Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors.
Trang 1pheromone complex
Scott F Cummins1,2, Fang Xie3, Melissa R de Vries1, Suresh P Annangudi3, Milind Misra4,
Bernard M Degnan2, Jonathan V Sweedler3, Gregg T Nagle1and Catherine H Schein4,5
1 Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA
2 School of Integrative Biology, University of Queensland, St Lucia, Australia
3 Department of Chemistry and the Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA
4 Sealy Center for Structural Biology, Department of Biochemistry and Molecular Biology, Sealy Center for Vaccine Design,
University of Texas Medical Branch, Galveston, TX, USA
5 Department of Microbiology and Immunology, Sealy Center for Vaccine Design, University of Texas Medical Branch, Galveston, TX, USA
Pheromones play an important role in coordinating
male and female reproductive behavior in many
aqua-tic species Strikingly, however, relatively few peptide
or protein pheromones have been characterized in
invertebrates or vertebrates, as they are often difficult
to isolate in sufficient quantities to characterize either
biochemically or behaviorally Furthermore, as we have seen with the marine mollusk Aplysia, protein pheromones require interaction with other proteins to exert their effect Egg laying in Aplysia, which can be reliably induced by injection of egg-laying hormone, induces secretion of at least four distinct proteins from
Keywords
enticin; fibrillin; epidermal growth factor-like
domains; Marfan’s syndrome; signaling
network
Correspondence
C H Schein, 218 Clay Hall, Department of
Biochemistry and Molecular Biology, UTMB,
Galveston, TX 77555-0857, USA
Fax: +1 409 747 6000
Tel: +1 409 747 6843
E-mail: chschein@utmb.edu
(Received 29 June 2007, revised 15 August
2007, accepted 28 August 2007)
doi:10.1111/j.1742-4658.2007.06070.x
Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aply-sia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein–cell surface contact dur-ing fertilization and blood coagulation In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using
a cold shock promoter and purified by RP-HPLC CD spectra confirmed a predominantly b-sheet structure Two disulfide bonds were determined via limited proteolysis and MS One internal disulfide (Cys57-Cys77) was pre-dicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca2+-binding, EGF-like domain of the extracellular matrix protein fibrillin Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a pro-posed metal-binding loop The water-borne pheromone attractin in egg cor-don eluates is complexed with other proteins Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors Gel shifts confirmed that temptin complexes with wild-type attr-actin These results indicate that temptin, analogous to the role of fibrillin
in controlling transforming growth factor-b concentration, modulates pheromone signaling by direct binding to attractin
Abbreviations
EGF, epidermal growth factor; HFBA, heptafluorobutyric acid; IPTG, isopropyl thio-b- D -galactoside; TEE, translation-enhancing element; TGF-b, transforming growth factor-b.
Trang 2the albumen gland that play a role in water-borne
pheromone signaling The pheromone signal must
radi-ate long distances (tens of meters from the secreting
Aplysia organism) to attract other Aplysia organisms
to fresh egg cordons [1,2] Binary blends of attractin
and either temptin, enticin, or seductin, three other
characterized Aplysia protein pheromones that are
released during egg laying, are sufficient to attract
ani-mals in T-maze assays [3,4] The first water-borne
pro-tein pheromone characterized in invertebrates was
the water-borne Aplysia pheromone attractin [5,6], a
58-residue protein released during egg laying that is
involved in forming and maintaining egg-laying and
mating aggregations in Aplysia [6–9] The
disulfide-bonding pattern and NMR solution structure indicated
that attractin has a novel helical fold that most
resembles the pheromones identified for unicellular
organisms [10–12] We have also characterized the
disulfide-stabilized helical fold of enticin as being
simi-lar to that of attractin [13] In this article, we provide
biophysical evidence that temptin, a third member of
the complex, resembles the disulfide-linked, primarily
b-strand structures of members of the epidermal growth
factor-like (EGF), family and functions in pheromone
signaling by binding to attractin in the protein bouquet
Native temptin, a 103-residue protein, was purified
from albumen gland extracts by RP-HPLC,
character-ized by N-terminal microsequence analyses of the
puri-fied protein and of tryptic fragments, which defined
the site of signal sequence cleavage, and cloned [3]
Temptin was 91% conserved between two
geographi-cally and physigeographi-cally distinct species, Aplysia californica
(Pacific coast) and Aplysia brasiliana (Gulf of Mexico)
[3] Sequence analysis of temptin indicated it would
have a fold quite different from that of other members
of the pheromone bouquet, such as enticin and
attrac-tin Fold recognition server results and a blast search
indicated that temptin would have a fold similar to the
Ca2+-binding, EGF-like domain of the extracellular
