Tài liệu Báo cáo khoa học: Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family docx

Molecular and cellular specificity of post-translational
aminoacyl isomerization in the crustacean hyperglycaemic
hormone family
Ce
´
line Ollivaux
1,2,3
, Dominique Gallois
4
, Mohamed Amiche
4
, Maryse Boscame
´
ric
4
and Daniel Soyez
4
1 Universite
´
Pierre et Marie Curie – Paris 06, UMR 7150 Mer et Sante
´
,E
´
quipe Physiologie Compare
´
e des Erythrocytes, Station Biologique
de Roscoff, France
2 Centre National de la Recherche Scientifique, UMR 7150, Station Biologique de Roscoff, France
3 Universite
´
Europe

´
enne de Bretagne, UEB, Rennes, France
4 Equipe Biogene
`
se des Signaux Peptidiques, ER3, Universite
´
Paris, France
Introduction
Modification of the chirality of a single aminoacyl resi-
due within a peptide chain is a subtle and intriguing
mechanism that remains poorly known to date, and
which leads to structural and functional diversification
of eukaryotic bioactive peptides. Subsequent to the
study by Montecucchi et al. [1] describing the presence
of a d-alanyl residue at the second position of dermor-
phin, a powerful opioid peptide isolated from skin
secretions of the tree frog Phyllomedusa sauvagei, such
a phenomenon has been reported in vertebrates, in
different opioid peptides [2], in antibacterial and
haemolytic peptides from frog skin [3], as well as in
venom from the mammal Platypus [4] (Table 1).
In invertebrates, d-amino acid containing peptides
(DAACPs) were isolated from the nervous system of
molluscs and crustaceans [5], and from venom of a
Keywords
aminoacyl isomerization; confocal laser
scanning microscopy; crustacean
hyperglycaemic hormone; MALDI-TOF MS;
vitellogenesis inhibiting hormone
Correspondence

C. Ollivaux, Universite
´
Pierre et Marie Curie
– Paris 06, UMR 7150 Mer et Sante
´
,E
´
quipe
Physiologie Compare
´
e des Erythrocytes,
Station Biologique de Roscoff, 29682
Roscoff, Cedex, France
Fax: +33 1 44 27 23 61
Tel.: +33 1 44 27 22 62
E-mail:
(Received 29 March 2009, revised 23 June
2009, accepted 26 June 2009)
doi:10.1111/j.1742-4658.2009.07180.x
d-aminoacyl residues have been detected in various animal peptides from
several taxa, especially vertebrates and arthropods. This unusual polymor-
phism was shown to occur in isoforms of the crustacean hyperglycaemic
hormone (CHH) of the American lobster because a d-phenylalanyl residue
was found in position 3 of the sequence (CHH and d-Phe3 CHH). In the
present study, we report the detailed strategy used to characterize, in the
lobster neuroendocrine system, isomers of another member of the CHH
family, vitellogenesis inhibiting hormone (VIH). We have demonstrated
that the fourth residue is either an l-orad- tryptophanyl residue (VIH
and d-Trp4 VIH). Furthermore, use of antisera specifically recognizing the
epimers of CHH and VIH reveals that aminoacyl isomerization occurs in

specialized cells of the X organ–sinus gland neurosecretory system and that
the d-forms of the two neuropeptides are not only present in the same
cells, but, importantly, also are co-packaged within the same secretory
vesicles.
Abbreviations
CHH, crustacean hyperglycaemic hormone; DAACP,
D-amino acid containing peptide; gp, guinea pig; PTM, post-translational modification;
r, rat; rb, rabbit; VIH, vitellogenesis inhibiting hormone; XO, X organ.
4790 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS
spider [6] and cone snails [7]. l-tod-aminoacyl
isomerization, or epimerization, comprises a true post-
translational modification (PTM) of peptides [8], as are
glycosylation or phosphorylation, where an l-residue,
which is always the same in a given peptide, is con-
verted into its d-counterpart [9]. This PTM occurs at a
late step of peptide precursor processing, after the
cleavage of the propeptide [10]. The nature of the d-res-
idue varies according to the peptides, although it has
consistently been found at the extremities of the
sequence at the second or third position, mostly at the
N-terminus and more rarely at the C-terminal end,
except in several conotoxins (e.g. the contryphan, an
octapeptide with a d-tryptophan in the fourth position)
[11] (Table 1). This intriguing modification is likely to
involve a specific enzyme, designated as peptidylaminoa-
cyl-l ⁄ d-isomerase or, more simply, isomerase [12] or
epimerase [13]. Amino acid sequences of putative
enzymes have been obtained from venom of the spider
Agenelopsis aperta [14], and more recently from the skin
secretion of Bombina frogs [15]. Unexpectedly, these

