CYCLOTIDES (Craik) The name cyclotides was introduced for ...

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CYCLOTIDES (Craik)

The name cyclotides

was introduced

for ribosomally
-
synthesized peptides from plants that are
characterized by a head
-
to
-
tail cyclic peptide backbone and a cystine knot arrangement of three
conserved disulfide bonds
1
.

They were originally discovered in plants of the Rubiaceae (c
offee)
and Violaceae (vi
olet) families but have since

been found i
n the Cucurbitaceae

and Fabaceae
(legume) families. They are expressed in many plant tissues, including leaves, stems, flowers and
roots. Dozens to hundreds of different cyclotides are

expre
ssed

in an individual plant

and there
appears to be

little
crossovers of cyclotides between different
species
, i.e., most plants have

a
unique set of cyclotides
.

Figure 1 shows the structure of the prototypic cyclotide
,

kalata B1, from the African herb
Oldenlandia affinis

(Rubiaceae).
2

It comprises 29 amino acids, including the six conserved Cys
residues that form the signature cyclic cystine knot (CCK) motif of the family. The backbone
segments between successive Cys residues are referred to as loops and the sequence variations of
cycl
otides occur within these loops.

Cyclotides have been classified into two main subfamilies,
Möbius or bracelet, based on the presence or absence of a cis X
-
Pro peptide bond in loop 5 of the
sequence but there is

also
a smaller subfamily that is referred to

as the trypsin inhibitor
subfamily. This third subfamily has high sequence homology to some members of the knottin
family of proteins from
squash

plants and
its members
are also referred to as cyclic knotti
ns
3
.

So far more than 200
sequences

of cyclotides have been reported and
they
are documented in a
database dedicated to circular
proteins called CyBa
se (
www.cybase.org.au
).
4

They range in size
from 28
-
37 amino acids and are typically not highly charged peptides.
Aside from their disulfide
bonds
5

and head
-
to
-
tail cyclised backbone, there appear to be no other post
-
translational
modifications in cyclotides.

Cyclotides appear to be ubiquitous in

Violaceae

family plants but
are more
sparsely

distributed

in the
Rubiaceae
, occurring in about 5% of
the hundreds of
plants
screened so far.

The reports of cyclotides in other plant families are more recent and there is
limited information available on the distribution of cyclotides in these families.

Nevertheless, it
appears that cyclot
ides
are

a very large family of plant proteins
, potential
ly numbering in the tens
of thousands
.
6

Cyclotides are gene
-
encoded peptides that are processed from
larger
precursor proteins.

The
precursors of Rubiaceae and Violaceae cyclotid
es are dedicated proteins, whose purpose appears
to be only to produce cyclotides. By contrast, cyclotides in
Clitoria ternatea
, a member of the
Fabaceae family
,

are produced from a chimeric precursor protein that also encodes an albumin
,
7
,
8

a situation similar to that recently

reported for

a small cyclic peptide trypsin inhibitor from
sunflower seeds.
9

Thus, there appear to be multiple
types of precursors leading

to circular
proteins

in plants
.


In the case of Rubiaceae and Violaceae family plants the leader

sequence

is
considered to comprise a pro
-
region and an N
-
terminal region (NTR) that is

repeated in some
genes along with the

adjacent core peptide region, as illustrated in Figure 1.


Figure x
. Structu
re of kalata B1 and cyclotide

precursor protein
s
.

A schematic
representation of three
precursor proteins

from
O.

affinis

is shown at the top of the figure. The
proteins

comprise a
n endoplasmic reticulum

signal sequence labeled ER, a
leader sequence
(comprising a pro
-
region and

a conserved repeated fragmen
t labeled NTR
)

and either one or
multiple copies of the
core

peptides; for example,
kalata
B1,
B2
,

B3 and B6. A s
hort
hydrophobic C
-
terminal recognition sequence

is
present in each precursor
.
At the bottom of the
figure t
he amino acid sequence of kalata B1

is shown, with the cysteine residues labeled with
Roman numerals. The cleavage sites
for

excision of a
core cyclotide

domain with an N
-
terminal
Gly and
a
C
-
terminal Asn, which
may be

subsequently linked by an asparaginyl endopeptidase,
are indicated by arrows.
The location of the ligation point which forms loop 6 of the mature
cyclic peptide is shown on the right. Parts of the precursor protein flanking the mature domain
are shown in
lighter shading. Figure adapted from Daly et al.
10


Biological activities of cyclotides

Cyclotides

are thought to be plant def
ence molecules, given their potent
insecticidal

activity
against

Helicoverpa

species
11
-
14
. However,

they also have a broad range of other
biological

activities, including anti
-
HIV
15
,
antimicrobial
16
,
cytotoxic
17
,
molluscicidal
18
, anti
-
barnacle
19
,
nematocidal
20
,
21

and haemolytic
activities
22
.