matrix protein fibrillin [2] This finding immediately
suggested a function for temptin in pheromone
signal-ing, as mutations in the multiple repeats of the
EGF-like domains in fibrillin have been linked to Marfan’s
syndrome, an autosomal dominant disease affecting
connective tissues [14,15] Whereas the disease was
originally thought to be due to loss of elasticity in
con-nective tissues, recent results show instead that fibrillin
specifically binds to and controls the available levels of
transforming growth factor-b (TGF-b), and that one
treatment for Marfan’s syndrome is based on
control-ling binding of TGF-b to its receptor [16] Here, we
present evidence that temptin has a similar role in the
pheromone complex to that of fibrillin in the
extra-cellular matrix, and mediates binding of attractin to sensory cells in the chemosensory rhinophores of target Aplysia
In the present study, we expressed and purified temptin and characterized its secondary structure with
CD measurements Recombinant full-length temptin was efficiently expressed in a soluble form using a recently developed ‘cold-shock’ method for protein expression in Escherichia coli [17,18], as demonstrated
by immunoblot analysis We determined temptin’s disulfide-bonding pattern using a combination of lim-ited proteolysis and MS The CD spectrum of the puri-fied protein indicated that temptin was largely b-sheet
in structure, consistent with the suggested homology to EGF-like domains of proteins involved in coagulation and interaction with the cell surface [2] One of the disulfide bonds, determined by limited proteolysis and
MS, was consistent with the previous alignment of the temptin sequences with EGF-like domains from other proteins The closest related known structure, detected
by fold recognition servers, was that of the type 1,
Ca2+-binding, EGF-like repeat domains that are con-served in the fibrillins, notch proteins, and clotting factors We generated a three-dimensional homology model for temptin, based on that of human fibrillin type I (Protein Data Bank file 1EMN), that was con-sistent with these data, and showed an extended, disul-fide-stabilized hairpin-like structure with a stabilized metal-binding loop We also found, using protein gel shifts, that recombinant temptin formed complexes with attractin that resembled those in egg cordon elu-ates Docking results with our model of temptin, and our previously determined NMR structure of attractin, indicated a preferred conformation for the complex that was consistent with these results Thus, temptin may play a role during pheromone detection by organizing the interaction of attractin with the cell membrane Further studies of temptin, e.g with multi-dimensional NMR, should reveal how similar the structure of this protein pheromone is to those of other EGF-like proteins, and yield more information about its role in signal transduction in Aplysia
Results
Recombinant expression of temptin using pCold III vector
Using temptin cDNA as template, the region enco-ding the mature temptin protein was amplified by PCR and the resulting PCR product was cloned into
a pCold III vector The insert was present as deter-mined by PCR analysis and nucleotide sequence
Trang 3analysis SDS⁄ PAGE analysis demonstrated that
temptin expression was induced by 0.1 mm isopropyl
thio-b-d-galactoside (IPTG) after 20 h of incubation
of bacteria at 15C, as judged by Coomassie Blue
staining (data not shown) Recombinant temptin was
soluble, as the protein was detected in the
superna-tant following sonication and centrifugation of E coli
lysates Following C18 Sep-Pak purification and C18
RP-HPLC purification of bacterial lysates containing
temptin (see below), the presence of temptin was
verified by Coomassie Blue staining (Fig 1A; left
panel) and western blot analysis using temptin
anti-serum (Fig 1A; right panel) Temptin was
sequen-tially purified on C18 Sep-Pak cartridges and by C18
RP-HPLC using heptafluorobutyric acid (HFBA) as
counterion An immunoreactive temptin peak was
obtained in pooled fractions 124–134 (Fig 1B) The
protein was further purified on a second RP-HPLC
gradient using trifluoroacetic acid as counterion
(data not shown) for CD spectra analyses and
disul-fide bond determination Temptin concentration was
estimated by densitometry (integrated density value)
following each stage of purification, and temptin was
purified 9.0-fold relative to the bacterial cell lysate
supernatant, with 23.9% recovery after the first
RP-HPLC purification
CD spectra of temptin The CD spectrum of purified temptin (Fig 2; 0.3 mgÆmL)1) was interpreted with the cdsstr program [19,20] This indicated that the protein was primarily b-strand⁄ turn, with < 10% helix This was consistent with our alignment with the predominantly b-strand EGF-like domains of fibrillins (Fig 3) A temptin-like expressed sequence tag cDNA that was recently isolated from the mollusk Haliotis [21] also aligns well with the temptin family Fold recognition servers suggested 1EMN (fibrillin 1 in the sequence alignment of Fig 3) as a potential template However, temptin contains only four cysteine residues, and only two of these aligned with cysteine residues involved in disulfide bonds in the conserved EGF-like fold
