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OCR for page 50
Colloquium
Efficient oxidative fooling of conotoxins and the
radiation of venomous cone snails
Grzegorz Bulaj*t, Olga Buczak*, lan Goodsell*, Elsie C. Jimenez*$, Jessica Kranskit, Jacob S. Nielsent, James E. Garrettt
and Baldomero M. Olivera*§
*Department of Biology, University of Utah, Salt Lake City, UT 84112; tCognetix, Inc., 421 Wakara Way, Salt Lake City, UT 84108; and "Department of
Physical Sciences, College of Science, University of the Philippines Baguio, Baguio City, Philippines
The 500 different species of venomous cone snails (genus Conus)
use small, highly structured peptides (conotoxins) for interacting
with prey, predators, and competitors. These peptides are pro-
duced by translating mRNA from many genes belonging to only a
few gene superfamilies. Each translation product is processed to
yield a great diversity of different mature toxin peptides (~50,000-
100,000), most of which are 12-30 aa in length with two to three
disulfide crosslinks. In vitro, forming the biologically relevant
disulfide configuration is often problematic, suggesting that in
viva mechanisms for efficiently folding the diversity of conotoxins
have been evolved by the cone snails. We demonstrate here that
the correct folding of a Con us peptide is facilitated by a posttrans-
lationally modified amino acid, y-carboxyglutamate. In addition,
we show that multiple isoforms of protein disulfide isomerase are
major soluble proteins in Con us venom duct extracts. The results
provide evidence for the type of adaptations required before cone
snails could systematically explore the specialized biochemical
world of "microproteins" that other organisms have not been able
to systematically access. Almost certainly, additional specialized
adaptations for efficient microprotein folding are required.
Chemical interactions between organisms, the major theme of
this symposium, are usually mediated through organic mol-
ecules that elicit physiological effects in the targeted organisms
that are of benefit to the producer. In this respect, a group of
predatory molluscs, the cone snails (see Fig. 1), use an idiosyn-
cratic strategy; small structured peptides, instead of conven-
tional natural products, are the primary agents used for inter-
acting with other animals. The cone snails belong to the genus
Conus, comprising SOD different species, all of which capture
prey by injecting a peptide-rich venom (1~. Some, if not most,
species of Conus also use venom to defend against predators and
for competitive interactions (24. Thus, the "front-line" genes for
the biotic interactions of Conus are those encoding their venom
peptides. The peptidic nature of the functional gene products
means that these can be directly elucidated and chemically
synthesized from the gene sequence. Consequently, for the cone
snails, the link between chemical ecology and genomics is
unusually straightforward.
Each Conus species has a distinct set of biotic interactions
characteristic of that species; this helps to rationalize why each
one has a different repertoire of 100-200 venom peptides (only
a subset of which are probably expressed at any one time). Thus,
the SOO different living species of cone snails (3) can potentially
produce ~50,000-100,000 different conopeptides in their venom
ducts, an enormous diversity of gene products reflecting the
complex chemical ecology of the genus as a whole (for a review,
see ref. 2~. Rapid advances in DNA sequence analysis have made
these chemical agents much more amendable to systematic
interspecific analysis than any comparably diverse set of natural
products produced by any genus or family of animals or plants.
14562-14568 1 PNAS 1 November 25, 2003 1 vol. 100 1 suppl. 2
The analysis carried out on Conus venom peptides suggests
that a majority of the estimated >SO,OOO peptides are encoded
by only ~12 conotoxin gene superfamilies. These superfamilies
have undergone rapid amplification and divergence, accompa-
nying the parallel radiation and diversification of Conus species
at a macroevolutionary level (~Conus is arguably the most species-
rich genus of living marine invertebrates). Each major Conus
peptide gene superfamily comprises thousands of genes, encod-
ing different peptides. This leads to the remarkable functional
diversity seen among the ~SO,OOO different peptides. A majority
of all Conus peptides exert a powerful effect on some specific ion
channel or receptor target (4~. It is fair to say that the snails likely
have evolved a greater diversity of ion channel-targeted phar-
macological agents than even the largest of pharmaceutical
companies (this diverse array includes peptides that are being
developed for use as human pharmaceuticals). These venom
peptides have allowed different cone snail species to specialize
on at least five different phyla of prey and defend themselves
against a spectrum of predators that might be even more diverse.
Most protein genes initially produce a translation product that
is >100 amino acids in length, a size sufficient to allow conven-
tional folding by multiple intramolecular interactions. Toxin
proteins found in venoms are generally smaller (50-100 act), with
additional stability provided by disulfide bonds. In effect, the
cone snails have extended this tendency one step further, with
some venom peptide superfamilies being the smallest highly
structured but functionally diverse classes of gene products
known (12-20 aa with two to three disulfide bonds). Conopep-
tide evolution has resulted in a large diversity of biological
function being generated in each conotoxin superfamily. Thus,
Conus peptide superfamilies are like other major classes of
proteins produced through gene translation: structural and
functional novelty can evolve, and thus, conotoxins are in many
respects "microproteins" and differ from more conventional
unstructured peptides.