enzymes appear to be totally unrelated with regard to
their structures and their target residues. In addition,
such an isomerase has been isolated from platypus
venom, but remains unsequenced [12,16].
Although d-amino acids have been mostly found in
the sequence of small (i.e. 3–40 residues) peptides,
remarkable exceptions include peptides belonging to
the crustacean hyperglycaemic hormone (CHH) family,
which are 72–83 residues long, elaborated in the major
crustacean neurosecretory system, the X organ–sinus
gland complex. In several crustacean species, two
epimers of CHH can be purified, which differ in the
configuration of the phenylalanyl residue at position 3
(i.e. either l or d). To date, the presence of both
CHHs (named CHH and d-Phe3 CHH) has been dem-
onstrated only in Astacoidea (crayfish and lobsters),
where CHH displays the same N-terminal aminoacyl
sequence, at least up to the tenth residue [17]. Phe3
isomerization has major physiological consequences on
CHH biological activity because the all-l-peptide is
strictly hyperglycaemic, whereas d-Phe3 CHH may
exhibit, in addition to higher hyperglycaemic potency,
other functions, such as moult inhibition [18] or osmo-
regulation [19]. At the present, it is unclear whether
functional differences between CHH isomers are
related to binding to specific receptors or to differences
in haemolymphatic clearance rate. Indeed, DAACPs
are known to be more stable because they are less sus-
ceptible to protease degradation. As noted above,
CHH constitutes the archetype of an original peptide

family mostly found in crustaceans, the CHH family,
which contains other essential neurohormones, such as
moult-inhibiting hormone, mandibular organ inhibiting
hormone and vitellogenesis inhibiting hormone (VIH;
also called gonad inhibiting hormone) [20].
In Homarus americanus, where d-Phe3 CHHs were
first identified [21], VIH is present in the neurohaemal
organs, the sinus glands, as two isoforms (VIH I and
II) with an identical 77-amino acid sequence, molecu-
lar mass (9135 Da) and pI (6.8) [22]. Only one cDNA
was cloned, encoding a precursor with a signal peptide
directly flanking the progenitor VIH sequence [23].
With regard to its biological functions, VIH may inhi-
bit vitellogenesis synthesis in ovary or at extra-ovarian
sites and also may inhibit vitellogenin uptake in
oocytes [24]. When American lobster VIHs were tested
in a heterologous in vivo assay, only VIH I, the hydro-
philic form, demonstrated significant inhibitory activity
with respect to repressing oocyte growth that had been
induced by eyestalk removal in grass shrimps [25]. To
date, no function has been assigned to VIH II, the
hydrophobic form.
VIH has also been detected in male American lobster
sinus gland [26], in the Norway lobster Nephrops nor-
vegicus [27] and in the woodlouse Armadillidium vulgare
[28]. In the latter species, sinus gland grafting experi-
ments have suggested that VIH could be involved in
androgenic gland growth [29].
In the present study, we describe an experimental
strategy that was applied to determine the difference

between VIH I and II from the X organ–sinus gland
system of the lobster H. americanus. We demonstrate
that VIHs differ in the chirality of the tryptophan at
position 4. This result has been exploited to develop
specific antisera recognizing specifically the N-terminal
end of VIH and d -Trp4 VIH, which has allowed
Table 1. D-amino acid containing peptides in animals. Bold and
underlined letters indicate the
D-residues. CHH, crustacean hyper-
glycemic hormone; VIH, vitellogenesis inhibiting hormone; OvCNP,
Ornithorhyncus venom C-type natriuretic peptide; DPL, defensin-
like peptide.
Organism Tissue Name Sequence Reference
Frog Dermal gland Dermorphin Y
AFGTPSNH2 [1]
– – Bombinin H I
IGPVLG [3]
Platypus Venom gland OvCNP L
LHDHPN [32]
– – DLP I
MFFEMQ [4]
Snail Ganglia ⁄ heart Achatin G
FAD [31]
Mussel Muscle FFRF amide A
LAGDHFFRFNH2 [52]
Aplysia Heart NdWamide N
WFNH2 [53]
Cone snail Venom duct Contryphan GChP
WEPWC [11]
Spider Venom gland Xagatoxin MEGL