Determination of the disulfide bond pattern
in temptin The full-length amino acid sequence of mature temptin predicted by the cDNA is shown in Fig 4; a 13-resi-due N-terminal translation-enhancing element (TEE) sequence encoded by the pCold vector is included in the final sequence We next determined the disulfide-bonding pattern of the protein experimentally Purified
Fig 1 Expression and purification of recombinant temptin (A) SDS ⁄ PAGE and western blot analysis of recombinant temptin expression Temptin expression was induced by 0.1 m M IPTG After 20 h of induction, bacteria were lysed and the supernatant was fractionated in paral-lel SDS-polyacrylamide gels, one for Coomassie Blue staining (left panel) and the other for western blot analysis using temptin antiserum (right panel) The amount of soluble protein added in each lane corresponded to 30 lL of an initial 10 mL culture The Coomassie Blue-stained gel in the left panel indicates the level of purity of temptin following C18 Sep-Pak Vac purification and two successive C18 RP-HPLC gradient purification steps using HFBA and trifluoroacetic acid, respectively, as counterions Arrow, temptin protein (B) RP-HPLC purification
of pCold-expressed lysate containing recombinant temptin The supernatant of a temptin-containing bacterial extract was purified on a C18 Sep-Pak Vac cartridge and fractionated by C18 RP-HPLC The sample was eluted with a linear gradient of 0.1% HFBA and 100%
CH3CN ⁄ 0.1% HFBA Fractions indicated by the solid bar were pooled and lyophilized Pooled fractions 124–134 from several RP-HPLC runs were repurified with a linear gradient of 0.1% trifluoroacetic acid and 100% CH 3 CN ⁄ 0.1% trifluoroacetic acid (data not shown) and analyzed
by Coomassie Blue staining and western blot analysis (A).
Trang 4recombinant temptin expressed using the pCold III
vector was assayed by MS and had a monoisotopic
mass of 12405.7 Da, which corresponded to the
pre-dicted mass of temptin with the addition of a
13-resi-due N-terminal bacterial TEE (resi13-resi-dues ) 13 to ) 1 in
Fig 4)
The pCold III-expressed temptin was successfully
used to determine the disulfide connectivity Initial
reduction and alkylation experiments resulted in the
addition of four alkyl moieties, indicating the presence
of two disulfide bonds in temptin (data not shown)
To deduce the bonding pattern, the protein was
ini-tially digested with trypsin, which cleaves after lysine and arginine residues The enzymatically digested peptides were analyzed further by reduction–alkyl-ation Figure 4 shows several MS spectra obtained fol-lowing enzymatic digestion and reduction–alkylation treatments that enabled determination of the disulfide linkages
Following trypsin digestion of temptin, four peaks were detected (1679.7 Da, 1814.7 Da, 2231.0 Da, and 6430.4 Da) that were consistent with four peptides containing free cysteines at positions 103, 57, 77, and
18, respectively In addition, two other key peaks were detected (4043.6 Da and 8106.8 Da) that were consis-tent with fragments containing disulfide bonds between Cys57 and Cys77, and Cys18 and Cys103, respectively This indicated that the first and fourth cysteines were disulfide-bonded and the second and third cysteines were disulfide-bonded To confirm this pattern, trypsin digests were subjected to reduction–alkylation Follow-ing this treatment, only four alkylated peaks (1737.5 Da, 1872.6 Da, 2289.2 Da, and 6488.7 Da) were detected, indicating that the disulfide bonds in those two peaks (4043.6 Da and 8106.8 Da) were bro-ken during reduction, and each peak was brobro-ken into the two corresponding alkylated peptides with free cysteines These results confirmed that the disulfide-bonding pattern in recombinant temptin was Cys18-Cys103 and Cys57-Cys77 in the mature protein
Three-dimensional modeling of temptin The sequences of both A californica and A brasiliana temptin aligned well with the EGF-like domains of the
Fig 2 The CD spectrum of HPLC-purified recombinant temptin.
The CD spectrum (+ signs) for temptin is shown overlaid with the
CDSSTR 2-reconstructed spectrum (rectangles), which is based on
the protein sample containing 35% b-strand, 24% turn ⁄ coil, and
34% disordered structure.
Fig 3 Alignment of the central 1 region of temptin (residues 38–91 of the recombinant protein) with the central conserved Ca2+-binding, EGF-like domains of fibrillin sequences from human, chicken, and pufferfish Residues that are identical in fibrillins, known temptins (A cali-fornica, A brasiliana) and a temptin-like expressed sequence tag sequence (Haliotis asinina) are shaded black; identical and similar residues within the temptin family are in dark gray (red in the online version), and those that are similar to conserved residues in the fibrillins are shaded light gray (green in the online version) Cysteines involved in the disulfide bond that is common to the fibrillins and temptins are marked by the top line, and the residues involved in the proposed metal-binding loop are shown by the bottom line; see Fig 5C The Trp52 and Trp79 residues that flank the two disulfide bonds in our model (shown in Fig 5A) align with two conserved cysteines in the fibrillins.