Some of these activities are of potential
pharmaceutical interest and because of their exceptional stability cyclotides have also attracted
attention as potential protein engineering or drug design templates
.
23
,
24

The

diverse range of activities
of cyclotides
seems to have a common mechanism that involves
binding to and disruption of biological membranes.
25

Electron
micrographs

of
Helicoverpa

larvae fed a diet containing cyclotide
s

at
a
similar concentration to that whi
ch occurs naturally in
plants for example
show

marked swelling and blebbing of mid
-
gut cells
11
.
Larvae that have
ingested cyclotides are markedly stunted in their growth and development, presumably as a
result of this disr
uption of
their
mid
-
gut membrane
s
.
A range of biophysical studies
26
-
28

have

confi
rmed m
embrane interactions for

both Möbius and bracelet cyclotides
,

and detailed
information is available on the residues involved in making contact
with membrane surfaces.
Furthermore,
electrophysiological and vesicle
leakage

studies have confirmed the
leakage

of
marker
molecules
through

membrane
s

treated with cyclotide
s
.

It appears that cyclotides are able
to self associate in th
e membrane
environment and form

large pores in membranes.


Cyclotide biosynthesis

As indicated in Figure x, c
yclotides are derived from precursor proteins that encode one or more
copies of the core (cyclotide)

peptide sequence
.

For example the
Oak1

(
O
ldenlandia
a
ffinis

k
alata B
1

gene) encodes an 11 kDa precursor
protein
that contains an
endoplasmic reticulum
(
ER
)

signal, leader peptide, kalata B1
core peptide
, and a C
-
terminal peptide region,

whereas the
Oak4

gene, encodes a precursor containing three copies of ka
lata B2
13
.

The single or multiple
copies of the cyclotide domain
s

are flanked
in ea
ch case
by N
-
terminal and C
-
terminal
recognition

sequences that are thought to be implicated in the processing reactions.

Because

of the presence of the ER signal it is thought that cyclotide
precursor
s are

probably
folded

in the ER prior to processing (excision and cyclization) of the cyclotide domain. Protein
disulfide isomerases are o
f
ten
involved

in t
he folding of disulfide
-
rich

proteins
29

and
in vitro

experiments

have

shown increased yiel
d
s of
folded

cyclotide
kalata B1
in the presence of PDI,
although so far there h
ave been no
definitive

studies

on the
involvement

of PDI for

in planta

cyclotide
folding
. A r
ec
ent st
u
dy has sh
o
wn that
cyclotides

are targeted to vacuoles in plant cells
and this is where the
excision and cyclization processes are

thought

to occur
30
.

The N
-
terminal repeat (NTR) region
of cyclotide precursors
has been
found

to adopt an α
-
helical
structure
as an
isolated

synthetic peptide,
31

but the significance, if any, of this is not yet known.
It has been proposed that it might
represent

a
recognition

sequence for cyclotide folding
by PDI,
but interestingly cyclotide precursors from
the

Fabaceae

plant
C.
ternatea

lack the NTR region
seem in Violaceae or Rubiaceae precursors. The
nature of the processing reaction
(s)

occurring at
the N
-
terminal end of the cyclotide domain is

still

essentially unknown.

However, t
here is strong evidence that asparaginyl endopr
otease (AEP) activity is responsible for
processing at the C
-
terminal end of
the

cyclotide

domain

including involvement

in

the
cyclization

process.
32
,
33

With just one exception (circulin F) all cyclotides have an Asn or Asp
residue as the C
-
terminal end of the core peptide
,

and

an AEP is principle able to cleave after this
residue. It has been proposed that
this

cleavage reaction occurs contemporaneously with
ligation
to the earlier
-
released

N
-
terminus

of the core
peptide region. Mutage
nesis
experiments
in
transgenic tobacco and

Arabidopsis

plants have shown a

vital

role for

the N
-
terminal Asn
residue
,

as well as other key fl
an
king
residues
.
32

Similarly
,

gene
silencing

experiments

in which
AEP
activity

is

knocked
down

show decreased production
of cyclic

peptide in transgenic
plants.
33

Overall, the processing of cyclotides can be
summarized

as involving excision and
cyclization

from a pre
-
folded precursor
.


Specific recommendations for the family

At present there is no common gene nomenclature for
cyclotide

precursor
s

and
given the limited
knowledge of the full complement of associated

biosynthetic genes encoding proteins involved
processing it see
ms

premature to propose one. A naming scheme for the mature
cyclic
peptides
that involves a fo
ur letter pronounceable acronym

based on the Latin name of the plant species in
which the cyclotid
e was first discovered has been proposed
34

but has not been uniformly
fol
lowed so far
.



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