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'#' Dissertation for the degree of Doctor of Philosophy (Faculty of Pharmacy) in
Pharmacognosy presented at Uppsala University in 2002
ABSTRACT
Göransson, U. 2002. Macrocyclic Polypeptides from Plants. Acta Universitatis
Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of
Pharmacy 270. 57 pp. Uppsala. ISBN 91-554-5279-5.
The aim of this work was to explore the structural and functional diversity of polypeptides
that are found in plants. Expanding knowledge of simililarities between plant use of these
compound and animal use promises exceptional opportunities for finding, from plant
research, new structures with biomedical and biotechnological potential.
A fractionation protocol was developed and applied to many plant species, providing
fractions enriched in polypeptides, amenable to chemical and biological evaluation. From
one species, the common field pansy (Viola arvensis), a 29-amino-acid residue polypeptide
was isolated, named varv A, which revealed a remarkable macrocyclic structure (i.e., N- and
C-termini are joined) stabilised by three knotted disulfides.
Varv A, together with an increasing number of homologous peptides, form the
currently known peptide family of cyclotides. Their stable structure makes them an
attractive scaffold for protein engineering. In addition, they display a wide range of
biological activities (e.g., antimicrobial, cytotoxic, and insecticidal). As a part of this
work, the cytotoxic effects of varv A and two other isolated cyclotides were evaluated
in a human cell-line panel: all were active in the low µM range. Most likely, these
effects involve pore formation through cell membranes.
Cyclotides were found to be common in the plant family Violaceae; with eleven
cyclotides isolated and sequenced from V. arvensis, V. cotyledon, and Hybanthus
parviflorus. For six members of the genus Viola, cyclotide expression profiles were
examined by liquid chromatography-mass spectrometry (LC-MS): all expressed
notably complex mixtures, with single species containing more than 50 cyclotides.
These profiles reflect the evolution of the genus.
To assess these mixtures, a rational strategy for MS based amino acid sequencing
of cyclotides was developed, circumventing inherent structural problems, such as low
content of positively charged amino acids and the macrocyclic structure. This was
achieved by aminoethylation of cysteines, which, following tryptic digestion, produced
fragments of size and charge amenable to MS analysis. This method was also modified
and used for mapping of disulfide bonds.
Methods for isolation and characterisation developed in this work may prove
useful not only for further studies on macrocyclic polypeptides from plants, but also
for other plant peptides and disulfide-rich peptides from animals.
Ulf Göransson, Division of Pharmacognosy, Department of Medicinal Chemistry,
Biomedical Centre, Uppsala University, Box 574, S-751 23 Uppsala, Sweden
© Ulf Göransson 2002
ISSN 0282-7484
ISBN 91-554-5279-5
Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2002
Acti labores jucundi
Utfört arbete är angenämnt.
Marcus Tullius Cicero
106–43 BC
To my family
3
PAPERS DISCUSSED
This thesis is based on the following papers, which are referred to in the
text by their Roman numerals (I-V).
I
Per Claeson, Ulf Göransson, Senia Johansson, Teus Luijendijk,
Lars Bohlin. Fractionation protocol for the isolation of
polypeptides from plant biomass. Journal of Natural Products,
Vol. 61, pp. 77-81 (1998)
II
Ulf Göransson, Teus Luijendijk, Senia Johansson, Lars Bohlin,
Per Claeson. Seven novel macrocyclic polypeptides from Viola
arvensis. Journal of Natural Products, Vol. 62, pp. 283-286
(1999)
III
Adriana M. Broussalis*, Ulf Göransson*, Jorge D. Coussio,
Graciela Ferraro, Virgina Martino, Per Claeson. First cyclotide
from Hybanthus (Violaceae). Phytochemistry, Vol 58, pp. 47-51
(2001)
IV
Petra Lindholm*, Ulf Göransson*, Senia Johansson, Per Claeson,
Joachim Gullbo, Rolf Larsson, Lars Bohlin and Anders
Backlund. Cyclotides - a novel type of cytotoxic agents.
Molecular Cancer Therapeutics Accepted
V
Ulf Göransson, Adriana M. Broussalis, Per Claeson. Peptide
profiling by LC-MS and MS sequencing of intercysteine loops:
Expression profiles of Viola cyclotides. Submitted
* Contributed equally to this work
4
CONTENTS
ABBREVIATIONS
1. INTRODUCTION
The cyclotides
The aim of present study
2.
PLANT PEPTIDE ISOLATION
A fractionation protocol
Isolation of varv A
Aiming for cyclotides
Adsorption chromatography on Sephadex LH-20
Butanol partition
Cation exchange chromatography
The protocol revised
Assessing cyclotide expression profiles
3. CHARACTERISATION OF CYCLOTIDES
Proof of the cyclic nature of the varv peptides
Sequence determination
MS sequencing of intercysteine loops
Chemical determination of disulfide bridges
The disulfide unfolding pathway of varv A
On the effects of cyclotides
4. DISCUSSION
Mass spectrometry in cyclotide analysis
Cyclotide sequence and structures
About the biosynthesis of cyclotides
Past and present use of cyclotides and cyclotide
bearing plants
7
8
10
11
11
12
13
14
16
16
17
17
20
20
21
23
25
28
28
31
31
34
41
43
5. CONCLUDING REMARKS
45
6. ACKNOWLEDGEMENTS
47
7. REFERENCES
49
APPENDICES
56
Amino acid abbrevations and Peptide MS fragmentation
Novascreen assays
5
ABBREVIATIONS
AcN
BuOH
CAM
CH2Cl2
ESI
EtOH
HPLC
IAM
IEC
iPrOH
LC
MALDI
MeOH
MS
NMR
RP
SCX
SEC
SPE
TCEP
TFA
TLC
TOF
6
acetonitrile
n-butanol
carbamidomethyl
dichloromethane
electrospray ionisation
ethanol
high performance liquid chromatography
iodoacetamide
ion exchange chromatography
isopropanol
liquid chromatography
matrix assisted laser desorption/ionisation
methanol
mass spectrometry
nuclear magnetic resonance
reversed phase
strong cation exchange
size exclusion chromatography
solid phase extraction
tris-(2-carboxyethyl)-phosphine
trifluoroacetic acid
thin layer chromatography
time of flight
1. INTRODUCTION
“When you find one, it's unusual, but when you find another,
it suggests there might be a lot more out there.”
C. Ryan, the discoverer of systemin, about plant peptides in Science, 1996 (1)
I
n tomato plants, a peptide triggers the defense against herbivores. This
peptide, named systemin, is transported throughout the plant via the
phloem, as a systemic wound signal. It binds to a receptor on the cell
surface, starting a cascade of events, ending with the conversion of linolenic
acid into jasmonic acid, a potent activator of defense gene transcription (2,
3).
In mammals, when stretch receptors in the right atrium detect changes
in blood volume, the cardiac hormone atrial natriuretic peptide (ANP) is
released to decrease the heart load. Injecting the same peptide in plants,
induces an analog effect: stomata are opened to regulate the osmotic
pressure of the plant cell (4, 5).
These are two examples of something that plants were considered
incapable of, until just recently—using peptides as transmitter substances.
The rigid plant cell wall was thought to effectively hinder such large
molecules as peptides and proteins. Then, with the discovery of systemin,
evidence was put forward that fundamentally revised this opinion, opening
up a whole new research area. Today, we are just beginning to explore this
fascinating field of science, and can anticipate an increasing number of
biologically active plant peptides to appear (6). The examples above also
illustrate functional similarities between peptides within plants and animals,
that is, the uses and roles of peptides within these two kingdoms. For
example, the pathway for systemin in plants parallels directly the animal
pathway of inflammation for the tumour necrosis factor-α; and in animals,
the counterpart of jasmonic acid is the family of oxygenated unsaturated
cyclic fatty acids called prostaglandins (2).
The occurrence of peptides in plants, however, has been known for a
long time. Pioneer work in this field includes Samuelssons isolation of toxins
from mistletoes during the 1950s (7, 8). These peptides are today regarded
7
as members of the thionin family, which together with the defensins are
known to be involved in host defense of the plant. These peptides also have
their equivalents in animals (9), with the innate immune system relying on
antimicrobial peptides (10, 11).
These and other functional analogies between plants and animals may
well be upheld, in the plant kingdom, by structurally diverse peptides and
proteins, suggesting possibilities for finding new chemical structures from
plants with potential in biomedical and biotechnological research (12). One
recently described family of plant peptides that fulfills these characteristics
is that of the cyclotides – cyclo- peptides (13, 14)— for they have a unique
structure, combined with potent bioactivities. These macrocyclic
polypeptides are the subject of this thesis.
THE CYCLOTIDES
The intriguing beginning of the cyclotide story was recently reviewed by its
leading protagonist, the Norwegian physician Lorents Gran (15). During his
service as a Red Cross physician in Zaire during the 1960s, he observed that
the native women used a decoction of the plant Kalata-Kalata (Oldenlandia
affinis DC., Rubiaceae) to accelerate childbirth (Figure 1). The effect was
drastic; extremely strong uterine contractions were induced.
Figure 1. Native use of cyclotides, as
illustrated on the cover of Lorents Gran's
thesis (1973): The uteroactive principles of
“Kalata-Kalata” (Tsjiluba language) (i.e.,
Oldenlandia affinis DC). The first report of
this use of the plant was made, however,
in The Central African Republic. There the
plant was known as “Wetegere” (Gbaya
language) (16).
Reprinted with permission of the author.
Since the constituents causing the effect on the uterus were unknown, a
project aimed at finding them was started; and the prototypic cyclotide
kalata B1 was isolated (17), guided by uterine activity. The exceptional
stability of this peptide was, by then, already observed, along with the fact
that its termini were blocked. Whether this could be explained by a cyclic
structure was being speculated, but having to rely on enzymatic tests only,
and lacking the possibilities of today’s techniques, the conclusion was that
no end-to-tail structure was possible (18). Thus, over 20 years later, when
the three-dimensional structure of kalata B1 was determined, the cyclic
8
structure of the cyclotides, knotted by three disulfides, was first established
(19).
In 1993 and 1994, the isolation of four additional cyclotides were
reported by three from each other independent groups, all guided by
different biological activities. This included the haemolytic violapeptide I
(20), the HIV inhibitory circulins A and B (21), and the neurotensin
binding inhibitor cyclopsychotride A (22). This was the initial context and
setting for my research and thesis.
Today more than 40 cyclotides have been described; yet their
occurrence is still confined to only three plant families: Rubiaceae,
Violaceae, and lately, Cucurbitaceae (Table 1). Of these, Violaceae is
striking in that cyclotides seem to be present in all genera and species,
hitherto examined. In this family, a single species can include a large
number of different cyclotides, as holds for some of the studied species
belonging to the family Rubiaceae; but the current number of known
cyclotide-bearing species in this latter family are few. For the third family,
Cucurbitaceae, only two known cyclotides are reported. With their sizes
ranging from 28 to 37 amino acids, the cyclotides are the largest
macrocyclic peptides known today.
Table 1. The known taxonomic distribution of cyclotides.
Family
Taxa
Violaceae
Hybanthus parviflorus Baill.
Leonia cymosa Mart.
Viola cotyledon Ging.