Trang 5fibrillin family, with 11 of the residues being conserved
in all five fibrillins (Fig 3) Most significantly, the
interior disulfide bond in temptin, determined
experi-mentally, was also consistent with the disulfide bond
between two of the EGF-like cysteines that these
resi-dues aligned with We generated a model based on the
fibrillin 1 domain (Protein Data Bank structure
1EMN), which allowed us to generate the interior
disulfide based on homology and insert the second by
minimization (see Experimental procedures) The
resulting model (Fig 5) had several interesting
fea-tures The first was that the two tryptophan residues in
temptin, which both aligned with conserved cysteine
residues in fibrillins, flanked the two disulfide bonds
This suggested that they might serve as an
electron-rich barrier to stabilize these disulfides from reduction
Trp79, near the interior disulfide, could also stabilize
a possible negatively charged metal-binding loop (Fig 5C) This metal-binding site is part of a ‘top face’
of temptin that could interact with the cell surface, consistent with that seen for the interaction of EGF with its receptor [22]
Fibrillins form part of the extracellular matrix; recent research has also revealed an active role for these proteins in signal transduction, in which they bind growth factors To learn more about the possible intermediary role of temptin in the pheromone signal-ing process, we docked our previous NMR structure
of attractin [10] to the temptin model Two possible complexes (Fig 5B,D) show a potential binding sur-face on the bottom sur-face of temptin for attractin In both configurations, the surface of the second attractin
2231.0
1814.7 1679.7
2289.2
1872.6 1737.5
m/z
(Met-ox)
8106.8
6488.7
5x
A
B
C
D
T 101 -C 116
*
*
*
*
*
Q 64 -R 79
S 80 -R 100
Q 64 -R 79 &
S 80 -R 100
M 1 -K 59
M 1 -K 59 &
T 101 -C 116
E
Fig 4 Summary of disulfide linkage determination showing diagnostic MALDI TOF MS spectra of enzymatic digests of recombinant temp-tin Following digestion with trypsin, six key peptides were observed in (A), (C) and (E) The peak at m ⁄ z 6430.4 corresponds to the tryptic peptide Met13–Lys46 which includes the 13-residue bacterial TEE (Met13–Glu1 from the vector; dashed underline in sequence) and an oxidized methionine Peaks at m ⁄ z 4043.6 Da and 8106.8 Da are dimers that are each composed of two disulfide-bonded peptides After further reduction–alkylation of the whole enzymatic digest, four alkylated peptides were observed in (B) and (D) that confirmed the disulfide bond pattern (Cys18-Cys103, Cys57-Cys77) labeled on the sequence (E) is an enlarged spectrum of the boxed area in (C) The MALDI matrix is a-cyano-4-hydroxycinnamic acid in (A), (C), and (E), and sinapinic acid in (B) and (D) The residues shown by an asterisk are the potential cleavage sites (C-terminal) for trypsin.
Trang 6helix, which we previously showed by mutagenesis to
be essential for pheromonal attraction [8], is turned
outwards, whereas the first helix forms a multitude of
contacts (atom–atom distances less than 3 A˚) to the
bottom face of temptin These complexes suggest how
temptin could mediate binding of attractin to
phero-mone signaling proteins in the rhinophores of Aplysia
Temptin complexes with attractin to form
complexes similar to those seen in egg cordon
eluates
We used gel shifts and immunoblotting to determine
whether temptin would indeed complex with attractin
(Fig 6) Essentially all the immunoreactive attractin
in the extracts from the egg cordon of A californica
is complexed with other proteins, as it runs signifi-cantly above the position of the free protein Adding recombinant temptin to attractin, at an equimolar ratio, was sufficient to shift all the immunoreactive protein to a higher position on the gel, similar to that seen for the egg cordon eluate This indicates that temptin can form complexes with attractin, which may enhance its binding to sensory cells in the target Aplysia (or prevent degradation of the protein) Attempts to determine which face of attrac-tin was involved in the binding, using our previously described mutant proteins [8], were unsuccessful, as both of these proteins were difficult to detect with the attractin antibody
A
C
B
D
W79 W52
C18-C103
C57-C77
Backbone
O-G65,D64, S61, G63 R66
OD2-D64 OD1,2-D62
W79
S67-O
Fig 5 Views of a three-dimensional model structure of temptin alone (A, C) and in docked complexes with attractin (B, D) (A) Ribbon dia-gram of the temptin model, showing the two disulfide bridges (Cys18-Cys103, Cys57-Cys77) and their flanking tryptophan residues (Trp52 and Trp79, respectively) (C) Solid view of the model, same orientation as (A), showing the proposed metal-binding loop stabilized by Trp79 (right side, labeled residues) (B, D) Ribbon diagram (B) and space-filling view (D) of possible complexes of temptin with attractin (the lowest and 10th lowest energy complexes from ZDock, respectively) Temptin is in approximately the same orientation as in (A) and (C), but is tilted slightly away from the viewer (rotation arrow to the right) to show the interacting face with the first helix of attractin; attractin residues that are in closest contact with temptin are Met15 and Cys20–Thr28 The side chains of interacting residues in (B) are colored blue for temptin and brown for attractin In (D), attractin atoms are colored according to the Corey–Pauling–Koltun convention (O, red; C, black; N, blue; S, yellow), whereas all atoms of temptin are colored khaki In both (B) and (D), the residues in the second helix of attractin, which constitute the conserved pheromone signaling site [8], face outwards towards the viewer.
