THE MANY ROLES OF PROTEINASES IN PLANTINSECT INTERACTIONS

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THE MANY ROLES OF PROTEINASES IN PLANT-INSECT
INTERACTIONS

• David Bown and John Gatehouse
• School of Biological and Biomedical Sciences
• University of Durham,
• South Road, Durham, United Kingdom

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PROTEINASES AND PROTEINASE INHIBITORS

• Plant proteinase inhibitors (PIs) are a
  well-established mechanism of defence against
  insect herbivores
• Proteinase inhibitors are present as a
  constitutive defence (accumulated in tissues such
  as seeds)
• Proteinase inhibitors are also induced by insect
  feeding (wounding response)
• Plant proteinase inhibitors used in defence have
  a specificity directed towards herbivore
  digestive proteinases
• Inhibitors primarily target chymotrypsin-type
  serine proteinases not used by plants for protein
  digestion
• (however, genes encoding proteins of this type
  are present in plants 7 potential genes in A.
  thaliana, but all encode large proteins targetted
  to organelles or membrane-bound likely
  involvement in protein folding/assembly)

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PROTEINASES AND PROTEINASE INHIBITORS
The accepted model for PI action insect
digestion is blocked, leading to nutritional
deprivation through inadequate levels of amino
acids/small peptides in the gut. Recycling of
essential amino acids incorporated into digestive
enzymes is also prevented.
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PROTEINASES AND PROTEINASE INHIBITORS

• How have insect herbivores dealt with the problem
  of overcoming the proteinase inhibitor defences
  of their plant hosts?
• Coevolution of plants and insects has led to
  different strategies being employed by different
  insect species, families and orders
• Insect feeding strategies can be broadly grouped
  into two types
• Monophagous/oligophagous - feed on one species,
  or a limited range of species
• Polyphagous - feed on a wide range of species
• Monophagous/oligophagous insect herbivores show
  adaptations which are specific to their chosen
  host plant species
• these plant-insect interactions lead to the
  classical (Ehrlich and Raven) model of
  coevolution of species
• Polyphagous insect herbivores show adaptations
  which enable them to deal with a wide range of
  host species

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DIVERSITY IN INSECT PROTEINASES IS A RESPONSE TO
PLANT PROTEINASE INHIBITORS

• Digestive proteinases in insect herbivores have
  to be adapted to deal with proteinase inhibitors
  in their plant hosts
• Also need to be able to digest a range of
  proteins efficiently
• Both considerations are significant, as insect
  herbivores are usually limited by nitrogen
  availability (most plant tissues have a low
  nitrogen content)
• Selection pressures have led to a diversity in
  insect digestive proteinases
• Diversity in insect digestive proteinases shown
  both in diversity within a proteinase type, and
  by the use of different types of proteinases

Colorado potato beetle (Leptimotarsa
decemlineata) adult and larva an insect which
uses multi-gene families of different types of
digestive proteinases
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM
Corn rootworm (Diabrotica spp.) - adult, larva,
and damage
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM
In common with many coleopteran insects, corn
rootworm has an acidic midgut, with a pH optimum
for proteolysis of approx. 5
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• Effects of specific inhibitors suggests that
  proteolysis is mainly due to cysteine
  proteinases, with aspartic proteinases playing a
  minor role.
• No evidence for serine proteinases playing a
  significant role in digestion proteolysis not
  affected by plant protein inhibitors of serine
  proteinases.

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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• Corn rootworm is adapted to the presence of
  serine PIs in its host by using different
  (non-serine) classes of proteinases for digestion
• What are the characteristics of the cysteine
  digestive proteinases?
• Cysteine proteinases in Diabrotica virgifera have
  been characterised by isolation of cDNA clones
  from a library representing larval gut RNA
• Gut contains a family of cathepsin L-like
  proteinases, and cathepsin B-like proteinases

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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• Do these clones encode digestive proteinases?
• Compare abundance of mRNA in total RNA from gut
  and body tissue using northern blotting (right)
• Cathepsin L-like enzymes are much more abundant
  in gut tissue than other tissues, providing
  evidence for a digestive role
• One cathepsin B-like enzyme is low abundance and
  not gut-specific, but the other is gut-specific
• Digestive cysteine proteinases are predominantly
  cathepsin L-like

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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• Do these cDNAs encode digestive proteinases?
• N-terminal sequence of major cysteine proteinase
  polypeptide partially purified from Diabrotica
  virgifera larval gut extract (Koiwa et al., 2000,
  FEBS Letters 471 67-70) matches sequence
  predicted by cDNA clone