V. arvensis Murr.
V. odorata Linn.
V. hederacea Labill.
V. riviniana Reichb.
V. biflora Linn.
V. tricolor Linn.
Reference
Paper III
(23)
Paper IV
Paper I and II (20)
(13)
(13)
Paper IV
Paper IV
Paper IV
Rubiaceae
Chassalia parvifolia Schum.
Oldenlandia affinis DC.
Palicourea condensata Standl.
Psychotria longipes Muell. Arg.
(21, 24)
(13, 17, 18, 25, 26)
(27)
(22)
Cucurbitaceae
Momordica chinensis Hort.
(28)
Throughout the peptide family of cyclotides, the number and positions of
cysteine residues are strictly conserved. Sequences between cysteines are
made up of variable loops that are presented at the surface of the molecule.
These structural features, together with additional activities that have been
attributed to cyclotides, including cytotoxic (IV) (29), antimicrobial (29),
insecticidal (26), HIV-inhibitory (21, 23, 24, 27), and trypsin inhibitory
(28), have made them interesting as a starting point material for molecular
engineering (30). The very stable and compact cyclotide molecule could
9
then, by means of such engineering, serve as a scaffold to present, to a
biological target molecule, otherwise labile peptide sequences in the
intercysteine sequence loops.
AIM OF THE PRESENT STUDY
This work has been conducted within a research programme at the Division
of Pharmacognosy, Uppsala University, with the overall aim of identifying
peptides of plant origin having novel chemical structures and biological
profiles, and having a potential for drug development, or use as
pharmacological tools. The idea has also been that such peptides could be
used for mapping occurrences of peptides in biologically diverse plant
genera, providing valuable markers for chemotaxonomic classification of
plants.
The following have been specific aims of my research and this thesis:
• to develop and establish a fractionation protocol for isolation of
polypeptides from plant biomass
• to further refine methods for separating cyclotides, and to apply
these methods to isolation and identification of additional members
of this novel peptide family
• to further characterise, chemically and biologically, the nature of this
particular family of plant peptides
• to develop methods for rapid analysis of their occurrence and
structure
10
2. PLANT PEPTIDE ISOLATION
Figure 2. Isolation of varv A from Fraction
P of the aerial parts of Viola arvensis by RPHPLC. (For details see I.)
Peptide isolation from plant biomass was at the start of the project, and still
is, an undeveloped field compared to isolation of low molecular substances
from plants, or peptides from animal sources. To address some major
problems encountered when dealing with plant materials, such as removal
of chlorophyll, polyphenols, and low molecular compounds omnipresent in
plants, a fractionation protocol was developed (I). The fractions produced
were amenable to chemical and biological evaluation of polypeptide
content, illustrated by the isolation of the cyclotide varv A (Figure 2).
(Polypeptide are defined here as peptides containing 10 to 50 amino acids.)
The naming of these peptides has varied considerably. We proposed
(III) that the trivial name be constructed as an indicative and pronounceable
acronym of the Latin binomial of the plant from which the cyclotide was
first isolated, followed by a letter indicating the order of appearance. This
principle has been used for all reported cyclotides from our group;
accordingly, the first peptide from Viola arvensis was named varv A.
A FRACTIONATION PROTOCOL
The fractionation protocol starts with a small amount (4g) of dried and
ground plant material. For natural product chemistry, a relatively small scale
was chosen, allowing increase of throughput for this first screening of
possible plant peptides. The way the individual steps were conducted was
also adapted to this, when possible. Chlorophyll and lipophilic substances
were then removed in a pre-extraction with dichloromethane, and after
drying of the plant material, the main extraction was done with 50%
aqueous ethanol. Procedures for these extractions were similar, with the
plant material in a soxhlet extraction thimble put in a flask containing the
solvent on a shaking table. Consecutive extractions (1 h each) with fresh
solvent, four times with dichloromethane and three times with ethanol,
11
were found to yield more than 90% of that obtained by exhaustive
extraction (continuous Soxhlet extraction for 8 h).
To remove tannins, ubiquitous in plants and known to interfere with
both chromatography and bioassays, the ethanol extract was filtrated
through polyamide (31, 32). Prior to the filtration, the ethanol was removed
in vacuo, and the aqueous remains acidified with acetic acid to a final
concentration of 2% to promote hydrogen bonding of tannins to the gel
(33). Peptides were then eluted using acetic acid (2%) followed by ethanol
(50%)/acetic acid (2%). The latter eluted possibly insoluble peptides, in 2%
acetic acid alone.
The tannin-free extract was then subjected to size-exclusion
chromatography (SEC) on a calibrated column packed with Sephadex G10. On this column, compounds with a molecular weight above
approximately 700 Da were eluted in the void volume. Then, mainly to
remove NaCl used in the especially designed buffer in the preceeding SEC,
but also to remove polysaccharides that were detected by NMR and TLC to
occur in the fraction, the collected void volume was subjected to solid phase
extraction (SPE) on reversed phase (RP) material. The thus desalted
fraction, named Fraction P, was directly amenable, after freeze-drying, to
analysis and/or biological testing.
Isolation of varv A
Applicability of this initial fractionation protocol was proven by the
isolation from V. arvensis of varv A, a macrocylic peptide, 29 amino-acidresidues long, containing three disulfides. This was one of the first examples
of the macrocyclic peptides later to be known as cyclotides. The presence of
varv A in Fraction P of V. arvensis was confirmed by several of the analytical
methods available at that time, including analytical diode array HPLC and
chemical reaction of amides after staining with o-tolidine on TLC, and also
by the release of free amino acids after acidic hydrolysis of the fraction.
Moreover, the presence of about 3 kDa substances was shown by SEC
(Superdex Peptide HR 10/30, Amersham Biosciences, Uppsala, Sweden)
and later confirmed by MS analysis.
The isolation of varv A was done at a larger scale than the initial
screening, starting from 130 g of plant material compared to the
standardised amount (in the protocol) of 4 g. The fractionation protocol was
equivalently scaled up; and the Fraction P, thus obtained, was subjected to
preparative RP-HPLC (Figure 2). After acidic hydrolysis of the pure
compound, molar ratios of amino acids were determined by automated
quantitative amino acid analysis.
The sequence of varv A, determined then by automated Edman
degradation, was highly homologous to kalata B1 (19) as well as to viola
peptide 1 (20), previously isolated from V. arvensis (Figure 3).
12
Among the polypeptides in V. arvensis, detected by HPLC and MS, varv
A was dominant.
Varv A
Kalata B1
Violapeptide 1
cyclo-(GETCVGGTCNTPGCSCSWPVCTRNGLPVC)
cyclo-(GETCVGGTCNTPGCTCSWPVCTRNGLPVC)
cyclo-(GETCVGGTCNTPGCSCSRPVCTXNGLPVC)
Figure 3. Sequence alignment of three almost identical macrocyclic polypeptides. Varv A differs
from kalata B1 only by an S/T substitution (marked in bold). The reported sequence of
violapeptide 1 contains a W/R substitution and an undetermined amino acid X (in italics). The
placing of R was based on sequencing of a tryptic fragment PVC (underlined) (20). This sequence is
repeated after the chymotryptic cleavage site L. The occurrence of this fragment may therefore be
the result of digestion with non TPCK treated trypsin, placing then the R on the wrong position. W
was undetected, but because it is known to hydrolyse during quantitative amino acid analysis,
violapeptide 1 might in fact be identical with varv A. (16)
Note that the starting point when writing the sequences of these macrocyclic peptides is
arbitrary; in this thesis they are generally written like above, according to the numbering of the
loops outlined in (13) and (V).
AIMING FOR CYCLOTIDES
With sequence homologies supporting the fact that cyclotides with
interesting structures and effects could be found in the Fraction P from V.
arvensis, there was a demand for more efficient means of isolating them.
The first method of choice was gradient RP-HPLC, in which the cyclotides
are characterised by eluting in a narrow window at a relatively high
percentage of organic modifier (Figure 4), due to their lipophilic surface.
This method was also superior in resolution: cyclotides differing in only a
Ser/Thr substitution (i.e., one additional methyl group) were easily
separated (e.g., varv D and E, Figure 6). On the other hand, peptides
showing less homologous sequences could coelute in the same peak (e.g.,
varv A and F, Figure 6). This problem could partially be solved by repetitive
chromatography in different solvent systems, that is, by changing to a more
shallow gradient and/or by including another organic modifier (e.g.,
isopropanol).
Although such repetitive chromatography was successful, purification is
normally more rationally approached, using a combination of
chromatographic techniques, which together exploit different properties of
the analyte. Nevertheless, with RP-HPLC used for final purification, several
methods were adapted for cyclotide capture and separation, as described
below.
13
36% AcN
35% AcN
34% AcN
33% AcN
Figure 4. Isocratic separations on RP-HPLC of a cyclotide-containing fraction from V. tricolor.
Slightly decreasing the concentration of organic modifier (AcN) enabled the separation of two
peptides from a peak initially appearing as one single compound. This drastic change in behaviour is
the result of the very narrow window of the adsorption/desorption process of peptides and
proteins on RP (34).
Adsorption chromatography on Sephadex LH-20
Seven additional cyclotides, designated as varv B-H, were isolated from the
Fraction P of V. arvensis, aided by an initial group separation on Sephadex
LH-20 (Figure 5), followed by RP-HPLC (II). This gel was originally
designed for gel filtration in polar organic solvents and aqueous solvent
mixtures, but adsorption has also been shown to be an additive separation
mechanism (35). In analogy with RP-chromatography, the adsorption is
suppressed with decreasing polarity of the solvents.
1
A206
2
0
14
10
20
T (hours)
Figure 5. Fraction P of V. arvensis was
split up in two cyclotide-containing
fractions, marked 1 and 2 [see (II) and
below] on Sephadex LH-20 (mobile
phase 30% MeOH/0.05% TFA).
Contrary to true size-exclusion
chromatography, in which an exclusion
limit of approximately 4 kDa could be
expected (35), the peptides eluted
after the low-molecular-weight range
of the column. Increasing the polarity
of the eluent by adding water further
increased the retention times of the
peptides, indicating that the separation
took place in a RP-like manner.
Sephadex LH-20 has been used in previous isolations of cyclotides (21, 23,
36). As the solvent, 100% methanol was used, which eluted the peptides in
the void volume of the columns; which means that the peptides were eluted
in true gel filtration mode. However, by adding water to the solvent, delay in
elution of the varv peptides was possible; and at 30% aqueous methanol,
they eluted after the total volume of the packed column bed, thus indicating
that the adsorption mechanism was predominant (Figure 5 and II).
Interaction between the dextran gel matrix and aromatic solutes has
been a proposed cause of the adsorption (37), and has been used extensively
for the separation of low-molecular-weight substances (35, 38). Results
from separation of the varv peptides indicate a similar behaviour for
polypeptides. Cyclotides containing one aromatic amino acid in addition to
the conserved tryptophan were also more strongly retarded (eluted in peak
2, Figure 5).
The isolation of individual varv peptides was then facilitated not only
by the removal of non-peptidic substances, but also by the separation of the
peptide mixture into two major groups (Figure 5 and 6). Thus, this
inclusion of chromatography on LH-20 simplified significantly the following
final separation on RP-HPLC, on C18-material, and also improved the final
yield of varv A by five-fold (II) [1750 µg from 100 mg of Fraction P,
compared to the previously described 150 µg from 42 mg (I)].