Trang 7Expression of temptin in other Aplysia tissues
The sequencing of the A californica genome (http://
www.ncbi.nlm.nih.gov⁄ ) revealed several isoforms of
temptin (temptins 2–5 in Fig 3) Northern blot
analy-sis (Fig 7) indicated these other isoforms may be
expressed in other Aplysia tissues, particularly in the
ovotestis, central nervous system, and buccal muscle;
the sizes of the cDNAs agreed with the predicted sizes
of the albumen gland transcript This suggests that
temptins may have similar, but not identical, functions
in other Aplysia tissues, and may indeed bind other
Aplysiagrowth factors in these tissues
To date, no molluskan fibrillin has been
character-ized A tblastn search of the A californica genomic
sequence database at NCBI GenBank (http://www
ncbi.nlm.nih.gov⁄ ), using the Homo sapiens sequence
of fibrillin 1 (accession number CAA45118) revealed
one candidate sequence, contig AASC01127679.1 This
encodes a protein similar to those of the Notch protein
family, conserved throughout the Metazoa, which are
membrane receptors that contain Ca2+-binding
EGF-like repeats similar to those of fibrillins [23] Notch
proteins use these repeats to control extracellular
signaling, in areas distinct from fibrillins This lends
further support to our suggestion that molluskan temptin is derived from a common ancestral gene that also yielded EGF and the EGF-like family of signaling proteins
Discussion
We originally identified temptin as one of four proteins that were expressed in the exocrine albumen gland and released during egg laying, and showed, using T-maze attraction assays, that temptin was an essential compo-nent of the pheromone bouquet that attracts Aplysia
to freshly laid egg cordons [3] Here, we expressed and purified recombinant temptin (Fig 1) to use in struc-tural and functional studies Our results indicate that temptin has a fold and function similar to those of repeat EGF-like domains in human fibrillins [24] The evidence for this is as follows: (a) the CD spectrum of the purified recombinant protein is predominantly b-strand (Fig 2); (b) the sequence alignment (Fig 3) shows the conservation of residues in both temptin sequences, and the other isoforms known, with those common to all fibrillins; (c) this alignment also predicts the correct position of a disulfide bond found for recombinant temptin (Fig 4); (d) modeling and
Fig 6 Gel shift experiments show that recombinant temptin forms complexes with recombinant attractin that are similar in size to the com-plexed forms of attractin seen in egg cordon eluates A western blot is shown, developed with antibody to attractin Recombinant attractin (1 nmol) was combined with a 1 : 1 or 1 : 5 molar excess of temptin, fractionated on a native gel, and transferred as described in Experi-mental procedures The picture is a composite of three experiments, with a repetition of the temptin–attractin equimolar complex From left
to right, attractin, temptin, and the complexes, a Coomassie Blue stain, followed by a blot of egg cordon eluates, and a repeat gel showing attractin, temptin and their 1 : 1 complex For Coomassie Blue visualization, proteins were loaded at 5 nmol each Arrow 1 shows the posi-tion of unshifted, native attractin, Arrow 2 indicates the middle of the shifted attractin–temptin complex (which migrates nonuniformly on the nondenaturing gel).