Protein A V E(X)V D W R E S A V L G V K D Q
G Q(X)G S . . A V E E V D W R D S A V L G V K D
Q G Q C G S . cDNA
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• Cathepsin L-like enzymes encoded by different
  Diabrotica cDNAs contain different
  specificity-determining residues at the bottom of
  the S2 substrate binding sub-site
• The predicted enzymes fall into two types
• those with a neutral, hydrophobic S2 residue
• those with an acidic S2 residue

C-terminal region of predicted cathepsin L-like
enzymes . N S W N T Y W G E E G Y L R I V R G K
N - Q C G I N E V A D Y P L L DvRS29 . N S W G
T S W G E Q G Y I R V A R G E N - L C G I N L M N
S Y P K L DvRS5 . N S W G A D W G M D G Y I W
M S R N K N N Q C G I A T D A T Y P T I
DvRS30 . N S W G V N W G M D G Y I R M S R N K N
N Q C G I T T D G V Y P N I DvRS33

asn of cys-his-asn catalytic triad
S2 residue
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• cDNA clones encode Diabrotica cathepsin L-like
  proteinases with predicted specificity towards
• substrates with neutral, hydrophobic residues at
  P2 (DvRS5, 29) or
• substrates with basic residues at P2 (DvRS30, 33)
• Using both types of proteinases could enable the
  insect to digest protein substrates more
  efficiently (c.f. use of serine proteinases of
  differing specificities in higher animals), and
  possibly decrease its sensitivity to cysteine
  proteinase inhibitors
• Confirm differences in specificity of proteolysis
  by producing functional recombinant proteins from
  cDNA clones
• Expression system based on the yeast Pichia
  pastoris used enables (his)6tagged recombinant
  protein to be secreted into culture medium, from
  which it can be purified by hydrophobic
  interaction chromatography and affinity
  chromatography.

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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• Representative Diabrotica cathepsin L-like
  proteinases expressed as recombinant proteins in
  Pichia pastoris
• Purified from culture supernatant in mg
  quantities (see gel left)
• DvRS30 protein was glycosylated by yeast
  expression system (sequence predicts
  glycosylation site in mature protein)
• DvRS5 protein was not glycosylated (no predicted
  glycosylation site in mature protein)
• Both proteins were functional, as determined by
  proteolytic activity towards synthetic and
  protein substrates

5
30
DvRS30 protein
DvRS5 protein
SDS-PAGE gel of purified recombinant Diabrotica
cathepsin L-like proteinases
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• N-terminal sequences of Diabrotica cathepsin
  L-like proteins produced in yeast showed that the
  prosequences had been removed, presumably as a
  result of autoactivation
• Comparison with N-terminal sequence of protease
  purified from Diabrotica gut extract suggests
  that further trimming of N-terminal sequence of
  mature protein takes place in vivo (see below)
• Cathepsin B-like protein from Diabrotica
  expressed in Pichia was inactive until activated
  by treatment with bovine trypsin - no
  autoactivation

Gut protein A V E(X)V D W R E S
A V L G V K D Q . DvRS5 cDNA . V A D P N V Q
A V E E V D W R D S A V L G V K D Q . DvRS5
Protein D P N X Q A V E X V . .
Predicted from
mammalian homologues DvRS30 cDNA . S L T P V K
D L P S K F D W R E K G A V T E V K D . DvRS30
Protein D(F)P S K F D . .
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM
Kinetic parameters for recombinant proteins show
that DvRS5 has cathepsin L-like specificity
(hydrolysis of Z-phe-arg-AMC, but no hydrolysis
of Z-arg-arg-AMC), but DvRS30 has cathepsin
B-like specificity (hydrolysis of both
Z-phe-arg-AMC and Z-arg-arg-AMC), as predicted by
S2 subsite amino acid residue. Together, these
enzymes account for hydrolytic activity in gut
extract towards synthetic substrates.
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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• Specificity of recombinant Diabrotica cathepsin
  L-like proteinases towards peptide substrates
  investigated by mass spectrometry
• Time course for peptide hydrolysis followed by
  analysis of products on SELDI instrument (right)
• Results for cleavage of insulin B chain (below)
  show expected specificity for neutral hydrophobic
  residues at P2 position both enzymes show this
  specificity
• DvRS30 (dashed arrows) shows one extra cleavage,
  with basic residue at P2 (residues 23-24)

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DIVERSITY IN INSECT PROTEINASES 1 CORN ROOTWORM

• Conclusions
• Corn rootworm uses multiple cysteine proteinases
  with differing specificities for digestive
  proteolysis
• Major digestive cysteine proteinase is similar to
  mammalian cathepsin L in substrate specificity
• Corn rootworm also contains digestive proteinases
  homologous to cathepsin L, which act as
  endopeptidases, but which have substrate
  specificity at P2 substrate position similar to
  cathepsin B
• Corn rootworm also contains cathepsin B-like
  enzymes, with structural features which suggest
  that these enzymes act as dipeptidases (I.e.
  presence of occluding loop. These enzymes have
  similar substrate specificity to mammalian
  cathepsin B
• Work in progress
• Characterisation of corn rootworm digestive
  aspartic proteinase - protein identified,
  functional recombinant enzyme has been produced

