Characterization of cucurbita maxima phloem serpin-1 (CmPS-1). A developmentally regulated elastase inhibitor

J Biol Chem. 2000 Nov 10;275(45):35122-8. doi: 10.1074/jbc.M006060200.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aphids
  • Binding Sites
  • Blotting, Western
  • Chymotrypsin / pharmacology
  • Cloning, Molecular
  • Cucurbitaceae / chemistry
  • DNA, Complementary / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Glycine / chemistry
  • Kinetics
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Serine / chemistry
  • Serine Proteinase Inhibitors / chemistry*
  • Serpins / chemistry*
  • Serpins / genetics
  • Serpins / metabolism
  • Thrombin / pharmacology
  • Time Factors
  • Trypsin / pharmacology
  • Valine / chemistry

Substances

  • DNA, Complementary
  • Recombinant Proteins
  • Serine Proteinase Inhibitors
  • Serpins
  • phloem serpin 1
  • Serine
  • Chymotrypsin
  • Trypsin
  • Thrombin
  • Valine
  • Glycine

Associated data

  • GENBANK/AF284038