Each conotoxin superfamily has a characteristic arrangement
of cysteine residues, which is assembled into a particular disul-
fide bonding configuration (the "disulfide frameworks. The
latter is the primary determinant of polypeptide backbone
structure. Despite hypermutation of the amino acids between
Cats residues, the disulfide framework generally remains con-
served within a superfamily, generating a characteristic scaffold.
In principle, for peptides with six Cys residues (characteristic of
at least four conotoxir1 superfamilies) there are 15 different
This paper results from the Arthur M. Sackier Colloquium of the National Academy of
Sciences, "Chemical Communication in a Post-GenomicWoricI," held January 17-19, 2003,
at the Arnoici anci Mabel Beckman Center of the National Acaciemies of Science anci
Engineering in Irvine, CA.
Abbreviations: PDI, protein disulfide isomerase; ER, enclopiasmic reticulum.
§To whom corresponcience should be addressecl. E-maii: olivera~biology.utah.edu.
2003 by The National Academy of Sciences of the USA
www.pnas.org/cgi/cioi/1 0.1 073/pnas.2335845100
OCR for page 51
Fia. 1. Shells of cone snails. Seven different Conus species (of the ~500 total)
are illustrated: the examples shown represent the three major feeding types
of Conus. Experimental data that are described in the text were obtained from
these species. (Top) The glory-of-the-sea cone, C gloriamaris (Left); the cloth-
of-gold cone, C textile (Right). (Middle) C omaria (Left); C consors (Center);
C aurisiacus (Right). (Bottom) The fly-speck cone, C stercusmuscarum (Left);
C. betulinus (Right). C betulinus is worm-hunting, whereas C censors, C
aurisiacus, and C stercusmuscarum are piscivorous (fish hunting). C textile, C
gloriamaris, and C omaria are molluscivorous (mollusc hunting). A PCR screen
of PDI was carried out on most of these species (see text). A comparison of the
spasmodic peptide of C textile and C gloriamaris provided the basis for the
work on the role of posttranslational modification in Conus peptide folding.
C textile was the species used for most of the experimental studies described
in this article.
disulfide-bonded arrangements possible, with only one being
biologically relevant. How does folding to this single structural
framework (instead of the 14 other possibilities) occur? This is
a specialized version of the more general protein folding prob-
lem, one of the central unsolved questions in biology.
-Efficient solutions to this specialized folding problem must
have been evolved by cone snails before they could effectively
explore (in an evolutionary sense) the alternative world of
microproteins that they have so efficiently exploited for their
diverse physiological purposes. In this article, data are presented
that are relevant to this previously unaddressed issue. There are
two levels at which solutions to the specialized folding problem
Bula; et a/.
Of Conus peptides could have evolved: (i) specialized intramo-
lecular interactions may stabilize conformation, and (ii) inter-
molecular interactions with extrinsic factors, perhaps within the
endoplasmic reticulum (ER), may promote appropriate folding
pathways within the ER. We explore both possibilities.
An unusual feature of Conus venom peptides is the high
degree of posttranslational modification observed (5~. We dem-
onstrate that posttranslational modification can generate in-
tramolecular interactions that facilitate conopeptide folding. For
potential intermolecular interactions, we show that multiple
isoforms of protein disulfide isomerase (PDI), which catalyze
protein thiol disulfide exchange reactions, are present within
Conus venom ducts, and we show that, apart from the conotoxins
themselves, these isoforms are the major soluble protein com-
ponents of the ducts.
Materials and Methods
Cloning of Conus PDI. Full-length Conus textile PDI cDNAs were
isolated by RT-PCR of venom duct RNA, using degenerate
primers based on the amino acid sequence of the highly con-
served thioredoxin-like active site motif found in PDI proteins
isolated from other organisms. PCR products were gel-purified
and cloned into a plasmid vector, and several cloned isolates
were sequenced. The DNA sequence of the cloned PCR product
contained a long ORF with significant homology to PDI proteins
from other organisms. The DNA sequence of this internal PCR
product was used to design nested PCR primers for 5' and 3'
RACE procedures to isolate the full-length cDNA. C. textile
venom duct cDNA was synthesized with 5' and 3' RACE
adapters (Ambion, Austin, TX) and used for RACE amplifica-
tions. Specific 5' and 3' RACE products were gel-purified,
cloned into a plasmid vector, and sequenced. The sequences of
each of these RACE products overlapped with the previously
isolated central portion of the C. textile PDI cDNA and together
these three PCR-generated cDNAs could be merged to give the
full-length cDNA sequence encoding the complete PDI protein.
To detect PDI expression in venom ducts of other Conus species,
RT-PCR was performed in the following species: C. textile,
Conus stercusmuscarum, Conus aurisiacus, Conus consors, Conus
betulinus, and Conus omaria. The PCR amplification was per-
formed by using primers based on the thioredoxin active site
sequence. Approximately 20 ng of venom duct cDNA and Taq
polymerase was used for the PCRs.