Figure 6. Analytical RP-HPLC of the Fraction
P before (A) and after separation on
Sephadex LH-20 (B and C). The
chromatogram of peak 1 in Figure 5 is shown
in (B), and the chromatogram of peak 2 is
shown in (C). Individual varv peptides are
marked by their letters. The difference in
selectivity of reversed-phase adsorption
chromatography on Sephadex LH-20 and
silica based C18 material is clearly
demonstrated.
Peaks overlapping each other (varv A and F)
in preparative HPLC on C18 material were
separated on Sephadex LH-20. Final
purification of the peptides was achieved by
rechromatography on RP-HPLC. The
analytical RP-HPLC was performed on a
Rainin Dynamax C18 column (250x4.6 mm, 5
µm, 300 Å) operated with a linear gradient
from 37 to 43% organic modifier
(AcN/iPrOH, 3/2) in 0.1% TFA.
The main part of the injection peak seen in
(A) corresponds to the substances eluting
before the low-molecular-weight region of
the Sephadex LH-20 column.
15
Butanol partition
The very lipophilic nature of the cyclotides was also exploited by the use of
a simple solvent-solvent partitioning between water and butanol. This,
during the isolation of the cyclotide hypa A from Hybanthus parviflorus
(III), replaced two steps in the original protocol, gel filtration on Sephadex
G-10 and solid phase extraction on RP material.
The cyclotide containing butanol fraction was then directly amenable to
reversed phase chromatography (on open columns, as in solid phase
extraction, or by HPLC) or to ion exchange chromatography, as described
below.
Cation exchange chromatography
While the above separation methods are based on hydrophobic interactions,
strong cation exchange (SCX) chromatography was successfully used in the
isolation of more basic cyclotides from V. odorata (e.g. cycloviolacin O2, see
IV). For preparative purposes, the dried butanolic extract, redissolved in
25% acetonitrile in 0.1% aqueous TFA, was pumped through a polymeric
Vydac SCX column (polystyrene-divinylbenzene beads with sulphonic acid
as exchange groups) until the binding sites of the gel were saturated. The
addition of acetonitrile in the mobile phase promotes the ionic interactions
and helps in solubilising the lipophilic peptides. Bound substances were
then eluted in a salt gradient, ranging up to 1M NaCl (Figure 7).
This combination of butanol partitioning and SCX proved to be very
powerful; polar substances were removed in the butanol partioning, thus
leaving very few unwanted charged substances in the subsequent SCX
capture.
Figure 7. Capture of
cyclotides by strong cation
exchange chromatography.
After elution of non charged
substances, the bound
peptides, here marked *, were
eluted in a NaCl gradient (0-1
M). A solvent composition of
25% AcN, 0.1% TFA in water
was used to promote the ionic
interactions.
16
THE PROTOCOL REVISED
With increasing experience of plant polypeptide fractionation in general,
and separation of cyclotides in particular, strategies directly targeting the
cyclotides could be adapted. Moreover, by exploiting subtle differences in
amino acid sequences within the peptide-family, efficient chromatographic
methods were developed for isolation of individual cyclotides. These
advances are summarised in the figure below (Figure 8).
Figure 8. Isolation schemes of described approaches for plant peptide isolation. The initial
extraction steps have remained the same, but the initial protocol for isolation of Fraction P have
been modified to simplify cyclotide isolation. To the left, the original protocol to produce Fraction P
is outlined, including the LH-20 and RP-HPLC chromatography that was used for isolation of the
varv peptides (I, II). In the middle, the route for capture by ion exchange chromatography is shown.
This was used for isolation of cycloviolacin O2 from V. odorata (IV). The BuOH extract is also
amenable to direct RP-chromatography, which was used in the isolation of hypa A (III). To assess
the cyclotide expression profiles by LC-MS (V), the BuOH-extract was passed through RP-SPE
before analysis (outlined to the right).
ASSESSING CYCLOTIDE EXPRESSION PROFILES
The isolation of the varv peptides showed that multiple cyclotides are
expressed in a single species. The same was also reported by other groups, as
for example, the report of 12 sequences from V. odorata (13) and 7
additional kalata peptides by Craik and co-workers (13, 26), and 6 circulins
by Gustafsson and co-workers (21, 24). Taking this a step further, a way of
profiling the whole expression was developed, exploiting the powerful
combination RP-HPLC and MS (V).
17
By such profiling, six species from the genus Viola were analysed,
representing four taxonomic groups recognised within the genus (39). In all
of them, complex peptide mixtures were revealed (Figure 9). These
analyses gave an estimation of the number of naturally occurring cyclotides
expressed in a single species; for V. arvensis, this was more than 50.
In addition, the analyses highlighted similarities and differences between
species in cyclotide expression. For example, for the two species with the
highest number of cyclotides reported, V. arvensis and V. odorata, the
expression profiles are more similar than previously recognized. From V.
odorata, the majority of cyclotides reported carried a positive net charge
(13), while those from V. arvensis had a neutral or slightly negative charge
(I, II). Of the reported sequences, only one has previously been shown to
occur in both species (i.e., cycloviolacin O12 is identical to varv E). By LCMS though, more positively charged cyclotides were shown to be present,
and also in V. arvensis. Moreover, peptides originally described from V.
arvensis were also detected in V. odorata (Figure 9, parts C and F).
Apart from these two species, four additional ones were examined: V.
tricolor, V. biflora, V. riviniana, and V. cotyledon, none hitherto analysed for
cyclotide content. Including these in the comparison, the expression profiles
seemed to correspond to the identified sections of the genus Viola, thus
indicating a relevance and possible use of this kind of data in the study of
the evolution of cyclotide containing plants. Conversely, the potential
relevance of systematics and evolution in the search for novel cyclotides and
cyclotide containing plants is made clear.
Figure 9. Basepeak chromatograms (m/z 1400-1640) of six Viola species from four infrageneric
sections, arranged after their systematic distance, with the most primitive species at the top (39).
All peaks in the figure show masses in the same range as previously reported cyclotides. In addition,
masses of some peaks are shown to illustrate the following: the dominance of vico A and B [3271
and 3237, respectively, in (A)]; the advantage of the combination LC and MS in resolving peaks with
identical masses [two peaks with (M) 2998 in (B)]; similarities between species, exemplified by the
occurrence of varv A [2878 in (C), (D), (F), and (G)] and the peak 3210 in (B) and (E). There is
also an example of a peak unique for a species [3180 in (B)], and of another peak unique for
different anatomical parts within a species [3109 in the underground part of V. odorata (G)].
The injected samples were prepared essentially according to the protocol outlined in Figure 8.
Before analysis, samples (BuOH extract redissolved in aqeous mobile phase) were passed through a
solid phase extraction column. The consumed sample corresponded to approximately a 15 mg
starting amount of plant material.
18
19
3. CHARACTERISATION OF CYCLOTIDES
Figure 10. The nanospray (40) ion
trap setup that was established as the
standard MS technique for cyclotide
analysis in our lab. The very low flow
(nl/min), fed directly into the orifice by
the nanospray needle (seen at the top
right corner), enables low sample
consumption (i.e., extended analysis
time).
The MS used was a ThermoFinnigan
LCQ ion trap that allows a wide range
of experiments, from zoom-scan mass
determinations to multiple MSn for
peptide sequencing.
Among the first observations made on the chemical character of the
cyclotides was their extreme stability: that is, they have here been shown to
maintain their structure over a wide pH interval (see Figure 11), and have
been reported to be stable in boiling water (17), and to withstand
enzymatic degradation (18). The presence of disulfides in the cyclotide
molecule was also established at an early stage, and was shown to be
important in that biological activity was lost after reduction (18). However,
the cyclic structure and the exact configuration of the three disulfides were
not established at that time; and even with today’s refined techniques in MS
(Figure 10), NMR and automated Edman degradation, proving or
demonstrating the existence of these structural features for cyclotides
remains an analytical challenge.
This chapter addresses the characterisation of cyclotide structure,
primary and secondary: new methodologies are described and applied for
MS based amino acid sequencing and for determination of disulfide
connectivities. Chemical proofs of the cyclic structure of cyclotides are
presented; and in addition, cyclotide biological activity and possible
underlying mechanisms are discussed.
PROOF OF THE CYCLIC NATURE OF THE VARV PEPTIDES
The macrocyclic nature of the varv peptides was established by a
combination of techniques. Masses determined by MS generally differed by
24 mass units compared to the ones calculated from quantitative amino
acid analysis (I, II). This agrees with expected values for macrocyclic
structures, attributed to the deficit of one water molecule (-18 Da) and six
20
15
10
CD
0
-10
-15
185
200
220
Wavelength[nm]
240
260
Figure 11. The pH stability of cyclotides shown by CD spectroscopy. The spectra of varv A at pH
2, 7, and 12 (phosphate buffer) nearly exactly superimpose, indicating no changes in secondary and
tertiary structure.
hydrogens (-6 Da), due to the absence of N- and C-terminals, and to the
formation of three disulfide bridges.
Another fact supporting ring structure is that single linear derivates
could then be produced by enzymatic cleavage of the peptide backbone.
Endoproteinase GluC was shown to be useful in this case, as most of the
varv peptides contained only one glutamic acid residue. The only exception
to this was varv H, which has two Glu in the sequence. This exception was
exploited, in combination with tryptic cleavage, to unambiguously prove
that the structure is macrocyclic (II and Figure 12). A similar approach was
used to prove the cyclic structure of circulin A (21), and likewise for
cyclopsychotride A (22). In the latter case, partial acidic hydrolysation was
used to produce overlapping fragments.
Figure 12. The sequencing of varv H as
an additional proof that the backbone of
cyclotides is macrocyclic (II). Edman
degradation of the tryptic digest (second
from the top) overlaps the fragments
obtained from cleavage with
endoproteinase GluC (below).
SEQUENCE DETERMINATION
Standard methods for sequence determination have been a combination of
automated Edman degradation, quantitative amino acid analysis, and MS
(MALDI-TOF or nanospray-ion trap MS), as in the case of varv A-H (I, II).
Together these three independent methods yield reliable sequence data. In
21
the sequencing of hypa A, however, MS2 sequencing was used for the first
time in the verification of a macrocyclic polypeptide (III).
The latter technique has the advantages of speed and sensitivity, but the
inherent structural features of the cyclotides complicate the analysis; the
cystine knotted cyclic motif requires that the disulfides be broken and
alkylated, and thereafter, requires cleavage of the cyclic peptide backbone.
Moreover, in the cyclotide sequence, positively charged amino acid residues
are few or clustered, yielding either enzymatic (i.e., tryptic) cleavage
products that are too large, or fragments with unsuitable charges for MS
sequencing. This was exemplified in the sequencing of the endoproteinase
GluC fragment of hypa A, which was less than straightforward (III).
As an analytical strategy to circumvent these problems, charges were
introduced at the most conserved parts of the sequences, that is, cysteines
were aminoethylated following reaction with bromoethylamine (V and
Figure 13 and 14). In addition, these derivates proved to be excellent targets
for enzymatic digestion.
