary; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C417246:phloem serpin 1; D012694:Serine; D002918:Chymotrypsin; D014357:Trypsin; D013917:Thrombin; D014633:Valine; D005998:Glycine