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM
Corn earworm (bollworm), Helicoverpa armigera
A highly polyphagous lepidopteran crop pest
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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Larvae of corn earworm, like many lepidopteran
  species, have strongly alkaline conditions in the
  gut and use serine proteinases as the major
  digestive enzymes.
• Serine proteinases form a large multi-gene family
  in which individual members are differentially
  regulated in response to dietary proteinase
  inhibitors (Bown et al.,1997, Insect Biochem.
  27 625-638).
• Carboxypeptidases with a broad alkaline pH
  optimum are detectable as components of total
  proteinases in larval gut extract.

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Synthesis of carboxypeptidases in gut tissue of
  larval corn earworm is strongly up-regulated by
  dietary proteinase inhibitors
• Increased use of carboxypeptidases for digestive
  proteolysis by corn earworm larvae may be a
  response to the presence of serine PIs, since
  serine proteinases are normally responsible for
  most proteolytic activity

Northern blot showing increased level of mRNA
encoding HaCM1 carboxypeptidase when larvae are
fed diet containing soybean Kunitz trypsin
inhibitor (S) compared to control diet (C)
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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Corn earworm digestive carboxypeptidases have
  been characterised through a cDNA library
  prepared using larval gut RNA as a template
• They form a family of related proteins,
  homologous to mammalian carboxypeptidase A/B. The
  cDNA clones represent four subfamilies of
  proteins (see below) with further minor variants
  (lt2 difference) within subfamilies.
• The corn earworm carboxypeptidases are all more
  similar to each other than to any Drosophila
  carboxypeptidase sequence

Phylogenetic tree of Helicoverpa armigera
carboxypeptidase protein subfamilies
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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM
DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM
Digestive carboxypeptidases in corn earworm
larval gut extract were identified by binding to
potato carboxypeptidase inhibitor (PCPI)
PCPI
Carboxypeptidase
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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• The digestive carboxypeptidases in gut extract
  from larval corn earworm were purified by
  affinity chromatography on a column of
  immobilised potato carboxypeptidase inhibitor
  (PCPI)
• PCPI column bound several proteins which were
  released under mild denaturing conditions
  (extreme pH eluted at pH12)
• Tight binding netween PCPI and carboxypeptidases
  required strongly denaturing conditions (6M
  guanidine hydrochloride) to release proteinases

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Bands were excised from blotted gel and subjected
  to N-terminal sequencing to identify polypeptides
• Bands eluted at pH 12 were not carboxypeptidases

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Bands were excised from blotted gel and subjected
  to N-terminal sequencing to identify polypeptides
• Bands eluted by 6M guanidine hydrochloride
  corresponded to three subfamilies of corn earworn
  carboxypeptidase

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Specificity of cleavage by carboxypeptidase
  A-type proteinases is based on amino acid residue
  in S1 substrate binding site (residue 255, human
  carboxypeptidase A). The C-terminal residue of
  the substrate binds here
• The four subfamilies of carboxypeptidases
  identified in corn earworm have different
  predicted specificities of cleavage, based on the
  S1 amino acid residues
• Subfamily represented by HaCA42 has an arginine
  residue at this position, and is predicted to
  show specificity towards C-terminal acidic
  residues

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Carboxypeptidase specificity can be assayed using
  synthetic substrates with C-terminal
  phenylalanine (N-(3-2-furylacryloyl)-Phe-Phe
  FAPP), C-terminal lysine (N-(3-2-furylacryloyl)-
  Phe-Phe FAAK) or C-terminal glutamic acid
  (N-(3-2-furylacryloyl)-Glu-Glu FAEE)
• Corn earworm larval gut extract contains activity
  against all three substrates
• Carboxypeptidase HaCM1 has been expressed as a
  recombinant protein in insect cells using a
  baculovirus expression system (Bown et al., 1998,
  Insect Biochem. Mol. Biol. 28 739-749)

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Corn earworm carboxypeptidase HaCA42 expressed as
  a recombinant protein in Pichia pastoris
• Purified from culture supernatant by hydrophobic
  interaction chromatography on phenyl-Sepharose
  followed by affinity chromatography on
  immobilised nickel ions
• Purified protein essentially a single band on
  SDS-PAGE, mol. wt. approx. 48,000
• No hydrolytic activity towards carboxypeptidase
  substrates until activated by treatment with
  trypsin

HaCA42Trypsin
Trypsin
HaCA42
M
Mr, kDa
67
45
36
29
25
20
14
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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM
Pro-
Mature
Activn.Peptide
0
5
20
40
60
80
100
120
C
min