1
2
3
+
NH3
S
+
NH3
+
S
NH3
N
4
N
+
NH3
Figure 14. Native and introduced positively charged enzymatic cleavage sites. The basic amino
acid residues were lysine (1) and arginine (4), along with aminoethylated (2) and aminopropylated
cysteines (3) [(3) was shown to be readily cleaved by trypsin; however, since the mass of this
modification coincides with carbamidomethylation, and is therefore unsuitable for MS analysis in the
method (here described) for disulfide mapping, the method was abondoned]. To confine cleavage
to intercysteine loops (i.e., digestion only after introduced sites), acetylation of lysines was used in
combination with exchange of trypsin for endoproteinase LysC (V). Note that only the side chains
are shown, the rings represents the peptide backbone.
Other reagents found in the literature and used for aminoethylation of proteins include
ethyleneimine and N-(ß-iodoethyl)triflouroacetamide (41). The latter is also marketed under the
trade name Aminoethyl-8, as a one-step modification reagent for sulfhydryl groups (Pierce
Chemical Co, Rockford, IL, USA), and was used in the first attempts to derivatise varv A. However,
complete removal of trifluoroacetyl groups was problematic for varv A, derivatised with this
reagent, in that hydrolysation, which is supposed to occur spontaneously (41), was unsuccessful
even after pyridine treatment for TFA removal (42): MS of HPLC purified peptides still showed the
presence of TFA. Because this would complicate the production and analysis of loop-specific
fragments, this reagent was replaced with bromoethylamine.
22
A
AV: 33 NL: 1.20E7
B
1091 3+
vcotF1ae #1-35 RT: 0.00-0.98
T: + p Full ms [ 500.00-2000.00]
100
100
95
95
90
90
85
85
Relative Abundance
Relative Abundance
020125vcf1 #1-33 RT: 0.02-1.00
T: + c ms [ 150.00-2000.00]
80
75
70
65
60
55
50
45
40
35
30
25
80
70
65
1179 3+
60
55
50
45
40
35
30
25
20
15
15
1636 2+
708 5+
75
20
10
885 4+
AV: 35 NL: 6.37E4
534 6+
1768 2+
10
5
5
0
0
600
800
1000
1200
1400
m/z
m/z
1600
1800
2000
600
800
1000
1200
1400
1600
1800
m/z
m/z
Figure 13. Introduction of charges by aminoethylation (Nanospray MS of vico A). For the native
peptide, the doubly and triply charged ions are predominant (A). Then, after aminoethylation of
cysteines, the distribution pattern undergoes a dramatic change, with the introduction of six artificial
basic amino acids (B).
MS sequencing of intercysteine loops
Already, in 1956, Lindley (43) pointed out the possibility of using such
aminoethylated cysteines as tryptic targets. Consequently, digestion of derivatised
cyclotides will then produce peptide fragments corresponding to the variable loop
regions between the cysteines.
All hitherto-known cyclotides also contain at least one native tryptic cleavage
site, giving rise to additional fragments that complicate the analysis. However, by
using a combination of protection of lysines and the exchange of trypsin for
endoproteinase LysC, this can be avoided.
The utility of this strategy was demonstrated in the sequencing of vico A and
B from Viola cotyledon (V). Quantitative amino acid analysis revealed the
presence of two Lys residues in each of them, which then were acetylated, prior to
aminoethylation with bromoethylamine, to confine possible cleavage sites to only
aminoethylated cysteines [referred to as Cys(AE)]. Thus, only six fragments, all
loop-specific, were produced (Figure 55 and Table 2). In the subsequent MS2
analyses, all peptide derivates produced fragmentation patterns that were easily
interpreted (Figure 16). To aid sequencing, a database contaning all cyclotide
sequences was constructed using the Sherpa program (44). Masses for
aminoethylation of cysteines (+44) and acetylation of lysines (+42) were added;
and to produce only intercysteine-loop fragments, the digest method was set to cut
after Cys(AE). Analogously, digests of non-protected peptides were analysed by
excluding the acetylation and changing the digestion settings. Masses of observed
fragments, both from MS and MS2 experiments, were then searched within these
matrixes. The order of the loops was then deduced based on sequence homology
and identification of partially digested peptides. Similar setups would be possible
with most other MS softwares for analysis of peptides and proteins.
23
2000
Table 2. Fragments of digested vico A. Calculated and experimental masses (MH +) of the tryptic
digests of aminoethylated vico A, with and without prior acetylation of Lys residues (cf. Figure 15).
Loop No.
Mw Exp.
Mw Calc.
MS2 Sequencing
#4
252.2
252.1
SC
a
261.1a
261.2a
NKa
a
a
a
264.1
264.1
VCa
#1
452.2
452.2
AESC
#2
637.3
637.3
VYIPC
#3
711.3
711.4
FTGIAGC
#5b
718.3b
718.4b
KNKVCb
#6
960.2
959.4
YYNGSIPC
a
b
Fragments of intercysteine loop #5 from cleavage, after unprotected Lys. Lys protected by
acetylation.
A
020121vcf1ae #1-160 RT: 0.01-2.66
T: + c Full ms [ 150.00-1000.00]
100
AV: 160 NL: 1.18E7
#22+
95
90
85
Relative Abundance
80
75
70
#32+
65
60
NK
VC
55
50
45
#62+
#3
40
#4
35
#2
30
25
20
2+
#1
*
#1
#6
*
*
15
10
5
0
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
750
800
850
900
950
m/z
B
020121vcf1aeac2 #1-173 RT: 0.01-3.00
T: + c ms [ 150.00-1000.00]
100
AV: 173 NL: 5.65E6
95
90
85
80
Relative Abundance
75
#52+
70
65
60
#5
55
50
45
40
35
30
25
20
15
10
5
0
150
200
250
300
350
400
450
500
550
600
650
700
m/z
Figure 15. Digests of derivatised vico A. (A) Tryptic digest after aminoethylation (cf. Table 2). Five
intercysteine loops were easily identified. Lys residues gave rise to additional fragmentation of loop
number 5 (K-NK-VC), of which two were found in the mass spectra (NK and VC). To yield loop
specific digestion only, Lys residues were acetylated before aminoethylation and digestion to yield
result (B). Here, loop number five was found intact, and sequenced by MS2 (see paper V). Figure 16
shows the sequencing of loop #6.
*Peaks originating from the digestion buffer.
24
Figure 16. Sequencing of the largest intercysteine loop of vico A by nanospray ion trap MS2.
Fragmentation of the doubly charged ion of loop no 6 [m/z 460.0 (M+2H)2+, c.f Table 2 and Figure 15]
gave the complete sequence. Note the doubly charged peaks corresponding to the loss of AE (-44) and
sulphur+AE (-76) from the aminoethylated C-terminal Cys residue. Equivalent peaks were seen when
fragmenting the doubly charged ions of other intercysteine loops (V). List of masses: a2, 299.0; b2; 326.9;
b3, 442.0; b4, 498.9; b5, 586; b6, 699.0; y2,262.0; y3, 375.1; y4, 462.1; y5, 519.1; y6, 634.1; y7, 797.1; [M(AE+S)]2+, 442.0; [M-AE]2+, 458.1; b60 and y60 are respectively fragments –18 (the loss of water); [C* is
Cys(AE); CID set to 35%; nomenclature according to (45), see also Appendix].
CHEMICAL DETERMINATION OF DISULFIDE BRIDGES
Classical elucidation of disulfide connectivity requires suitable enzymatic or
chemical cleavage sites in the sequence between cysteine residues. After
cleavage of the native peptide, disulfide-containing fragments are identified,
reduced, and analysed by Edman sequencing or MS; but peptides that failed
to meet these demands (i.e., contained no suitable intercysteine cleavage
sites), were impossible to analyse under controlled conditions. For tightly
disulfide-knotted peptides, partial acid hydrolysis was then the only possible
method. This latter method is also used in the only existing example of
chemically elucidation of the disulfide bridges of native cyclotides—circulin
A and B (46)—showing a disulfide arrangement, which was later verified by
NMR (47) and chemical synthesis (48, 49).
25
In 1993, Gray introduced a strategy that circumvented the abovedescribed problems: peptides were only partially reduced and, following
alkylation of reduced thiols, provided a marker in the subsequent Edman
analysis. Thus, the cysteines that originally formed a disulfide could be
identified (50). Use of the reductant tris-(2-carboxyethyl)-phosphine
(TCEP) made this possible, through its ability to act as a reducing agent at
low pHs (51). At these conditions, keeping the reactivity of the thiolate
anion to a minimum, rearrangement of disulfides (reshuffling) can be
avoided.
After incubation with TCEP, partially reduced disulfide species were
isolated by RP-HPLC (a disulfide species is defined as a peptide with a
particular pairing of cysteines). For varv A, two partially reduced disulfide
species were isolated, each containing one (1S), and two remaining (2S)
disulfides (Figure 17). These species were then labeled with iodoacetamide
(IAM), using an oversaturrated solution of this alkylator. After termination
of the reaction (the alkylation is done in 20-30 s), the mixture was
immediately injected, and separated again on RP-HPLC.
Figure 17. The reductive unfolding of varv A, from the native (N) to the fully reduced peptide (R). To
the left, the reaction is monitored by RP-HPLC at four different time intervals: 3, 5, 10, and 15 minutes
(from bottom to top). Interestingly, the reduced peptide is less hydrophobic than the native one,
indicating the importance of the disulfide knot to present the hydrophobic amino acids at the surface of
the molecule. This quite remarkable behaviour has previously been shown for a delta-conotoxin, which
contain a similar disulfide knot (52).
The best time for collection of partially reduced disulfide species was 3 min, when their relative
amount was highest. To the right, their presence at that time is shown in the enlarged chromatogram.
These peaks were collected and analysed as described in the text.
Because the pH is raised to 8 during the alkylation, the risk of disulfide
reshuffling of the isolated species is high. The use of an excess of IAM
partly solves this problem, but to unambiguously identify the correct
disulfide species, parallel experiments were run with ten-times lower
concentration of IAM, thus favouring reshuffling (53). Comparing the
26
results from the two experiments identifies the true species, distinguishing it
from the reshuffled ones.
Remaining disulfides were then reduced, after which Grays original
strategy relied on a secondary labelling with vinylpyridene, followed by
Edman sequencing to identify the position of the different alkylators. The
approach presented here uses, instead, bromoethylamine as an alkylating
reagent in this step. Analogous to the strategy outlined for intercysteine
loop sequencing, positively charged enzymatic cleavage sites are thus
introduced. Then fragments, corresponding to the numbers and positions of
aminoethylated cysteines, are produced after tryptic digestion, again of size
and charge amenable to MS analysis. For varv A, the 1S species was Cys9Cys21, and the 2S was des(Cys4-Cys16) (Table 3 and Figur 18). Combined,
the identification of these two disulfide species disclosed the pattern
CysI–CysIV, CysII – CysV and CysIII-CysVI, a result in agreement with
previously determined disulfide connectivities of cyclotides. (For curiosity’s
sake, the latter nomenclature of disulfides, based on their order of
appearance in the sequence, gives the correct disulfide pattern independent
of which cysteine one chooses to start with—another result of the
fascinating macrocyclic structure of cyclotides.)
Table 3. Determination of native disulfide bonds by MS
Disulfide species
Exp.
Calc.