Trang 8docking results show a structure that could mediate
the interaction of attractin with the cell surface of
tar-get Aplysia (Fig 5), and gel shift results show the
for-mation of a stable, high molecular weight complex of
temptin with attractin (Fig 6) Although the disulfide
bonds are fewer than those that stabilize other
EGF-like proteins, our model suggests that those in temptin
are stabilized by a novel mechanism Individual
trypto-phan side chains overlay both disulfides, suggesting
that they could serve as electron-rich barriers to
reduc-tion in seawater A region between the two cysteines
that form the internal disulfide (Fig 5C) has a
nega-tively charged surface ideal for binding hard metals
such as Ca2+or Mg2+[25] Temptin might thus
repre-sent a homolog of the EGF family One could propose
that the mechanism of structure stabilization by
over-lapping disulfides that stabilize the EGF fold [26]
evolved from a simpler architecture in more oxidizing
environments Further evidence for this is that the
pufferfish homolog of fibrillin (Fig 3) has a valine in
place of one of the conserved six cysteine residues
characteristic of mammalian EGF repeats
Our results are particularly important, as they
assign a role for temptin in the pheromone signaling
pathway that may have evolutionary significance Binary combinations of attractin and temptin are sufficient to stimulate mate attraction and are thought to act in concert with enticin and seductin, which are also released during egg laying [2,3] The presence of an expressed temptin-like cDNA in the abalone Haliotis (Fig 3) and several temptin-like iso-forms within the genome of the limpet Lottia (data not shown) suggests that a temptin-like protein could play a similar role in coordinating pheromone signal-ing in other mollusks Blends of soluble pheromones have also been characterized in vertebrates, e.g the salamander Plethodon, which contains a pheromone blend consisting of plethodontid-modulating factor, plethodontid receptivity factor, and sodefrin precur-sor-like factor [27] Plethodontid-modulating factor is
a hypervariable courtship pheromone that is a mem-ber of the three-finger protein superfamily, all of which share a relatively simple three-dimensional structure consisting of three adjacent ‘finger-like’ loops extending from a small, hydrophobic core that
is crosslinked by a common disulfide bond bridging pattern All plethodontid-modulating factor sequences contain a conserved 20-residue signal sequence and
a pattern of eight cysteines that is also found in cytotoxins and short neurotoxins from snake venoms,
as well as xenotoxins from Xenopus [27]
Expression and biophysical characterization of recombinant temptin
Temptin was efficiently expressed in a soluble form using the pCold bacterial expression system (Fig 1A) Although temptin has been previously expressed in insect cells [3], the present study demonstrates a better method for expressing the protein, as: (a) larger amounts of temptin protein are obtained relative to the insect cell expression system (data not shown); (b) temptin comprises a significant percentage of the expressed peptides and protein, as judged by RP-HPLC (Fig 1B); and (3) the labeling of bacterially expressed proteins in defined media for NMR struc-tural studies is relatively inexpensive as compared to using the insect cell expression system
An important question is whether the recombinant protein folded in a similar way to the native protein The same disulfide folding was observed when using native and recombinant Aplysia enticin [13] In the case of enticin, the additional 13-residue tag at the N-terminus of recombinant protein, which contained a bacterial TEE, did not affect the activity or pattern of Cys-Cys bonding, as the disulfide-bonding patterns were identical in native and recombinant enticin Thus,
Fig 7 Tissue distribution of Aplysia temptin mRNAs The blot was
overexposed to detect the presence of isoforms in tissues outside
the albumen gland Total RNA (10 lg) was isolated from the
albu-men gland, atrial gland, large hermaphroditic duct (LHD; combined
red and white hemiduct), small hermaphroditic duct (SHD),
ovotes-tis, pooled central nervous system (CNS; pooled cerebral, pleural,
pedal, buccal, and abdominal ganglia) and buccal muscle, and
frac-tionated on agarose ⁄ 1% formaldehyde gels, and the membranes
were hybridized with radiolabeled cDNA probes for temptin RNA
size markers (Invitrogen) are indicated Equivalent amounts of RNA
were loaded in each lane and confirmed by ethidium bromide
stain-ing Autoradiography was performed for 24 h.