Protein Analysis of Venom Ducts. The venom duct from C. textile
was dissected and immediately divided into four equal parts.
Each part of the venom duct was ground under liquid nitrogen.
Extraction was performed in 1 ml of 10 mM Tris HCl, pH 7.8
containing 0.25 M sucrose and 5 mM EDTA at 4°C. After
homogenization, the solution was centrifuged. The 50 ,ul of
resulted supernatant was lyophilized and dissolved in 30 ,ul of
SDS-electrophoresis buffer, boiled for 5 min, and applied on
4-20% Tris-glycine gel. Proteins were then electroblotted onto
Immobilion poly~vinylidene difluoride) membrane (0.45 ,um)
(Millipore) for 1 h at 50 V. Proteins were visualized by using
Coomassie blue staining, and the protein band of 55 kDa was cut
out from the membrane. Amino acid sequencing was performed
by the Edman degradation method. After protein separation,
polyacrylamide gel was also stained with Coomassie blue.
Peptide Synthesis and Folding. Peptides were synthesized on solid
support by using standard Fmoc [N-~9-fluorenyl~methoxycar-
bonyl] chemistry. All cysteines were protected with S-trityl
groups. The peptides were removed from the resin and purified
by using a semipreparative reversed-phase C~s HPLC column in
a linear gradient of acetonitrile. After purification, the linear
peptide was lyophilized. Folding reactions were initiated by
injecting a resuspended linear peptide into a 200-,ul solution
PNAS 1 November 25, 2003 1 vol. 100 1 suppl. 2 1 14563
OCR for page 52
A
B
1 2 3 4 5
._
"A..' . I
~~ 'a _
J _
expected
conotoxin
region
Conus PDI-1
Conus PDI - 2
Human PDI
Conus PDI-1
Conus PDI - 2
Human PDI
Conus PDI-1
Conus PDI - 2
Human PDI
Conus PDI-1
Conus PDI - 2
Human PDI
Conus PDI-1
Conus PDI - 2
Human PDI
Conus PDI-1
Conus PDI - 2
Human PDI
Signal sequence 1 ~ Trx 50
MKFSS CLVLTLLVFVSA EDVEQEENVHVLTKKNFDS FITDNEFVLVEFYAPW ~APEYA}CAATTLE2;1EKSNIKLAKVDATVEGDLASKFDVR
MKFSSCLVLTLLVFVSA EDVKREEGVYVLTEKNFDAFITDNEFVLVEFYAPW "GHCkALAPEYAKAATTLEEEKSNI KLGKVDATVEVNLATKFEVR
MLRRALLCLAVAALVRA DAPEEEDHVLVLRKSNFAEAI~LVEFYAPW ~PEYAKAAGKLKAE GSEIRI`AKVDATEESDLAQQYGVR
100 ~ row 150
GYPTI KFFRKEKPDG - PAI)YSGGRQAKD IVDWL]CKKTGPPAECELKEKDEVKAFVSKDEWVIGFFKDQESTGALAFKKAAAGIDDI PFAITSEDHVF
GYPTI KFFHKEMPAGS PADYSGGRQAPD IVGWLKKKTGPPAKELKI~CDEVKIFVEKDEVWIGFFKDQESTGALAFKKAAAG IDD I PFAITSEDHVF
GYPTI KFFRNGDTAS - PKEYTAGREADD IVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQA~EAIDD I PFGITSNSDVF
200 ~ - 0~ 250
KEYKMDKDGIVLLKKFDEGRNDFEGNLEEEEAIVKHVRENQLPLVVEFTQESAQKI FGGElI}WHILLFLKK- -EGGEDTIEKFRSAAEDFKGKVLFI
KEYKMDKDGIVLLKKFDEGRNDFEGNLEEEEAIVKHVRENQLPLVVEFTQESAQKI FGGEVKNHILLFLKK- -DGGEDTI EKFRG~AEDFKGKVLFI
SKYQLDKDGVVLFKKFDEGRNNFEGEVT - KENLLDFIKHNQLPLVIEFTEQTAPKI FGGEIKTHILLFLPKSVSDYDGKLSNFKTAP.ESFKGKILFI
YLDTDNEENGRI TEFFGLKDDEI PAVRLIQLAEDMSKYKPESSDLETATI ~ /QDFLDGKLKPHLMSEDVPGDWDAKPVRVI VGKNFKEVAMDKSK
YLDTDNEENGRI TEFFGLKDDE I PAVRLI QLAEDMSKYKPESSDLETATI KKFVQDFLDGKLKPHLMSEDVPGDWDAKPVK~LVGKNFKEVAMDKSK
FIDSDHTDNQRI LEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDRQPVKVLVG=FEDVAFDEKK
Trx 400 450 ~ - r~
AVFVEFYAPW CGH*QLAP IWDELGEKYKDS E~)IWAKMDATANEIEEVKVQSFPTLKYFPKDSE - EAVDYNGERTLDAFVRFLESGGTEGAGVQE -
AVFVEFYAPW CGH~CQI,AP IWDELGEKYKDS KDIWAKMDATANE IEEVKVQSFPTLKYFPKDSE - EAVDYNGERTLDAFVKFLESGGTEGAGVQE -
NVFVEFYAPW CGX(,CQLAPIWDKI`GETYKDXENIVIAXMDSTANEVEAmCVHSFPTLKFFPASAD~TV7DYNGERTLOGFKKFLESGGQDGAGODDD
ER retention signal
- -DEEEE - - EEDEEGDDEDLP {DEL
- -DEEEE- - EEDEEGDDEDLE lDEL
LEDLEE:AE]3PDMEEDDDQICA~ ~
Fig. 2. PDI from C. textile venom duct. (A) SDS/PAGE of extracts from different parts of a venom duct. Lanes 1-4 represent equal fragments from distal to
proximal parts of the venom duct. Lane 5 is a bovine PDI. The ~55-kDa band from lane 3 was extracted, and its N-terminal sequence was determined (see text)
(R. ,;tr'~'res Of PDI-1 and PDI-) from C textile. The signal seauence. thioredoxin active sites (Trx), and the ER retention signal are marked. Domains a, b, b', a'
and c are based on the assignments for human PDI (9, 10). T he shaded residues represent differences between PDI-1 and PDI-2.