Identified fragments
1S
1409.4
1409.5
NTPGC(CAM)SC(CAM)SWPVC(AE)
1565.6
1565.6
NGLPVC(CAM)GETC(CAM)VGGTC(AE)
2S
533.2
645.3
880.3
925.5
533.2
645.3
880.3
925.3
NTPGC(AE)
NGLPVC(AE)
SC(CAM)SWPVC(AE)
GETC(CAM)VGGTC(AE)
GETCVGGTCN10TPGCSCSWPV20CTRNGLPVC
Figure 18. The disulfide connectivities as chemically determined for varv A. The disulfide for the
1S species is drawn with solid lines (Cys9-Cys21; this is also the disulfide that is threaded through
the hole formed by the other two). The 2S lacked the disulfide marked with dotted lines [des(Cys4Cys16)]. Hence, by deduction, the third disulfide is the one marked with dashed lines (Cys14Cys29).
27
The disulfide unfolding pathway of varv A
Besides confirming the disulfide configuration, the reductive unfolding also
discloses the disulfide-bond susceptibility order, as illustrated in Figure 19
for varv A (at these experimental conditions). Interestingly, the intertwining
disulfide appears to be the most stable one.
N
2S
1S
R
Figure 19. A schematic molecular presentation of the reductive unfolding of varv A, based on the
isolation and identification of the partially reduced 2S and 1S species, as described in the text. Thus, the
order of reduction of individual disulfides is Cys4-Cys16, Cys14-Cys29, and Cys9-Cys21. (The structure
is schematically drawn as the one determined by NMR for kalata B1; PDB-code: 1kal (19).)
ON THE EFFECTS OF CYCLOTIDES
As listed in the introduction, a wide array of pharmacological effects have
been attributed to the cyclotides. Most intriguing were two compounds,
each apparently responsible for a specific mechanism: kalata B1, causing
uterine contractions (17, 25), and cyclopsychotride A, a possible inhibitor of
neurotensin in a radioligand binding assay (22).
Trying to evaluate possible specific interactions, varv A was screened in
a relatively high number of enzyme and radioligand-binding assays (run by
Novascreen, Hanover, Maryland, U.S., see Appendix). Assays were chosen to
represent a broad range of pharmacologically relevant targets, including ones
giving possible clues to explaining the cyclotides ability to control smooth
muscle contractions. However, no significant effects were seen for any of the
targets, at 1 µM, a concentration considered to be the highest reasonable for
a specific ligand-receptor interaction.
A plausible explanation to previously reported cytotoxic (i.e.,
haemolytic and antimicrobial) effects of cyclotides has been built on their
resemblances to other antimicrobial peptides, and on their mechanisms of
action (14, 21, 29, 54). One such family of peptides, the defensins, also
shares some structural properties with the cyclotides; members of the
family contain approximately the same number of amino acids, and they are
organised in ß-sheets reinforced by three disulfide bridges. The defensins are
widely distributed in plants and animals, where they are critical constituents
of the host defence (9-11, 55, 56). Anti-tumour effects have also been
assigned to the defensins (57, 58), which have several characteristics that
coincide with observations for varv A, varv F, and cycloviolacin O2 when
these cyclotides were tested in ten different cancer cell lines (IV). These
28
characteristics include the concentration interval at which effects are seen,
and the very sharp profile of the dose response curve (Figure 20 and IV).
Cycloviolacin O2
Varv A
Varv F
140,0
120,0
Survival Index (%)
100,0
80,0
60,0
40,0
20,0
0,0
-2,0
-20,0
-1,0
0,0
1,0
2,0
Concentration (log µM)
Figure 20. Cytotoxic effects of the cyclotides varv A, varv F, and cycloviolacin O2 on one of the
cell lines tested (CCRF-CEM, see paper IV). The effects on the other nine cell lines showed
equivalent dose response curves. Note the very sharp profile, similar to the ones described for
defensins, which in comparable concentrations (58) are known to disrupt cell membranes by
forming pores that penetrate through the lipid bilayer (10, 59, 60). Interestingly, all three tested
cyclotides lysed solid cell lines as well as the ones known to be more sensitive, with little or no
differences in IC50 (for the ten cell lines, IC50 ranged from 0.1 to 1.3 µM for cycloviolacin O2, 2.7 to
6.4 µM for varv A, and 2.6 to 7.4 µM for varv F). This capability has also been observed for the
defensin HNP 1 (57).
The initial interaction between cyclotides and microbial cell membranes is
salt dependent, suggesting that electrostatic interactions are the major
driving force (29). The positive charge of defensins is proposed to regulate
the selectivity between bacterial membranes rich in negatively charged
lipids and the more neutral eukaryotic cells (60). Recently, Huang and coworkers described the lipid composition of the cell membranes as another,
equally important, regulatory factor of the action of lytic and antimicrobial
peptides (61). They showed that peptides in low concentrations tend to
bind to the head-group region of the membrane lipids in a functionally
inactive state. As the concentration increases above a threshold value,
depending on the composition of lipids in the cell membrane, the peptides
form the pore state lethal to the cell. Thus, peptide selectivity is a function
of differences in lipid compositions of different cell membranes.
The results obtained for the cyclotides in the cell line panel (IV) accord
with the first theory that cationic amino acids are important for the potency
of the interaction between peptide and cell membrane: throughout the
panel, cycloviolacin O2, with a net charge of +2, was approximately ten
times as potent as varv A and F, both with a net charge of ±0 (Figure 20 and
IV). However, whether the selective effect observed for haematological
29
chronic lymphocytic leukemia cells, as compared that observed for healthy
lymphocytes, depends on differences in their lipid membrane composition
remains to be seen (both varv A and cycloviolacin O2 were approximately
eight times more potent against this cancer-cell type compared to the
healthy lymphocyte).
The pore-forming hypothesis may in fact also explain the apparent
specific effect of kalata B1. If cyclotides form these ion channels through
the cell’s membrane, Ca2+ will be one of the ions to move across the
membrane, thus mediating muscle contraction—in this case of the uterus.
A similar explanation may underlie the effect seen for cyclopsychotride
A. To determine whether this peptide expresses functional antagonist
activity, its effects on neurotensin-induced elevation of cytosolic Ca2+ levels
were examined (22). These levels did increase, and could not be blocked by
a known NT antagonist. In addition, cyclopsychotride A expressed a similar
behaviour in two unrelated cell lines, suggesting that the peptide acted
through another receptor (22). With today’s knowledge of cyclotides, these
results were conceivably due to the same pore forming mechanisms as
described above.
30
4. DISCUSSION
The first examples of cyclotides were found by bioactivity-guided isolation
(17, 20-22). From these studies, only the active ones occurring at high
concentrations were reported, although the presence of additional peptides
was indicated. When for the first time, then, we examined a plant, Viola
arvensis, with the specific aim of finding cyclotides, we isolated eight novel
ones (I, II). Following studies have used increasingly sophisticated methods,
targeted to cyclotides only (III, IV, V). Today the family has dramatically
expanded, and at the time of writing this thesis 46 members have been
reported in the literature.
Thus, during the course of this work, much effort has been directed to
the development of appropriate methods for separating complex peptide
mixtures, such as the cyclotides have proven to be. This effort includes
development of a protocol (I) that can provide fractions useful in the search
for natural products of a specific substance class—polypeptides.
Development of such a protocol exemplifies the benefits of standardised
protocols to provide fractions suitable for biological testing (62). Analytical
methods to identify and assess the structure of single cyclotides, or mixtures
thereof, have also been developed, most of them involving mass
spectrometry (MS) (III, V, Chapter 3).
MASS SPECTROMETRY IN CYCLOTIDE ANALYSIS
After the introduction of the MALDI (63) and ESI (64) ionisation
techniques, MS has become a standard method in peptide and protein
analysis. Apart from just determining molecular weights, today’s MS
techniques offer information about sequence and structure. To simplify
gathering of such data for the cyclotides, methods involving
aminoethylation of cysteines were developed (Figure 21, Chapter 3 and
paper V).
31
CAM
CAM
CAM
CAM
AE
AE
Figure 21. Schematic
presentation of the method used
for assigning disufide bonds. After
reduction by TCEP at pH 3,
partially reduced disulfide species
Isolation and capture of
are captured by
partially reduced peptides
carbamidomethylation, marked
CAM in the Figure. These peptide
derivates are then completely
reduced, and thus, the remaining
thiols are subsequently
aminoethylated (AE). (This latter
reaction proved unable to replace
existing CAM modified cysteines.)
Reduction of remaining disulfides
Introduction of charged cleavage sites The peptide is then enzymatically
cleaved and anlysed by MS.
An analogous strategy is used
for the intercysteine sequencing
AE
(see Chapter 3 and V), starting
AE
from fully reduced peptides. Thus
all cysteines are aminoethylated to
produce, after digestion, a
complete set of intercysteine loop
Enzymatic cleavage followed by
MS analysis of peptide fragments
fragments.
CAM
AE
AE
CAM
AE
AE
Charge derivatisation of peptides is normally accomplished by modifying
either the N- or C-terminal of the peptide (65-67). The fragmentation
during MS2 experiments is then directed to the derivatised end of the
peptide, and a predictable and simply interpretable fragmentation pattern is
obtained (65-67). For the cylotides, such derivatisations can only be
conducted after cleavage of the cyclic peptide backbone. In addition,
reduction and alkylation of disulfides are needed to gain useful information.
Nevertheless, large and/or unpredictable fragments will form due to low
abundance and clustering of suitable cleavage sites (i.e., basic amino acids),
making total sequence coverage exceedingly difficult to obtain (III, V).
To circumvents all these problems, the simple reaction of
aminoethylation can be used (the same protocol as for carbamidomethylation of cysteines, together with a prolonged reaction time, and with
the exchange of iodoacetamide for bromoethylamine). This derivatisation
32
successfully produces intercysteine peptide fragments of size and charge
suited for MS analysis when followed by enzymatic digestion. This
approach, as described above, has been successfully used for both
sequencing and assignment of disulfide connectivities.
This approach is not the first to be based on mass mapping of
intercysteine loops for determination of disulfide connectivities: Wu and
Watson have introduced a similar strategy using chemical cleavage after
cyanylation of the thiolate anion (68, 69). The cyanylation, taking place at
low pHs, is highly compatible with TCEP, and thus suited to trap partially
reduced disulfide species. After isolation, by changing to an alkaline pH at
which the corresponding iminothiazolidine (itz) derivates are formed, the
peptide is cleaved at the N-terminal of cyanylated cysteines. Disulfide
connectivities may then directly be read out from found masses.
For varv A, however, the cyanylation strategy proved unsuitable. First,
the unfolding of the peptide produced very small amounts of partially
reduced disulfide species, compared with those of fully reduced and native
peptides; and to recycle the fully reduced peptide, derivatisation before
isolation must be avoided. Secondly, varv A contains only one basic amino
acid residue (Arg), which is charged during MS analysis. The Arg containing
fragment then suppresses other ions, and particularly so, since the Nterminal Cys of all fragments is converted to (mainly uncharged) itzderivates. Thirdly, for this very tightly knotted peptide, cyanylation requires,
to work satisfactorily, rather rough conditions compared to those described
for the original protocol (i.e., 1 h incubation at 37°C, as opposed to 15 min
at room temperatur). Consequently, the risk of reshuffling disulfides is
increased. Fourthly, the amino acid on the N-terminal side of the cyanylated
Cys affects the yield of the cleavage reaction, with bulky or rigid side chains
acting as inhibitors. This, together with the commonly observed ßelimination of the cyanylated cysteine (loss of SCN, which is also the
cleavage site), further complicate the analysis (68).