Trang 9the pattern indicated by trypsin digestions⁄
MALDI-TOF MS analysis from the recombinant temptin (I–IV
and II–III) is most likely the correct one
Role of temptin as the glue in the attractin
complex
Our model of temptin, based on the class 1 EGF-like
domain of fibrillin [24], and the gel shift experiments
(Figs 5 and 6), indicate that this protein could serve to
organize the pheromone complex and facilitate
signal-ing, by binding to both the pheromone and a cell
sur-face receptor A crystal structure of the complex of
EGF with the extracellular domain of its receptor
indi-cated that EGF can bring two receptor domains
together, from two different binding surfaces [19] Our
docking and gel shift results indicate a similar role for
temptin in the signaling complex, whereby attractin
would bind with one conserved face to temptin, while
still displaying the residues on the second helix that
are essential for pheromone activity [8] Temptin could
also mediate the activity of another pheromone
com-plex protein, enticin [13]
This role for temptin would be consistent with a
direct role for fibrillin in mediating TGF-b signaling
that has direct therapeutic implications Data
accu-mulated since 1991 from Marfan’s syndrome patients
have shown mutations in fibrillins, particularly those
that affect the structure and number of EGF-like
domains Whereas this was taken to mean that the
symptoms were due to a weakness in the
extracellu-lar matrix that contains fibrillin, more recently it was
shown that mutations also decreased the ability of
fibrillins to bind and control the activity of TGF-b;
many symptoms of Marfan’s syndrome can also be
found in patients with mutations in the receptor for
this factor [28] Reagents that antagonize TGF-b
binding (such as losartan) can completely reverse the
symptoms of fibrillin mutants in a mouse model;
these results form the basis for a clinical trial in
humans with Marfan’s syndrome [16]
In an analogous fashion, we propose that temptin
modulates attractin signaling during the mate
attrac-tion process, and either enhances its ability to bind to
cells or controls its free concentration Alternatively,
complexation with temptin may be necessary for
attractin to bind to the host cell For example, the
parasitic protozoan, Eimeria tenella, synthesizes a
microneme protein (EtMIC4) containing multiple
extracellular Ca2+-binding EGF-like repeats [29] that
forms a high molecular mass ([EtMIC4]8[EtMIC5]4;
> 2 MDa) complex with a soluble protein EtMIC5
This complex, secreted to the protozoan surface,
mediates the binding of the parasite to mammalian cells [30]
Does temptin serve as Aplysia fibrillin?
Although our primary purpose was to determine the role of temptin in pheromone signaling, the northern blot (Fig 7) indicated expression of temptin isoforms
in muscle and other tissues We are only beginning
to understand the full functions of the extracellular matrix proteins, such as fibrillins [31], in mammalian intercellular signaling and control If temptin does indeed represent the mollusk fibrillin, then it may play
a more general role in the mollusk temptin isoforms expressed in the central nervous system and buccal muscle (Fig 7) of Aplysia, and support a comparable role in the binding of other proteins, such as growth factors or those involved in connective tissue organiza-tion
Conclusions
These results indicate that temptin has a disulfide-stabilized structure that resembles that of metal-bind-ing, class 1 EGF-like domains found in mammalian factors that play a major role in formation of the extracellular matrix As with these proteins, temptin could regulate the activity of the attractin complex
by mediating the binding of the potent pheromone signaling molecule, attractin, to chemosensory cells in the sensory rhinophores of target Aplysia Possibly, the signaling complex contains many copies of temp-tin, and further structural characterization of the Aplysia pheromone bouquet proteins should provide more details about how these molecules function in the signaling that initiates reproduction in mollusks The recombinant protein isolation methods developed during this work will aid in determining the three-dimensional structure of temptin, as we previously did for attractin [10]
Experimental procedures
Cloning of temptin in a pCold expression vector PCR was used to amplify residues 23–125 of the temptin precursor using A californica temptin cDNA as template (GenBank Accession number AY309079; bases 89–397); this corresponds to the mature full-length temptin protein [3] The sense primer (5¢-ACTTCTCGAGTACCCCCAAT ACCAG-3¢) was synthesized with an XhoI site (underlined) The antisense primer (5¢-TACTGAATTCTTAGCAC GATACTGTAGC-3¢) was synthesized with a stop codon
Trang 10followed by an EcoRI site (underlined) Samples were
heated at 94C for 3 min and amplified for 36 cycles
(94C, 1 min; 50 C, 1 min; 72 C, 1 min), and this was
followed by a 7 min extension at 72C PCR products were
cloned into the bacterial expression vector pCold III