containing 0.1 M Tris HCl (pH 8.7), 1 mM oxidized glutathione,
and 2 mM reduced glutathione. The folding reactions contained
CaCl2 or MgCl2 at concentrations indicated in the text or 1 mM
EDTA. The final peptide concentration was 20 ,uM. After an
appropriate folding time, the reactions were quenched by acid-
ification with formic acid (10% final concentration). The sam-
ples were separated by reversed-phase Cog analytical HPLC in
the gradient of acetonitrile (from 9% to 31.5% acetonitrile in
0.1% trifluoroacetic acid). The accumulation of the native form
was calculated relative to other folding species from integration
of the HPLC peaks.
Assays for Biological Activity of Synthetic Peptides. The activities of
synthetic peptides were evaluated by using 13- to 14-day-old
mice, weighing 5.9-6.4 g; the intracranial injections were carried
out as described (6~.
Results
PDI Is the Major Soluble High Molecular Weight Polypeptidic Compo-
nent of Conus Venom Ducts. The oxidative folding of secreted
proteins with disulfide bonds is thought to be promoted by PDIs
(7, 8~. These enzymes contain four thioredoxin-like domains,
two of which have the sequence -CXXC-, that are believed to
catalyze formation and isomerization of protein disulfide bonds.
The unprecedented density of disulfide linkages in conotoxins
(up to 50% of all amino acids can be Cys residues involved in
disulfide bonding) led us to initiate the characterization of PDI
from cone snail venom ducts. Protein extracts from C. textile
venom ducts were prepared (see Materials and Methods), and the
crude extracts were analyzed by SDS/PAGE. As shown in Fig.
2A, the expected low molecular mass gene products consistent
with conotoxins are prominently stained on the gel, but in
addition, a major protein band with a molecular mass of
~55 kDa stands out. This molecular mass is consistent with that
of PDIs characterized from other organisms (see marker band
in Fig. 2A).
The identity of the major polypeptide in the 55-kl~a band was
investigated by microsequencing: a clear N-terminal sequence,
-EEVEEQENVY-, was obtained; as will be discussed below,
this sequence is consistent with the conclusion that the ~55-kDa
band is PDI, possibly a mixture of different isoforms. Thus, apart
from the conotoxins themselves, PDI appears to be the major
soluble protein in Conus venom ducts. Such a high level of PDI
along the whole length of the duct is consistent with conotoxin
14564 1 www.pnas.org/cgi/doi/10.1073/pnas.2335845100
synthesis in both distal and proximal portions of the duct and
with a major role for PDI in the oxidative folding of conotoxins.
PDI expression was further evaluated in a variety of Conus
species by using a PCR screen, with primers matching conserved
sequences in PDIs (see Materials and Methods). The PCR
fragment obtained from cDNA prepared from a variety of
different Conus species representing the major feeding types of
the genus, including C. textile, C. stercusmuscarum, C. aurisiacus,
C. consors, C. betulinus, and C. omaria (see Fig. 1) was similar in
size and intensity to that obtained from C. textile. To characterize
Conus PDIs further, we analyzed a cDNA library from C. textile,
the cloth-of-gold cone. The sequences of two full-length cDNA
clones encoding two different PDI isoforms, PDI-1 and PDI-2,
were determined (Fig. 2B). There is a high degree of sequence
identity between the two isoforms, but there are two regions of
high divergence: amino acids 4-19 (40% sequence divergence)
and amino acids 71-96 (32% divergence). Both proteins are
~75% homologous to the well characterized human PDI se-
quence shown in Fig. 2.