Hence, subjecting varv A to the cyanylation strategy yielded incomplete
results. The above-described problems, in combination with the low
positively charged peptide, resulted in predominant peaks of the Arg
containing fragments, making clear interpretation impossible. In
comparison, the clean chemistry of enzymatic reactions and the balancing of
the native charged amino acids with introduced ones, is attractive. Thus,
even if the history of aminoethylation goes back to the ’50s (43), its
possibilities, when combined with MS, have yet to be fully exploited, as this
work and thesis demonstrate.
33
CYCLOTIDE SEQUENCE AND STRUCTURE
Besides the cyclic peptide backbone and the cystine knot, the cyclotides
contain another well-defined structure, a triple stranded antiparallell ßsheet. The combination of the cystine knot and the ß-sheet is commonly
found in toxins: inhibitory cystine knots (ICK) from plants and animals, and
in peptide transmitters: growth-factor cystine knots (GCK), only found in
animals, so far. Aptly, the cyclotide family has been referred to as the cyclic
cystine knot (CCK) (13, 30).
The structural similarity of peptides from these three peptide families
(ICK, GCK and CCK) has been described in detail in the literature (30, 70,
71). In comparing the three-dimensional structure of ω-conotoxin GVIA,
the squash trypsin inhibitor CMTI-I and the cyclotide kalata B1, Pallaghy
and coworkers in 1994 (71) suggested that the primary role of such cystineknotted motifs is “to provide a compact and stable framework for the
presentation of active residues for specific binding interactions.” This
suggestion remains valid.
The similarities of the cyclotides with the ICK peptides also encompass
the way peptides are expressed. Congruous with a number of toxic
peptides, a cocktail of cyclotides is found in a cyclotide expressing plant
species. These mixtures, still using the same cystine knot as a structural
scaffold, are maintained by diversification of the sequences presented in the
loops between cysteines. Such a hypervariable behaviour within certain
parts of a sequence has been described in detail for the conotoxins (72, 73).
This evolutionary strategy is also used in mammals. The immunoglobulins
are one obvious example of hypervariable sequences adapted to a specific
target interfoliated by conserved scaffold regions. Another example is the
recent discovery of several gene clusters expressing ß-defensins (considered
members the ICK family) in both mice and humans (74).
Cyclotides may also have functional similarity to the defensins
(discussed in Chapter 3 and Paper IV), in that their activity seems to
involve interaction with cell membranes. The defensins are known to
disrupt the lipid bilayer by forming pores that pass through it, thus acting as
ion channels (10, 59, 60). Their ability to do so is linked with their
amphipatic structure, displaying distinct hydrophobic and hydrophilic
surfaces.
These properties are also shown in the cyclotide structure. Instead of
burying its hydrophobic amino acids in the interior of the peptide, they are
forced by the tight disulfide knot to be exposed on the surface of the
molecule. This remarkable behaviour for a peptide or protein also explains
some cyclotide behaviour during isolation: (a) long retention time on RPHPLC, (b) the ability to partition to the organic phase when extracted
between butanol and water, and (c) the reversed order of elution after
reduction of disulfides compared to other peptides and proteins (Figure 17).
34
The hydrophilic patches on the surface are mainly due to the
presence of charged amino acids; all cyclotides have at least one basic
amino acid in loop 5 or 6, and there is a fully conserved Glu in loop 1
(Table 4 and Figures 23 and 24) (because they have atypical sequences,
palicourein, MCoTI-I, and MCoTI-II are excluded from the following
discussion). Of these, the positively charged amino acids seem
especially important for antimicrobial and cyctotoxic activity: Tam and
co-workers found that masking the single basic amino acid of kalata B1
(Arg) significantly decreased the antimicrobial activity (29). In
addition, they found that circulin A and B, and cyclopsychotride A, all
containing three basic amino acids, and a more pronounced clustering of
charge and hydrophobic residues, were more potent against microbes
(29). Correspondingly, cycloviolacin O2, having the highest number of
basic amino acids (the most pronounced amphipatic sequence) of the
peptides tested in (IV), proved to be the most potent cytotoxic agent.
Thus, besides the strictly conserved cysteines, the pattern of
hydrophobic and hydrophilic amino acids in cyclotide sequences is
maintaned throughout the family, with substitutions in the variable
regions (the intercysteine loops) seeming to occur for amino acids of
similar polarity (Figure 23 and Table 4). Loops differ though in degree
of variability. Those directly involved in the cystine knot (Figures 24, 25
and 26), which are loops 1 and 4, are highly conserved, along with the
region in loop 6, which is the place for ring-closure of the linear
precursor (26) (Figures 23, 25 and 26 and Table 4).
The cyclotides have been further divided, into two subfamilies, the
bracelet and the Möbius. Primarily, this division is based on the
presence of a cis peptide bond in a Trp-Pro sequence in the Möbius
cyclotides (13), but it also coincides with a difference in net charge and
total sequence homology (Figure 22 and Table 4). The naming of the
Möbius subfamily originates from a topological curiosity: the cis bond
results in a single twist of the peptide backbone, which, if considered a
ribbon, shows just a single side (13), in likeness to the fascinating
Möbius strip.
The number of cyclotides expressed was estimated by RP-HPLCMS to be more than 50 in Viola arvensis alone. Similar results were
observed for additional species from the genus (V and Figure 9). Most
likely, this holds for the other genera from which cyclotides have been
isolated: the number of known kalata peptides is now 8 (13, 17, 26),
and in all, 6 circulins have been reported from Chassalia parvifolia (21,
24). These mixtures may be used by plants to provide an arsenal
targeted at different types of organisms and/or cell membranes, but may
also mediate a synergistic effect that has been demonstrated previously
for antimicrobial peptides (75).
35
Table 4. Aligned sequences of known cyclotides, see text to the right.
1
2
3
4
5
6
Loop number
I
II
III,IV
V
VI
Cysteine number
AESCVYIPC-FTGIAGCSCK-NKVCYYNGSIP-C
vico A
AESCVYIPC-ITGIAGCSCK-NKVCYYNGSIP-C
vico B
AESCVYIPCTITALLGCSCK-NKVCY-NG-IP-C
hypa A
AESCVYIPCTVTALLGCSCS-NRVCY-NG-IP-C
cycloviolacin O1
GESCVFIPCTVTALLGCSCK-SKVCYKN-SIP-C
cyclopsychotride A
GESCVWIPCTITALAGCKCK-SKVCY-N-SIP-C
cycloviolacin O7
GESCVWIPC-VTSIFNCKCE-NKVCYH-DKIP-C
circulin D
GESCVWIPC-LTSVFNCKCE-NKVCYH-DKIP-C
circulin E
GESCVFIPC-LTTVAGCSCK-NKVCYRNG-IP-C
cycloviolin C
GESCVWIPC-LTSAIGCSCK-SKVCYRNG-IP-C
cycloviolacin O3
GESCVWIPC-LTSAVGCSCK-SKVCYRNG-IP-C
cycloviolacin O9
GESCVYIPC-LTSAVGCSCK-SKVCYRNG-IP-C
cycloviolacin O10
GESCVYIPC-LTSAIGCSCK-SKVCYRNG-IP-C
cycloviolacin H1
GESCVWIPC-ITSVAGCSCK-SKVCYRNG-IP-C
circulin C
GESCVFIPC-ISAAIGCSCK-NKVCYRNGVIP-C
cycloviolin A
GESCVFIPC-ISAAIGCSCK-NKVCYRNG-FP-C
cycloviolin D
GESCVWIPC-ISAAIGCSCK-NKVCYRA--IP-C
circulin F
GESCVWIPC-ISAALGCSCK-NKVCYRNG-IP-C
circulin A
GESCVFIPC-ISTLLGCSCK-NKVCYRNGVIP-C
circulin B
GESCVWIPC-ISSAIGCSCK-SKVCYRNG-IP-C
cycloviolacin O2
GESCVWIPC-ISSAIGCSCK-NKVCYRNG-IP-C
cycloviolacin O4
GESCVWIPC-ISAAVGCSCK-SKVCYKNGTLP-C
cycloviolacin O6
GESCVWIPC-ISAVVGCSCK-SKVCYKNGTLP-C
cycloviolacin O11
GESCVWIPC-ISSVVGCSCK-SKVCYKNGTLP-C
cycloviolacin O8
GESCVWIPC-ISSAVGCSCK-NKVCYKNGT-P-C
cycloviolacin O5
GESCVYIPC-ISGVIGCSCT-DKVCYLNGT-P-C
kalata B5
GESCYVLPC---FTVGCTCT-SSQCFKNGTA--C
cycloviolin B
GETCVGGTC---NTPGCSCS-WPVCTRNG-LPVC
varv A
GETCVGGTC---NTPGCSCS-WPVCTRNG-LPVC
kalata S
GETCVGGTC---NTPGCTCS-WPVCTRNG-LPVC
kalata B1
GETCVGGTC---NTPGCTCS-WPVCTRDG-LPVC
kalata B4
GETCVGGTC---NTPGCSCS-WPVCTRNG-LPIC
varv E
GETCVGGTC---NTPGCSCS-WPVCTRNG-LPIC
cycloviolacin O12
GETCVGGSC---NTPGCSCS-WPVCTRNG-LPIC
varv D
GETCVGGTC---NTPGCSCS-WPVCTRNGV-PIC
varv C
GETCFGGTC---NTPGCSCDPWPMCSRNG-LPVC
varv B
GETCFGGTC---NTPGCSCDPWPVCSRNGV-PVC
varv G
GETCFGGTC---NTPGCSCETWPVCSRNG-LPVC
varv H
GETCFGGTC---NTPGCSCT-WPICTRDG-LPVC
kalata B2
GETCFGGTC---NTPGCTCDPWPICTRDG-LPTC
kalata B3
GETCFGGTC---NTPGCSCSSWPICTRNG-LPTC
kalata B6
GETCTLGTC---YTAGCSCS-WPVCTRNGV-PIC
varv F
GETCTLGTC---YTQGCTCS-WPICKRNG-LPVC
kalata B7
Atypical cyclotide sequences
GETCRVIPVCTYSAALGCTCDDRSDGLCKRNGDPTFC
palicourein
RCRRDSD---CPGACICRGNGYCGSGSDGGVCPKILQ
MCoTI-I
KCRRDSD---CPGACICRGNGYCGSGSDGGVCPKILK
MCoTI-II
36
References
(V)
(V)
(III)
(13)
(22)
(13)
(24)
(24)
(23)
(13)
(13)
(13)
(13)
(24)
(23)
(23)
(24)
(21)
(21)
(13)
(13)
(13)
(13)
(13, 30)
(13)
(13)
(23)
(I)
(13)
(17, 19)
(13)
(II)
(13)
(II)
(II)
(II)
(II)
(II)
(13)
(13)
(26)
(II)
(26)
(27)
(28)
(28)
Table 4. Aligned sequences of known cyclotides. The numbering of the loops are as outlined in
(13). Interestingly, among the larger and (in sequences) atypical cyclotides, MCoT-I, MCoT-II, and
palicourein, the latter shows a higher degreee of similarity to the “normal” ones; probably a
reflection of its origin from the Rubiaceae plant family. The namegiving, which has varied
considerably but always somehow been connected to the name of the species, is explained as
follows, together with the number of cyclotides isolated from each species and family: Violaceae,
27: varv, Viola arvensis ,8; vico, V. cotyledon , 2; cycloviolacin O, V. odorata , 12, cycloviolacin H, V.
hederaceae , 1; hypa, Hybanthus parviflorus , 1; cycloviolin, Leonia cymosa , 4; from Rubiaceae, 16:
kalata, Oldenlandia affinis , 8; circulin, Chassalia parvifolia , 6); cyclopsychotride, Psychotria longipes ,
1; palicourein, Palicourea condensata , 1; from Cucurbitaceae, 2: MCoTI, Momordica chinensis , 2.