• Activation of purified HaCA42 carboxypeptidase
  with trypsin (left panel above) shows that
  cleavage of pro-sequence occurs very rapidly, but
  mature enzyme relatively stable to proteolysis
• Kinetics of activation (right panel above) show
  that cleavage of activation peptide is not
  sufficient to activate the enzyme (e.g. after 5
  min) activation peptide must be degraded to give
  full activity, implying that the pro-peptide is
  an inhibitor of the enzyme
• This is observed in other carboxypeptidases
  pro-peptide blocks activation site by binding to
  enzyme

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Purified recombinant HaCA42 carboxypeptidase,
  after activation by trypsin, is specific for
  FAEE substrate as predicted
• Different corn earworm carboxypeptidases show
  complementary specificities of hydrolysis

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Other properties of HaCA42 carboxypeptidase are
  consistent with role as digestive proteinase -
  e.g. pH optimum (right)
• This is the first enzyme with homology to
  carboxypeptidases of clan MC with specificity for
  C-terminal acidic residue
• Specificity for C-terminal glu similar to
  carboxypeptidase G (glutamate carboxypeptidase),
  but unlike carboxypeptidase G, HaCA42 does not
  hydrolyse methotrexate
• Drosophila genome contains 7 enzymes with
  homology to clan MC carboxypeptidases, but none
  with HaCA42 specificity

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DIVERSITY IN INSECT PROTEINASES 2 CORN EARWORM

• Conclusions
• Larval corn earworm uses several different
  carboxypeptidases with different specificities as
  digestive exopeptidases
• All these enzymes are metalloproteases, clan MC,
  and their synthesis is up-regulated by dietary
  serine protease inhibitors
• One enzyme shows a novel specificity for
  carboxypeptidases of clan MC in hydrolysing
  substrates with C-terminal glutamic acid
• Corn earworm carboxypeptidase expressed in Pichia
  pastoris as a recombinant enzyme must be
  activated by treatment with bovine trypsin
• Pro-sequence is an effective inhibitor of the
  enzyme, and must be degraded to give activity
• Work in progress
• Further characterisation of specificity of
  recombinant enzyme, using specially synthesised
  peptide substrates
• Identification of enzyme responsible for activity
  towards carboxypeptidase B substrate FAAK

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OTHER ROLES FOR PROTEINASES IN PLANT-INSECT
INTERACTIONS

• Plant proteinases are induced by wounding as part
  of the wounding response - but their role(s) are
  largely uncharacterised (protein turnover?)

Identified in cell culture. Other
miscellaneous genes of unknown roles are also
activated.From Ryan, C.A., BBA 1477 112-121
(2001)
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OTHER ROLES FOR PROTEINASES IN PLANT-INSECT
INTERACTIONS

• The wounding response is triggered by proteolysis
  of precursors of plant peptide hormones these
  are characterised in tomato/potato/pepper/ black
  nightshade and in tobacco
• Proteolysis does not occur at paired basic
  residues as in many animal prohormones

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OTHER ROLES FOR PROTEINASES IN PLANT-INSECT
INTERACTIONS

• Arabidopsis thaliana has 57 genes encoding serine
  proteinases of the subtilisin type - more than
  the number of genes coding for other types of
  proteinase. (See Prof. Thomas Altmanns web site
  http//www.bio.uni-frankfurt.de/botanik/mcb/AFGN/a
  ltmann.htm.) These proteinases are present in
  other plants also (cucumisins). Their roles are
  largely uncharacterised.
• Proteinases of this type are involved in
  proprotein processing in other kingdoms
  (proprotein convertases)
• It has been suggested that these proteinases are
  involved in regulating developmental processes
  via processing of (uncharacterised) peptide
  hormones
• Endogenous plant proteinasesare responsible for
  cleaving prosystemin and initiating the
  signalling process in the wounding response,
  since mechanical damage can initiate the response
• Is this proteolysis carried out by specific
  subtilisin-type proteinases, or by non-specific
  proteolysis as a result of damage to cells?
• Prosystemin is degraded by proteinases in
  apoplastic fluid, or vacuoles, but systemin is
  not produced under these conditions - suggests
  specific cleavage.

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PROTEINASES IN PLANT-INSECT INTERACTIONS

• With thanks to
• Co-workers in Durham
• Hillary Wilkinson (technical support)
• James McGregor(Diabrotica cDNAs)
• Xavier Foissac
• Jin-Ping Du(rice brown planthopper cDNAs)
• Martin Edwards
• John Gilroy(protein sequencing)

• Collaborators
• Maarten Jongsma (Wageningen)
• Francesc X. Aviles(Barcelona)
• Angharad Gatehouse(Newcastle)
• Funding
• European Union (FAIR)
• Syngenta plc
• BBSRC