The cleavage site for the removal of the signal sequence was
identified from the microsequencing results for the mature
protein detected in the SDS gel. Based on homology to the
human PDI, five domains can be distinguished: a, b, b', a', and
c (9, 10~. Two of these (a, a') are domains that have sequence
similarity to thioredoxin (a small protein cofactor in ribonucle-
otide reduction), whereas two (b, b') show little sequence
similarity but have a thioredoxin-like fold. The two putative
active-site -CGHC- sequences are located in domains a and a';
thus, the two regions of high divergence sandwich the -CGHC-
site of the a domain. The C-terminal domain is characterized by
a high degree of negatively charged Asp and Glu residues,
postulated to serve as the Ca2+ binding domain (11, 12~. The
C-terminal sequence RDEL- is a standard ER retention signal.
Thus, the Conus PDI isoforms cloned contain all characteristic
features of PDIs from other organisms. The results with the
directly sequenced protein band in Fig. 2A suggest that there
may be additional venom duct PDI isoforms. Preliminary mo-
lecular evidence for additional PDI isoforms has been indepen-
dently obtained (I.G. and J.E.G., unpublished results). We are
presently defining all Conus PDI isoforms and have expressed
the two shown in Fig. 2B. Enzymatic properties of the expressed
Conus PDIs need to be assessed to determine whether these may
exhibit specificity for particular conotoxin substrates, relative to
other Cys-rich polypeptides.
Bulaj et at.
OCR for page 53
y-Carboxvlation of Glutamate to y-Carboxvglutamate (Gla or y!
GLUTAMATE
Glu Gamma- glutamyl
CARBOXYLASE
iNH~ Vit. K, =2, O2
do.
Peptide Source
"Gla-domain" region/pep/ides
h-FIX Human
Sequence
K L ~ F V Q G N L y R ~ C M y K C S F y ~
Conantokin-G(27) C. geographus GEy
Multiply disulf1de-bonded~ non-Gla domain Conus peptides
txSa (28) C. textile
Bromosleeper (29) C. radiates
Spasmodic-Tx C. textile
Spasmodic-Gm C. glonamans
(non-y~ontaining)
Gamma-carboxyglutamate
Gla
rN iN H
O ~COO'
COO
~C=DGWCCT ' MO
yVFyNTyKTTy
~L - Q7LIRY
KS
WATID)/CaTCNVTFFKTCCGOOGDWQCV7ACPV
GCNNSCQ yHSDCySHC ICTFRGCGAVN#
SCNNSCQSHSDCASHCICTFRGCGAVN#
Fig. 3. Comparison of Gla-containing peptidesTrom Conus species.The posttranslational modification of glutamateto y-carboxyglutamate (Gla or ~y) is shown,
and a comparison of the sequences of four Gla-containing Conus venom peptides to the N-terminal sequence of human blood clotting factor IX is shown. Note
that one Conus oentide, conantokin-G from C qeooranhus, has a Gla domain motif that has similar soac~na to factor IX (see boxed seouences), whereas the three
Other Gla-conta~ning peptides do not have an arrangement ot Gla residues characteristic ot a Gla domain. #, amidated C terminus; W. 6-bromotryptophan; T',
glycosylated threonine; ~y, ~carboxyglutmate; h-FIX, human factor IX.
A Role for y-Carboxyglutamate in Conotoxin Folding. A Conus
peptide recently characterized from C. textile, the "spasmodic
peptide" (6), has serendipitously presented an opportunity to
evaluate the function of an unusual posttranslational modifica-
tion found in Conus peptides, the conversion of glutamate to
y-carboxyglutamate (see Fig. 3~. A closely homologous peptide
identified by RT-PCR from a closely related species, the glory-
of-the-sea cone, Conus gloriamaris (see Fig. 1) exhibited con-
siderable sequence identity (24/27 amino acids identical) to the
C. textile peptide (13~. However, no ry-carboxyglutamate residues
are present in the C. gloriamaris peptide. Thus, two very closely
related natural peptides from different Conus species are largely
identical in sequence, but one has two ~y-carboxyglutamate
residues, whereas the other has none (see Fig. 4 a and b).
Both the C. gloriamaris and C. textile peptides were chemically
synthesized on solid support and folded in the presence of
glotathione as described in Materials and Methods. The biological
activities of the two purified synthetic peptides were evaluated by
intracerebroventricular injection into mice; despite the differ-
ences in ~y-carboxylation, no difference in biological activity
could be detected. When injected into mice, both peptides
elicited the response characteristic of the native spasmodic
peptide, hypersensitivity to touch at a dose of 2 nmol and
Bulaj et a/.
hyperactivity and convulsions at 5 nmol. These results indicate
that the Gla residues do not play a major role in the molecular
interactions between the mature folded peptide and the physi-
ological target of the C. textile peptide.
