Plant Science 159 2000 243 – 255
Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant
defensin
Ratna Wijaya
a,b
, Gregory M. Neumann
a
, Rosemary Condron
a
, Andrew B. Hughes
b
, Gideon M. Polya
a,
a
Department of Biochemistry, La Trobe Uni6ersity, Bundoora, Vic.
3083
, Australia
b
Department of Chemistry, La Trobe Uni6ersity, Bundoora, Vic.
3083
, Australia Received 15 March 2000; received in revised form 15 July 2000; accepted 17 July 2000
Abstract
A novel trypsin inhibitor was extracted from the seeds of Cassia fistula by a process successively involving soaking seeds in water, extraction of the seeds in methanol, and extraction of the cell wall material at high ionic strength. The protease inhibitor
PI was subsequently purified by chromatography on carboxymethylcellulose, gel filtration and reversed phase HPLC RP- HPLC. Electrospray ionization mass spectrometry ESMS of the oxidized from of the PI yielded an average molecular mass of
5458.6 9 0.8 Da. Edman sequencing of the PI yielded a full-length 50 amino acid sequence inferred to contain eight cysteines and with a calculated average molecular mass fully oxidized form of 5459.3 Da, in agreement with the observed mass. The C. fistula
seed PI is homologous to the family of plant defensins g-thionins, which have four disulfide linkages at highly conserved locations. The C. fistula PI inhibits trypsin IC
50
2 mM, and is the first known example of a plant defensin with protease inhibitory activity, suggesting a possible additional function for some members of this class of plant defensive proteins. C. fistula seeds also
contain a 9378 Da lipid transfer protein LTP homologue, other LTPs, a 7117 Da protein copurifying with PI activity and a 5144 Da defensin which does not inhibit trypsin. The complete sequence of the 5144 Da defensin was determined by Edman sequencing,
yielding a calculated average molecular mass oxidized form of 5144.1 Da, in agreement with the mass observed by ESMS. The likely trypsin inhibitory residue on the 5459 Da defensin is Lysine-25, the corresponding amino acid being Tyrosine-25 in the
homologous 5144 Da defensin that is not a trypsin inhibitor. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Cassia fistula; Seeds; Protease inhibitor; Defensin www.elsevier.comlocateplantsci
1. Introduction
Plant defence against herbivores and fungal pathogens involves elaboration of a variety of
bioactive secondary metabolites and defensive proteins [1 – 5]. Plant antifungal proteins include
enzymes such as glucanases and chitinases that can hydrolyse fungal cell walls [1,6], and a variety of
proteins that can interact with membrane compo- nents, for example lipid transfer proteins LTPs
[7 – 9], thaumatin-related proteins [10,11], a- and b-thionins [2,3,5], g-thionins defensins [4,12],
napins [13,14] and napin-like proteins [15,16]. In addition to providing a nitrogen-rich nutrient
store in plant seeds, the napins can be protease inhibitors PIs [13,14] and a variety of other small
plant defensive proteins are also PIs that can variously act as insect anti-feedants and as anti-
fungal proteins [1,17].
Abbre6iations
:
BAPNA, N-benzoyl-arginine-p-nitroanilide;
BYPNA, N-benzoyl-tyrosine-p-nitroanilide;
CaM, calmodulin;
CDPK, Ca
2 +
-dependent protein kinase; ESMS, electrospray ioniza- tion mass spectrometry; LTP, lipid transfer protein; PI, protease
inhibitor; PTH, phenylthiohydantoin; RP-HPLC, reversed phase HPLC;
TFA, trifluoroacetic
acid; TLCK,
Na-p-tosyl-
L
-lysine chloromethyl ketone; TPCK,
L
-1-tosylamido-2-phenylethylchloro- methyl-ketone.
Corresponding author. Tel.: + 61-3-94792157; fax: + 61-3- 94792467.
0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 4 8 - 4
It is important to determine the precise bio- chemical interactions involved in the action of
antifungal and anti-insect proteins, noting that such defensive proteins may have more than one
mode of action. The antifungal napins provide a good example of defensive proteins that have evi-
dent multiple functions and which have been shown to interact with more than one biochemical
entity. Thus napins are glutamine-rich storage proteins and can also be PIs [13,14], cause fungal
membrane changes and act synergistically with other membrane-altering antifungal proteins [18].
Napins and the napin-like proteins have a het- erodimeric subunit structure [15,16] and it has
been found that particular napin [16] and napin- like protein [14] small and large subunits are sub-
strates
for phosphorylation
by plant
Ca
2 +
-dependent protein kinase CDPK, although the biochemical significance of this has not been
established. In addition, some of these oxidized complexes and their constituent small and large
subunits can bind to calmodulin CaM, as de- tected
through inhibition
of CaM-dependent
myosin light chain kinase MLCK or through their
effect on
Ca
2 +
-dependent dansyl-CaM
fluorescence [16,19]. Some other plant antifungal proteins are also
substrates for plant CDPK [19], including various LTPs [20,21], soybean Bowman – Birk PI BBI-I
[22], potato tuber carboxypeptidase inhibitor protein [23] and plant defensins g-thionins [12].
Some plant defensins can interact with Ca
2 +
-CaM [12] in addition to being CDPK substrates and
antifungal proteins [4,5] and it is conceivable that further specific biochemical interactions may con-
tribute to the defensive function of this group of plant bioactive proteins.
PIs have a major role in anti-insect plant de- fence [1,17]. Inhibition of serine proteases by plant
protease inhibitory proteins or protease inhibitory secondary metabolites interferes with insect diges-
tion and can inhibit larval growth and develop- ment [1,17]. As part of an investigation into the
anti-insect components of various Indonesian Cas- sia species, the nature and structure of Cassia
fistula PI proteins have been investigated. The present paper describes the purification and se-
quencing of a PI protein from the seeds of C. fistula and its identification as a plant defensin. To
the authors’ knowledge this is the first demonstra- tion of a protease inhibitory function associated
with a representative of this major class of plant defensive proteins.
2. Materials and methods