(Takara Shuzo Co Ltd., Shiga, Japan) and transfected into
Origami (DE3) bacteria Recombinant plasmids were
sequenced to confirm that no base changes had been
intro-duced during PCR amplification
Expression and purification of recombinant
temptin and native temptin
Expression of recombinant temptin was optimized in
pCold III-transfected Origami bacteria by controlling both
temperature and IPTG concentration Single colonies of
bacteria were used to inoculate 10 mL of overnight cultures
and incubated at 37C in LB medium containing
ampicil-lin, with shaking An aliquot of the overnight culture was
inoculated into fresh LB medium (1 : 100) containing
ampi-cillin Bacteria were incubated at 37C to a D600 of 0.6,
chilled for 30 min at 15C, induced with 0.1 mm IPTG,
and grown for an additional 20 h at 15C Cells were
cen-trifuged (using a GLC-2B Sorvall centrifuge and HL-4
rotor at 4C), the pellets were frozen () 70 C) and
resus-pended (10 mm Tris⁄ HCl, pH 8.0, 2 mm EDTA) at 4 C,
lysozyme (100 lgÆmL)1) and Triton X-100 (0.1% v⁄ v) were
added, and the lysates were incubated for 15 min at 30C
Lysates were sonicated and centrifuged (20 000 g using a
J2-21 centrifuge, Beckman, rotor type JA-20 for 30 min;
4C), and the supernatant was heated (5 min; 95 C) and
fractionated by SDS⁄ PAGE to examine temptin expression
Supernatants were also purified on C18 Sep-Pak Vac
car-tridges (5 g; Waters Corp., Milford, MA, USA) prior to
RP-HPLC Peptides were eluted with 70% acetonitrile
(CH3CN)⁄ 0.1% HFBA and lyophilized, and the
lyophiliz-ate was resuspended in 0.1% HFBA and purified on a
semipreparative Vydac C18 RP-HPLC column (1· 25 cm)
using a two-step linear gradient (0–10% CH3CN⁄ 0.1%
HFBA in 5 min; 10–65% CH3CN⁄ 0.1% HFBA in
195 min) Fractions were pooled, lyophilized and repurified
using the same gradient conditions, except that 0.1%
tri-fluoroacetic acid was the counterion Native albumen
gland temptin was purified using RP-HPLC as previously
described [3]
SDS⁄ PAGE and western blot analyses
Protein was quantified using the Protein Coomassie Blue
Assay Kit (Bradford method; Bio-Rad, Hercules, CA,
USA) Sep-Pak- and RP-HPLC-purified samples of
temp-tin were mixed with sample buffer (75 mm Tris⁄ HCl,
pH 6.8, 20% glycerol, 10% 2-mercaptoethanol, 4% SDS,
0.25% Bromophenol Blue) and fractionated on 12%
SDS-polyacrylamide gels Gels were either stained with
Coomassie Brilliant Blue R-250, or were used for immu-noblot analyses, performed essentially as previously described [3]; membranes were incubated with temptin antiserum (1 : 500 dilution) Details of temptin antiserum production have been previously described [3] As a nega-tive control, the primary antiserum was replaced with temptin antiserum preincubated with the corresponding antigen (20 lgÆmL)1) Detection of temptin was performed using the ECL western blotting detection system (Amer-sham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions Band density on SDS⁄ PAGE gels was quantified using an Alpha Imager (Alpha Inno-tech Corp., San Leandro, CA, USA)
Gel shift analysis of complex formation between recombinant attractin and temptin
Proteins were resuspended in filtered seawater (1 nmol of attractin, 1 nmol of temptin, 1 nmol of attractin⁄ 1 nmol
of temptin, 1 nmol of attractin⁄ 5 nmol of temptin, 20 lg of egg eluate), and then mixed with native gel sample buffer (75 mm Tris⁄ HCl, pH 6.8, 50% glycerol, 0.25% Bromophe-nol Blue) to a final volume of 5 lL Preparations were applied to a 12% Tris⁄ glycine gel (375 mm Tris ⁄ HCl,
pH 8.0) and run at constant voltage (120 V) for 60 min Gels were used for immunoblot analyses, performed essen-tially as previously described [3] Details of attractin antise-rum production have been previously described [3] As a negative control, the primary antiserum was replaced with attractin antiserum preincubated with the corresponding antigen (20 lgÆmL)1) Blots were incubated with Super-Signal chemiluminescence reagent (Pierce, Rockford, IL, USA); light emission was detected with autoradiography films For Coomassie stain visualization, proteins were pre-pared as described above and loaded onto the same gel (5 nmol)
CD
To characterize the secondary structure of temptin, CD analysis was performed using a 0.1 cm path length cell at 0.2 nm intervals with two scans (190–250 nm) averaged for each at 25C (Jasco J-715 spectropolarimeter, JASCO, Easton, MD, USA) Lyophilized temptin was slowly dissolved in 10 mm sodium phosphate buffer (pH 6.5), centrifuged to remove precipitated protein at 15 000 g using
a 5415D Eppendorf centrifuge and GE 009 rotor, and then concentrated by centrifugation (7000 g using a J2-21 centri-fuge; Beckman, and rotor type JA-20) in a Centricon 3 con-centrator (Amicon, Beverly, MA, USA) to a volume of 0.5 mL After one more addition of buffer and concentra-tion, the protein was diluted with sodium phosphate (pH 6.5) to a final concentration of 0.3 mgÆmL)1 Far-UV
CD spectra were taken at a protein concentration of 0.1 mgÆmL)1 The resultant spectra were corrected for the