[The alignment was done with Clustal W(1.7) (76)]
Figure 22. A
phylogenetic tree to
further illustrate amino
acid sequence homologies
in the cyclotide family.
Notably, the division into
the bracelet and Möbius
subfamilies still holds after
alignment of whole
sequences, and thus has a
wider descriptive meaning
than a single cis WP
bond. (According to the
latter MCoTI-I, MCoTI-II,
and palicourein may
belong to the bracelet
family; here however they
are put outside both
subgroups because of
their atypical sequences.)
Cyclotides from
Rubiaceae and Violaceae
are found in both
structural subfamilies; and
one example of identical
sequences from the plant
families is known, so far,
to exist in varv A and
kalata S.
One more pair of
cyclotides (varv E and
cycloviolacin O2) is
known from sequence
data, but as indicated by
the LC-MS profiling
(Figure 9 and Paper V),
these similarities are
probably very common.
[The tree was displayed
by the program NJPLOT
(77).]
37
Identical cyclotides have appeared previously in different species (i.e., varv
E and cycloviolacin O12), even in different plant families (i.e. varv A and
kalata S) (Table 4 and Figure 23). These similarities, highlighted by the use
of LC-MS, are best illustrated by the almost identical profiles of two closely
related species, V. arvensis and V. tricolor (Figure 9 and V). V. cotyledon, V.
biflora, and V. riviniana show distinct differences from the other species, and
thus appear to be good targets in the search for new, diverse cyclotide
sequences. Although only six species were investigated in the research
reported in Paper V, the differences and similarities of their cyclotide
profiles, corresponding to sections within the genus Viola, indicate a
relevance and possible use of this kind of data in the study of the evolution
of cyclotide containing plants, and conversely, show the potential of plant
systematics to pharmacognosy.
38
Loop #1
#2
#3
I
I
II
II
#4
III
III IV
#5
#6
IV
V
VI
V
VI
Figure 23. The pattern of conserved and variable parts as visualised by a sequence logo (78) of the
alignment in Table 4 [the atypical palicourein (27), and MCoTI-I and -II (28), were excluded]. In addition, the
coloration of the residues shows the degree of preservation of amino acids with similar characteristics:
hydrophilic residues in pink, hydrophobic in yellow, acidic in red, and basic in blue (cf. Figure 24,).
The most conserved loops are also the most structurally important: loop #1 (aa 1-4) and loop #4 (aa 1819) are the ones involved in the cystine ring through which the third disulfide is threaded (cf. Figure 25, 26);
loop #6 contains the GLP sequence which seems to be important in the ring closure of the linear proprotein
(26).
The brackets illustrate the cyclic amide backbone of the cyclotides and their disulfide connectivities. The
intercysteine loops are as outlined in (13).
Figure 24. The variable hot spots of the cyclotides. Identical amino acids are in yellow (the cysteine
residues) and dark green (glutamic acid and a proline residue); highly conserved amino acids, in light green;
and the variable ones, in red. SwissPDBviewer was used create the structures from the PDB entry of kalata
B1: 1kal (79).
39
Figure 25. A ribbon presentation of
cycloviolacin O2 to visualise the cyclic peptide
backbone and the disulfide knot of the
cyclotides. The disulfide threaded through the
hole formed by the other two is marked in
red here (cf. Figure 26). The triple ß-sheet
structure is shown as arrows in the peptide
backbone.
This particular structure is cycloviolacin O1
from V. odorata, whose knotted disulfides was
determined by NMR (13) (Available at PDB,
ID 1df6). Four additional cyclotide structure
may be found at the PDB: kalata B1, ID 1kal
(19); circulin A , ID 1bh4 (47); MCoTI-II, ID
1ib9 (80, 81). SwissPDBviewer was used to
visualise the structure (79).
Figure 26. A stereo view of the cystine knot. The disulfide that is threaded through the hole is
marked in red (cf. Figure 25). The importance of this arrangement was shown by Daly and Craik
(82). Acyclic permutants, cleaved at either of the peptide chains (loop #1 and #4, marked here in
grey) involved in the knot, resulted in the loss of both structure and activity. SwissPDBviewer was
used to visualise the structure (79).
40
ABOUT THE BIOSYNTHESIS OF CYCLOTIDES
During biosynthesis of cyclotides, the plant successfully handles two critical
steps: cyclisation of the peptide backbone and formation of the disulfide
knot. The combination of these two structural features, named the cyclic
cystine knot motif (13), is unique to the cyclotides; and until recently,
nothing was known about its biosynthetic pathway. Today we know that the
cyclotides are true gene products, expressed in a single linear sequence
within a larger preproprotein (26). These data, obtained from cDNA clones
of Oldenlandia affinis, describe the precursors to carry a 20-aa signal
sequence for the endoplasmatic reticulum, followed by an N-terminal
prosequence of 46-68 amino acids, and one, two, or three cyclotide domains
separated by regions of about 25 amino acids.
Another striking feature of the finding of these genes (five in all) was
that they have a common tripeptide at the start and the end of all cyclotide
sequences but one. Thus, in addition to determining the site of cyclisation,
this special sequence repeat—GlyLeuPro—seems to be crucial for the
process to take place (26). Examining additional cyclotide sequences reveals
that this is one of the most conserved regions (loop no 6 in Table 4, Figure
23). Obviously, these amino acids seem important for cyclisation, but if the
reaction itself is then self-mediated, by an intein-like mechanism (83) or
some completely new type of reaction, or catalysed by a hitherto unknown
enzyme, remains to be shown.
Moreover, a 25 amino acid part of the N terminal prosequence closest
to the cyclotide domains was found to be relatively well conserved, and was
proposed to have implications for cyclisation or for folding to the mature
cyclotide (26). The latter suggestion—including that of the peptide seeming
to require some kind of stabiliser during the folding process, acting in the
manner of a chaperon during protein folding—has been shown also during
chemical synthesis of cyclotides. Without some kind of lipophilic solvent
present during the oxidative disulfide folding of either linear or cyclised
peptides, the yield of cyclotides with native conformation was low (84).
This behavior is likely connected to the exposure of hydrophobic residues
on the surface of the cyclotides.
To clarify this part of the biosynthesis further, the same methodology as
used for chemical determination of the native disulfide bridges was applied
to folding intermediates isolated during the oxidative refolding of cyclic
varv A (Figure 21). Preliminary data show that oxidation in buffer alone
(i.e., reduced glutathione in ammoniumbicarbonate, pH 8) results in several
non-native, three-disulfide species, and in only a small fraction of the native
species. In contrast, by including isopropanol as a possible stabiliser of
hydrophobic surfaces (84), the reoxidation buffer yielded varv A with
native structure only.
41
Strikingly, the same initial events seem to occur under both
experimental conditions, involving coupling of cysteines in the middle of
the linear cyclotide sequence (as known from the cDNA clones).
Combining these data with the above described genetics of the cyclotides,
the following hypothesis seems plausible: the linear proprotein starts to fold
from the middle of the cyclotide sequence, driven by the oxidative
formation of disulfide bonds in the endoplasmatic reticulum, and ending up
in the native conformation, which in turn places the GlyLeuPro sequences
in a spatially favorable position for autocatalytic or enzymatic ligation.
During these events, the hydrophobic cyclotide surface is stabilised by the
conserved prosequence (Figure 27).
However, without question, the biosynthesis of cyclotides is different
from that of other known cyclic peptides. The small ones, produced
commonly in fungi and bacteria (e.g., cyclosporin and bacitracin), are
synthesised nonribosomatically via large enzyme complexes—so called
peptide synthases. The late example of the rhesus theta defensins, RTD 1-3,
a series 18 amino acid residue peptides found in Rhesus macaque leucocytes,
are true gene products, but constructed by the double ligation of two linear
9-residue sequences (85-87).
Figure 27. A schematic drawing of the
hypothetic biosynthesis of a cyclotide: the
linear proprotein starts to fold from the
middle of the cyclotide sequence, driven by
the oxidative formation of disulfide bonds
in the endoplasmatic reticulum, and ending
up in the native conformation, which in
turn places the GlyLeuPro sequences in a
spatially favorable position for autocatalytic
or enzymatic ligation.
42
PAST AND PRESENT USE OF CYCLOTIDES AND CYCLOTIDE BEARING PLANTS
The ethnopharmacological observations that led to the isolation of the
first cyclotide, the uterocontractive kalata B1, naturally gives rise to the
following question: are there any similar observations (on effects or use)
of plants from the family of Violaceae? A search in the literature and
relevant databases, including NAPRALERT (the largest database of natural
products, extract level included, and their biological activities and
ethnomedical use), gives the poignant answer: Yes! Several reports from
geographically distant places say that plants from Violaceae have been
used for similar or related gynecological and obstetrical purposes
(summarised in Table 5). Of the species found, only one, Rinorea
anguifera, belongs to a genus whose individuals have not been shown
experimentally to contain cyclotides.
Perhaps even more interesting, in the context of this thesis and the
reported cytotoxic activities in Paper IV, is the use of plants of the
genus Viola against cancer: both European and Chinese folk medicine
have made use of them for this purpose (88). In addition, a large
number of reports indicate that presently described activities and
structural features (i.e., amphipathic structure) of isolated cyclotides
can be used to advantage.
Table 5. Ethnopharmacological observations of the use of Violaceae plants for gynecological and
obstetrical indications.
Plant species
Plant part/
Indication
Place
Reference
formulation
Viola species
Used in the diet
Facilitate
New Guinea
(89)
(unnamed)
childbirth
V. odorata
Hot water
Abortifacient
Argentina
(90)
extract of
flowers
V. humboldtii
Juice of the
To increase
Nortwestern
(91)
whole plant
lactation
Amazonia
V. adunca
Roots and leaves Chewed during
USA
(92)
labor
Rinorea anguifera
Root decoction
Protective after
Malaysia
(93)
childbirth
Hybanthus brevis
Decoction of
Treat
Mexico
(94)
entire plant
menorrhagia and
hemorrhage
between
menstrual periods
H. enneaspermus
Tonic before
Africa
(95)
childbirth and
after delivery
H. concolor
Infusion of roots Veterinary aid for USA
(96)
and stems of
mares to expell
mixed with feed injured fetuses
Connected to this list are then the two ethnopharmocological observations of the use of
Oldenlandia affinis, to facilitate childbirth (16, 17).
43
These include the use of Violaceae plants as fungicides and antiseptics for
wound healing and treating of various skin disorders, that is as
antimicrobials; and for several throat and respiratory disorders, such as
asthma, soar throat, coughing, and for mucus release, taking advantage of
the surfactant properties of the amphipathic cyclotide structure.