We then assessed whether the Gla residues might affect
peptide folding. Under our standard Ca-free folding conditions,
yields of the properly folded C. gloriamaris peptide were slightly
higher than for C. textile peptide (Fig. 4 c and d). Because
~y-carboxyglutamate residues chelate Ca2+, we evaluated the
effects of Ca2+ on the folding of the two peptides. As shown in
Fig. 4e, the addition of Ca2+ results in a significant increase in
the yield of properly folded C. textile peptide; strikingly, Ca2+
elicited no increase in the yield of the C. gloriamaris peptide,
which lacks y-carboxyglutamate (Fig. 4f). Above 10 mM Ca2+,
the C. textile (Gla+) peptide is more efficiently folded than the
C. gloriamaris (Gla-) peptide, a reversal of the situation in the
absence of Ca2+ (Fig. 4 g and h). Furthermore, the magnitude of
the increase in yield of correctly folded peptide is not observed
when Mg2+ is added over the same concentration range (Fig. 4
i and j). Moreover, Ca2+ at 10 mM concentration did not
significantly change the reductive unfolding kinetics of C. textile
(Gla+) peptide (data not shown). These results are consistent
with the conclusion that once the peptides are correctly folded,
PNAS 1 November 25, 2003 1 vof. 100 1 suppl. 2 1 14565
OCR for page 54
a
b
C. textile peptide C. gloriamaris peptide
GCNNSCQ~yHSDC-ySHCICTFRGCGAVN# SCNNSCQSHSDCASHCICTFRGCGAVN#
.
i mM EDTA ~ /\
*
~ d
*
| lOmMC~++
e~1~
-
~E 34
0 2'
a)
.' 2(
ct
c 1!
o 1(
._
~ C
-
° 25
—' 5 ~1 1
EDTAO.1 1 10 100 .. =
C a++ [mM] 00
500 1 000 1 500
time [mind
It i
I_
TO
°E- 30 ~
0 25 rh
.> 20 .
0 1 5 it=
.o 1 0 -if/
, 5~ ~
~ O
2000 ~~ ( ~
a
_
*
b
early folding (2 min)
GANNSCQJHSDA7SHAlCT#
L
__- 0~ ;A -Ca
. ·~"4 . _Cg
500 1000 1500 2000
time [mind
- 25
20 j
s L 1111~ it ~
EDTA 0.1 1 10 100 Mg
C a++ [mM] 100 mM
Fig. 4. Effect of Ca2+ on the oxidative folding of peptides from C textile and
C gloriamaris. (a and b) Sequences of peptides from C textile and C glo-
riamaris. (c-f) HPLC analysis of oxidative folding of the peptides. The folding
mixtures contained reduced and oxidized glutathione, and the reactions were
carried out in the presence of 10 mM CaCI2 or 1 mM EDTA. Experi mental detai Is
are described in Materials and Methods. The correctly folded species (*) has
the shortest retention time. (c) Folding of C textile peptide in the absence of
Ca. (d) Folding of C gloriamaris peptide in the absence of Ca. (e) Folding of C
textile peptide in the presence of Ca. (fl Folding of C gloriamaris peptide in
the presence of Ca. (g and h) Kinetics of forming the native peptides from C
textile (g) and C gloriamaris (h). The folding reactions were performed as
described in Materials and Methods. Dashed lines, +10 mM Ca2+; solid lines
+ 1 mM EDTA. (i and JO Accumulation of the native peptides from C textile (i)
and C gloriamaris (i) in the folding reactions at 24 h, with increasing concen-
tration of calcium ions. Two other bars represent accumulation of the native
peptides in the presence of 1 mM EDTA or 100 mM Mg2+. All experimental
points are averages from duplicate experiments.
~y-carboxyglutamate residues make no significant contribution to
biological activity or stability; however, the y-carboxyglutamate
residues clearly facilitate oxidative folding when Ca2+ is present,
as would be the case in the ER under in viva folding conditions.
To further investigate a mechanism of the Ca2+-assisted
oxidative folding of the Gla-containing peptide, we synthesized
a model peptide, which represents a fragment of the conotoxin
from C. textile, but containing only one pair of cysteines and two
Gla residues (Fig. 5a). The second pair of Cys was replaced by
Ala. The rationale for using this model peptide is to assess
whether the presence of the Gla residues accelerates formation
of a disulfide bond in the presence of Ca2+. As shown in Fig. 5
b and c, the presence of 10 mM Ca2+ in the reaction mixture
increased the accumulation of the oxidation product both at
early (2 min) and later (10 min) time points. These results
provide a mechanistic rationale, at least in part, for the observed
14566 1 www.pnas.org/cgi/doi/ 10.1 073/pnas.2335845 100
c late folding (10 min)
Ox L
:~ -Ca
;: _ ~ +Ca
Fig. 5. Ca2+-assisted oxidation of the Gla-containing model peptide. The
peptide (a) was designed based on the sequence of peptide from C. textile.