Today, increased knowledge of the importance of antimicrobial peptides
in host defense, in both plants and animals, further strengthens connections
between the traditional uses of cyclotide bearing plants and modern
scientific explanations. The antimicrobial peptides (e.g., defensins, thionins,
cecropins) are known to disrupt cell membranes by forming pores that pass
through the lipid bilayer (10, 59, 60). Their ability to do so relates to their
amphipatic structure, a property reflected in the cyclotide structure, as
discussed previously. That cyclotides—and hence cyclotide bearing
plants—are involved in the creation of such pores, proposed to explain both
the uterocontractive and cytotoxic activity (Chapter 3), may provide, in
addition, a plausible explanation for the use of these plants in tradional
medicine.
44
5. CONCLUDING REMARKS
When starting this work, the aim was to explore the undeveloped field of
finding and analysing polypeptides from plants, with expectations of
discovering and characterising new biologically active substances. The initial
use of a fractionation protocol, focusing on plant polypeptides, ended in
isolation and structural characterisation of cyclotides (I). These macrocyclic
polypeptides were found to occur in complex mixtures in a single species
(II); for instance, Viola arvensis was estimated to express more than 50 such
polypeptides (V).
To assess such cocktails of closely related peptides is an intriguing
analytical challenge, and thus, one of the major achievements of this work is
the development of appropriate separation and identification methods (II,
III). This includes profiling of total cyclotide expression within a species by
LC-MS, which might find applications in phylogenetic studies of cyclotide
bearing plants. Another development was that of a strategy for intercysteine
loop MS sequencing. In combination, these two methodological advances
provide a rational approach that is highly suited to cyclotide analysis, and to
analysis of other cysteine-rich peptides as well (V).
The loop-specific MS sequencing, also adapted for mapping of
disulfides, was applied to varv A, a member of the Möbius cyclotide
subfamily. The result was consistent with previous reports for both
chemically and spectroscopically determined disulfide configurations for
bracelet cyclotides (CysI–CysIV, CysII–CysV, and CysIII-CysVI) (46, 47,
49). This answers the question recently raised as to whether the cystine
knot is present in both subfamilies. Based on NMR data and the lack of
chemical proofs for the Moebius cyclotides, kalata B1 had been suggested to
have an unknotted disulfide configuration (CysI-CysIV, CysII-CysIII and
CysV-VI) (97), but the results presented in this thesis show that this is not
the case.
45
Cyclotides are today known to be expressed as single linear precursors
(26), but the exact mechanism of cyclisation is unknown. The formation of
the cystine knot, the other characteristic structure of the cyclotides, is yet to
be described, but can be approached with the same strategy for disulfide
mapping as described above. Such knowledge about the biosynthesis of the
cyclotides can in the future lead to new ways of stabilising other peptide
and protein structures.
With the finding of the first cyclic peptides from mammals, the rhesus
theta defensins RTD 1-3 (85-87), cyclic backbones now emerge as
widespread, natural, structural stabilisers for biologically active peptides.
Lower organisms are the most well-known source of peptides showing this
characteristic structural feature, exemplified by the recent isolation of the
21-amino-acid-residue-long microcin J25 (98), and by substances with great
importance as drugs (e.g., cyclosporin). These peptides are all involved in
host defence, which is likely the function of cyclotides within the plant.
Recent data, of Jennings and coworkers (26) support this conjecture. They
proved that kalata B1 is an potent insecticide, and suggested that by means
of genetic modifications, cyclotides can be useful in crop protection (99).
Apart from this, a wide array of biological activities have been ascribed to
the cyclotides, including their cytotoxic effects against human cancer cell
lines, as shown in this work (IV). Most likely, their effects have a common
explanation that involves pore formation through cell membranes.
In addition, with the unique structural motif serving as a scaffold to
present otherwise-labile peptide sequences (in the variable loop regions) to
target molecules, the highly stable and compact cyclotide molecule is
regarded an interesting starting point for protein engineering (30). This has
already proven to have practical implications in the design of an analogue to
the conotoxin MVIIA (which in its native state has passed Phase III clinical
trials against chronic pain (14, 100)). The analogue incorporates the
cyclotide structure and the activity of the toxin (14, 101). As peptide
chemistry, especially addressing the synthesis of cyclotides, has been
developed (49, 102, 103), future exploitation of structural scaffolding seems
likely, and very promising.
The function of the scaffold, to present variable loops, was likely critical
in the evolution of cyclotides, with adaptation taking advantage of the
structural motif, along with sequence diversity, to mediate activities towards
specific targets. The expression of a cocktail of peptides in a single plant is
an indication of this strategy, which is known to be used by other organisms
as well. Similar scaffolds, rich in disulfides, include toxins (e.g., from snakes
and conus snails) and defensins (from plants and animals).
Thus, the structural, biochemical, and functional diversity of peptides
and proteins found in nature has exceptional opportunities for future
research (12). In this context, there is a distinct place for the macrocylic
polypeptides found in plants.
46
6. ACKNOWLEDGEMENTS
This work was carried out at the Division of Pharmacognosy, Department of
Medicinal Chemistry, Faculty of Pharmacy, Uppsala University.
I wish to express my gratitude to the following people who in various ways
contributed to this thesis:
Per Claeson, min handledare, för många goda och intressanta diskussioner
genom åren, för att du lärt mig god vetenskap och för att du är en god vän;
Lars Bohlin, för din entusiasm och ditt intresse för forskning, och för att du
tillsammans med Per var förutseende och startade peptidprojektet på
avdelningen;
Teus Luijendijk, for teaching me a lot about laboratory work and for being a
good friend;
Åke Engström, för det du lärt mig om peptidkemi, Edman-sekvensering och
mass-spektrometri
47
Alla mina doktorandkollegor under tiden på avdelningen, som alla blivit
mina goda vänner, dvs:
Senia Johansson, för att du även varit en vetenskaplig vapendragare,
alltid lika glad och pigg, och som tillsammans med Therese Ringbom stått
ut med mig från början på bästa sätt. Therese ska också ha särskilt tack för
att du lotsat mig genom avhandlingsbyråkratin och allt stöd på slutet av mitt
arbete. Det senare gäller också Erika Svangård, som även varit en mycket
god samarbetspartner på laboratoriet. Martin Sjögren, för gott samarbete
och trevliga dagar – bland annat på västkusten; Åke Stenholm för mycket
intressanta samtal och diskussioner; Ulrika Huss, för många glada skämt- och
kafferaster. Stort tack även till Lotta Flodmark, Petra Lindholm, Sonny
Larsson, Wimal Pathmasiri, Ylva Roddis, Hans Naumann och Håkan Tunón.
Premila Perera och Mervi Vasänge, för att ni väckte intresset för
farmakognosi under min första termin på avdelningen;
Alla mina fördjupningsstudenter genom åren;
Adriana Broussalis for fruitful collaborations; Hesham El-Seedi, Regina
Bruggisser, Thomas Idowu, Rilwan Adewunmi, Laura Segura, Shisheng Li,
Hemanthi Ratnayake, Tiina Ojala, Franz Bucar and Jayantha Welhinda for
fruitful discussions and good companionships;
.
Kerstin Ståhlberg, Maj Blad, Jan G. Bruhn och Anders Backlund för
stimulerande diskussioner, trevliga stunder, och annan stödjande
verksamhet;
Finn Sandberg och Gunnar Samuelsson, för ert intresse för min forskning,
men också för praktiska tips och spännade berättelser;
Ernst Oliw and Chao Su, för all hjälp med mass-spektrometern;
Mina medförfattare på uppsatserna som ingår i avhandlingen, och alla andra
samarbetare under arbetets gång: Mikael Hedeland och Erland Holmberg
för hjälp med CD-spektroskopi; Bo Ek för vissa Edman sekvenseringar.
Paul Johnson, for excellent linguistic revivion of this thesis;
Och till sist Camilla, puss och kram.
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55
APPENDICES
Amino acid residues
Trivial name, abbreviations and their masses
Alanine
Ala
A
Arginine
Arg
R
Asparagine
Asn
N
Aspartic acid
Asp
D
Cysteine
Cys
C
Glutamine
Gln
Q
Glutamic acid
Glu
E
Glycine
Gly
G
Histidine
His
H
Isoleucine
Ile
I
Leucine
Leu
L
Lysine
Lys
K
Methionine
Met
M
Phenylalanine
Phe
F
Proline
Pro
P
Serine
Ser
S
Threonine
Thr
T
Tryptophan
Trp
W
Tyrosine
Tyr
Y
Valine
Val
V
Unspecified aa
Xaa
X
71
156
114
115
103
129
128
57
137
113
113
128
131
147
97
87
101
186
163
99
MS fragmentation of peptides
Fragments normally encountered during ion trap peptide fragmentation include the a- and b-ion
series, carrying their charge at the N-termini, and the y-series, carrying its charge on the C-termini.
56
Novascreen assays
Tested compound varv peptide A, concentration 1 µM. Assays were conducted in August 2000.
Neurotransmittor related
Second messengers
Adenosine, Non-selective
Adenylate Cyclase, Forskolin
Adrenergic
Inositol Triphosphate, IP3
A1 Non-selective
Nitric Oxide Synthase, Neuronal-binding
A2 Non-selective
Protein Kinase C, PDBu
ß, Non-selective
Benzodiazepine, Peripheral
Growth factors, hormones
Dopamine, Non-selective
Atrrial natriuretic peptide A
GABA A
Corticotropin Releasing Factor
Agonist site
Epidermal Growth Factor
BDZ, Central Site
Oxytocin
Glutamate
Platelet Activating Factor
AMPA Site
Thyrotropin Releasing Factor
Kainate Site
NMDA
Brain/gut peptides
Agonist Site Glycine Strychn.-insens. Site
Angiotensin II
Glycine
AT1
Strychnine-sensitive
AT2
Melatonin
Bradykinin, BK2
Muscarinic
Cholecystokinin
Central, Non-Selective
CCKA
Peripheral, Non-Selective
CCKB
Opiate, Non-Selective
Endothelin
Serotonin, Non-Selective
ETA/Human recombinant
Sigma, Non-Selective
ETB/Human recombinant
Galanin
Immunological factors
Neurokinin
Complement C5a (human)
NK1
NK2 (NKA)/Human recombinant
Ion channels
NK3 (NKB)
Calcium
Neuropeptide Y, Non-Selective
Type L (Dihydropyridine site)
Neurotensin
Type L (Benzothiazepine site)
Somatostatin, Non-Selective
Type N
Vasoactive Intestinal Peptide, Non-Selective
GABA
Vasopressin 1
Chloride, TBOB
Glutamate
Chemokine
Chloride Dependent Site
Chemokine, CCR5 (Human recombinant)
NMDA, MK801 Site
NMDA, Phencyclidine Site
Enzymes
Potassium
Acetylcholinesterase
ATP-Sensitive (KATP)
Choline acetyltransferase
Ca 2+ Activated, Volt-Sensitive (SkCA)
Elastase (Human)
Ca 2+ Activated, Volt-Insensitive (KV)
Esterase (Human)
Sodium
Glutamic acid decarboxylase
Site 1
Monoamine oxidase A
Site 2
Monoamine oxidase B
NOS, Constitutive-neuronal
57