Cys-2, Cys-12, and Cys-16 were replaced by Ala residues, and Thr-19 had an
amidated Cterminus (labeled #). The disulfide bond between Cys-6 and Cys-18
is also shown. A hypothetical model illustrating how a coordination of Ca2+ by
the two Gla residues in the peptide may favor formation of a disulfide bridge
(-SS-) by bringing two Cys thiols in close proximity. (b and c) HPLC analysis of
oxidation ofthe model peptide with a mixtureof oxidized (1 mM) and reduced
(2 mM) glutathione and in the presence of either 10 mM CaCI2 or 1 mM EDTA.
The oxidation mixtures were quenched by acidification after 2 or 10 min and
analyzed by reversed-phase Cal analytical HPLC. L, linear (reduced) peptide;
Ox, oxidized form.
effects of Ca2+ on folding of the conotoxin from C. textile (see
Discussion).
Discussion
In the course of their evolutionary history, the venomous cone
snails have developed an unprecedented number of diverse,
highly structured peptides that are expressed in their venom
ducts. These peptides as a class are the smallest ribosomally
translated functional gene products (microproteins). Most of
these peptides potently interfere with the function of a specific
ion channel target. To have the potent and selective pharma-
cological activity required, the peptides have to be properly
folded with the correct disulfide connectivity. Clearly, cone
snails have generated mechanisms to facilitate small-peptide
folding; this article begins to address such mechanisms. There
are at least two motivations for embarking on this general
research direction: the first (rather pedestrian) reason is that
understanding factors that facilitate proper conotoxin folding in
Bulaj eta/.
OCR for page 55
vivo should help in the routine production of properly folded
Conus peptides in vitro. However, our long-term hope is that by
elucidating how cone snails are able to fold these small peptides
properly, we will also gain insights into the more general problem
of protein folding.
All Conus peptide precursors have highly conserved N-
terminal signal sequences that direct the newly translated pre-
cursor to the ER membrane, transferring it from a highly
reducing, low Ca2+ environment (the cytosol) to an oxidizing,
high Ca2+ environment (the ER matrix). In the oxidizing
environment of the ER (14), the high density of cysteine residues
in most Conus peptides would be expected to lead to rapid
formation of disulfide bonds. We provide evidence for high
levels of multiple PDI isoforms in Conus venom ducts; these
enzymes generally regulate the formation of disulfide bonds and
prevent them from being formed randomly. PDI appears to be
the most highly expressed high molecular weight polypeptide
component of Conus venom ducts. PCR amplification of PDI is
only intermittently successful when other Conus tissues are
similarly analyzed, presumably because of a much lower fre-
quency of PDI transcripts. These observations are consistent
with a key role for PDI in the folding of the major secreted
products of these cells, the conotoxins. An obvious possibility to
be explored is whether different Corvus PDI isoforms have
evolved specialized features to facilitate proper folding of the
specific disulfide frameworks in the various conotoxin super-
families. An additional question is whether Conus PDIs have
synergistic interactions with other specialized chaperones in the
ER that could lead to even greater facilitation of conotoxin
folding.
The many posttranslational modifications observed in Conus
peptides are believed to enhance the affinity and/or pharma-
cological specificity of modified conopeptides; however, the
results presented above suggest an additional function for some
Conus peptide posttranslational modifications: to facilitate
proper folding. In the example presented (^y-carboxylation of
glutamate residues), formation of the correctly disulfide-bonded
conopeptide was enhanced by the Gla residues in the presence
of physiological concentrations of Ca2+, but did not appear to be
important for activity of the folded peptide. The results pre-
sented suggest that the presence of "y-carboxyglutamate residues
facilitate folding of the C. textile peptide in vivo.
A role for Ca2+ in oxidative folding was previously suggested
for secreted proteins such as the low-density lipoprotein (LDL)
receptor (15, 16), Notchl receptors (17) or c'-lactalbumin (18~.
For the LDL receptor, a "calcium box" rich in Asp or Glu was
postulated to direct folding by chelating Ca2+ and nucleating
early folding intermediates as the LDL receptor entered the ER.
Similarly, as disulfide-rich y-carboxylated Conus peptides are
translocated through the ER membrane, we suggest that a
compact calcium mini-box with ~y-carboxyglutamate residues
may either facilitate a favorable spatial orientation of C5rs
residues for correct disulfide bond formation at early stages of
folding, leading to the final biologically active conformation, or
alternatively, inhibit formation of undesirable disulfide bonds.
The experiment on a model peptide (Fig. 5) provides support for
the former, but does not eliminate the possibility that the latter
may contribute as well. ~y-Carboxylation does not appear to be
required for the biological activity of the mature, properly folded
peptide gene product, nor is it required for maintaining the
structure of peptide once correct disulfide bonds are formed.
y-Glutamyl carboxylases, the enzymes that convert glutamate
to ~y-carboxyglutamate (Gla), were previously shown to be highly
conserved, clearly predating the divergence of vertebrates, ar-
thropods, and molluscs (19~. Thus, an ancestral role (more
general than the specialized uses of the modification in blood
clotting in mammals and venom peptides in Conus) that provided
selective pressure for enzyme conservation as animal phyla
Bulaj et a/.
diverged is strongly indicated. The present results raise the
possibility that the proper folding of proteins, directed by the
presence of Gla residues at strategic loci in the polypeptide, may
have been that ancestral role. We suggest that this putative
ancient role of Gla was recapitulated to facilitate folding of some
of the very rapidly evolving, unusually small Conus peptides.
Such a folding mechanism for proteins may have been more
generally important earlier in evolution, but it was probably
largely supplanted later by other mechanisms for facilitating
folding of larger polypeptides, such as specialized molecular
chaperones (20, 21~.
The role of Gla suggested above provides an attractive general
mechanism for folding small polypeptides, perhaps even includ-
ing the primordial proteins. Based on structural work on signal
recognition particle peptides, it was recently suggested (22) that
the first proteins evolved as membranes formed, when RNA still
dominated biochemistry. Specifically, the first functional
polypeptide-like chains in incipient life forms were created to
deal with a membrane surrounding the catalytic/informational
RNA. If this view is correct, then the possibility is raised that
~y-carboxyglutamate, with its capacity both for interacting with
membranes and directing folding may have been preserlt in the
earliest functional polypeptides, which were presumably much
smaller than present-day proteins. Once a Ca2+-free cytosol
evolved, however, a doubly negatively charged amino acid might
become a liability for intracellular protein function, and in most
taxa at the present time, ~y-carboxyglutamate is probably largely
a relict amino acid in a few secreted proteins. This modification
remains prominent only in those present-day phylogenetic sys-
tems where more specialized uses have evolved (such as mam-
malian blood clotting and Conus venom peptides) (Fig. 3~.
The canonical organization of conopeptide precursors, into an
N-terminal signal sequence, an intervening pro region, and a
single copy of the mature toxin region at the C terminus was first
established a decade ago (23~. The striking contrast found
between the conserved signal sequence and propeptide regions
versus the hypermutable mature toxin region led to the sugges-
tion that the conserved regions of the precursor were essential
for proper folding "such that the single biologically active
disulfide-bonded configuration can be specifically formed." It
was pointed out in the same paper that in vivo, additional factors
to facilitate the folding process might be required, such as PDI;
in this work, we provided the characterization of PDI from
Conus venom ducts. Evidence that posttranslational modifica-
tion enzymes may also be extrinsic factors that facilitate proper
folding was presented above. For both PDI and posttranslational
modification enzymes, the propeptide region may play a key role
by providing anchor sites, so that these factors can act efficiently
on the mature toxin region. This has been directly demonstrated
for the posttranslational modification enzyme that carboxylates
glutamate to ~y-carboxyglutamate, a specific sequence present in
the conotoxin precursor propeptide region is recognized by the
enzyme (24~. Evidence that PDIs facilitate proper folding of a
conopeptide precursor was also recently obtained (O.B. and
G.B., unpublished results). Thus, the relatively conserved
propeptide regions may have a function in establishing the
specific disulfide bonding conformation of Conus peptides.
~An obvious question is why y-carboxygiutamate in C texti/e peptide is not present in C
g/oriamaris pepticle. C texti/e peptide is from a tropical Conus species from a warm,
shallow-water habitat. C g/oriamaris peptide is from a deep-water species (~100 fath-
oms); the significantly cooler habitat may minimize the pressure to facilitate folding bythe
presence of Gla. An alternative/adclitional rationale is that C texti/e peptide is a major
venom component, whereas C gloriamaris peptide was never directly detectecl in venom,
but only from a cDNA clone (implying that it may be a quantitatively minor venom
component). A higher production rate woulcl provicle adclitional selective pressure for
mechanisms that facilitate folding.
PNAS 1 November25, 2003 1 vol. 100 1 suppl. 2 1 14567
OCR for page 56
The high expression levels and multiple isoforms of PDI in
Conus venom ducts and the involvement of a posttranslational
modification enzyme are by no means the only adaptation that
cone snails have made to efficiently fold the small, highly
structured, rapidly evolving peptides in their venoms. The studies
described here only serve to identify candidates in what is likely
a long list of molecular adaptations necessary for the genus
Con us to implement their unusual biochemical/pharmacological
strategy for interacting with the prey, predators, and competitors
in their environment.
In closing, we address the link between the amplification and
diversification of conotoxins and macroevolutionary trends in
Conus. The fossil record indicates that the genus had its origins
after the Cretaceous extinction, with a modest initial radiation
in the Eocene (25~; molecular data are consistent with several
early lineages of Con us diverging from each other at this time (2,
26~. Together, the fossil record and molecular data suggest that
one of these early Conus lineages expanded rapidly at the
beginning of the Miocene, a radiation that basically continues to
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This work was supported by National Institute of General Medical
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Bulaj et al.
Representative terms from entire chapter:
venom ducts