SFA [50]
Lobster Sinus gland CHH pQEV
FDQAC [21]
– – VIH ASA
WFTN Present
Study
C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells
FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4791
identification of VIHs from the European lobster
Homarus gammarus sinus gland extracts [30].
In such a complex system as this, where l- and
d- epimers of different neuropeptides are present in the
neuroendocrine organ, a puzzling question remains
unanswered: how are these peptides distributed in the
different cells of the X organ–sinus gland system? In
other words, what is the cellular specificity of the isom-
erization process? To answer this question, the distri-
bution of VIH and CHH isomers within the lobster
X organ–sinus gland complex was studied by immuno-
fluorescence ⁄ confocal microscopy. This technique was
supplemented by an immunogold-electron microscopy
study of the neuronal endings in the sinus gland to
determine the subcellular localization of the different
epimers. Taken together, these results allow a discus-
sion of the existence of enzyme(s) converting l-to
d- residues in the peptide chain in terms of substrate
specificity and availability.
Results
Purification and characterization of VIH I and II
VIHs were purified from H. americanus sinus gland

extract by RP-HPLC. Figure 1 shows a typical elution
pattern resulting from the fractionation of an extract
of 30 lobster sinus glands. The major peptides were
eluted between 38% and 40% acetonitrile, and were
identified as CHH B, d-Phe3 CHH B, VIH I, VIH II,
CHH A and d-Phe3 CHH A, respectively, according
to their elution order, by reference to a previous study
[21]. These assumptions were confirmed by a direct
ELISA performed on aliquots of the different frac-
tions, using antisera anti-4 (recognizing both VIHs)
and the two antisera discriminating CHH and
d-Phe3 CHH, guinea pig (gp)-anti-pQl and rabbit
(rb)-anti-pQd, respectively (not shown). Examination
of chromatograms from different sinus gland batches
shows a constant abundance ratio of the different pep-
tides, with the VIH I peak being half the size of the
VIH II peak (Fig. 1).
The rationale of our experimental approach for
identifying a putative d-residue in VIH was reliant on:
(a) DAACPs being generally more hydrophobic than
their l-counterparts in most DAACPs from eukaryotes
known to date, including CHH, and (b) the d-residue
being found near the N-terminal end, between the sec-
ond and the fourth position of the sequence. Conse-
quently, we hypothesized that the d-residue was
present in the N-terminal heptapeptide of VIH II, the
hydrophobic form. In a first attempt to identify the
putative d-residue in VIH, we considered that, in
CHH A and B, the d-amino acid is the phenylalanine
at position 3 [21]. We assessed whether the nature of

the modified residue (Phe3 in CHH and Phe
5
in VIH)
or its position (Phe3 in CHH and Ala3 in VIH) may
be conserved in CHH and VIH. Thus, heptapeptides
corresponding to the N-terminal sequence of VIH were
synthesized with different configurations (i.e. Hep-l,
Hep-dA3 and Hep-dF5). Samples of VIHs (ten sinus
gland equivalents, i.e.  50 pmol VIH I and
 100 pmol VIH II) were cleaved with endoproteinase
Asp-N and the resulting fragments were separated by
RP-HPLC. After fractionation of VIH I hydrolysate,
one fragment was eluted with the same retention time
as the synthetic peptide Hep-l (Fig. 2A). The mass of
this fragment was determined by MALDI-TOF MS to
be 796 Da, which is a value identical to the mass of
Hep-l. Because no other predicted fragment from
endoproteinase-Asp-N digestion of VIH displayed a
similar mass, this fragment could be identified as the
N-terminal heptapeptide of VIH I. After similar cleav-
age of VIH II by endo-Asp-N and RP-HPLC, MS
analysis indicated that a peptide with a molecular mass
of 795.9 Da was eluted at 31.5 min (i.e. later than
Hep-l, Hep-dA3 and Hep-d F5; not shown).
In a next step, to determine which residue may be in
the d-configuration in the fragments resulting from
VIH II digestion, we considered that numerous
DAACPs display a d-residue at position 2 (in snail
excitatory peptides [31], in frog opioid peptides [1] and
in platypus venom [32]). In addition, the tryptophan

residue at position 4 may be a good candidate because
contryphan, a conotoxin isolated from gastropod
venom has a d-Trp4 [11]. To test these hypotheses, the
synthetic peptides Hep-dS2 and Hep-dW4 were used
Acetonitrile (%)
39
39. 5
40
0.1 u AU
220 nm
CHH B
CHH A
D-Phe
3
CHH B
D-Phe
3
CHH A
49 53
Retention time (min)
VIH I
VIH II
45
41
Fig. 1. RP-HPLC profile of an acetic acid extract of 30 lobster sinus
glands. Only the part of the chromatogram where CHHs and VIHs
are eluted is shown. The nature of the ultraviolet absorbance peaks
was assessed by ELISA as well as by comparison with previously
published similar analyses [26].
Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al.

4792 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS
as standards. Figure 2B shows that the elution time of
Hep-dW4 is identical to that of the VIH II fragment.
By MALDI-TOF MS, the mass of this fragment was
found to be 795.8 Da, similar to the synthetic peptide.
Moreover, co-injection on RP-HPLC of the VIH II
fragment and Hep-dW4 resulted in a single symmetri-
cal peak (not shown). Therefore, the results obtained
indicated that the two VIH isomers differed by the
configuration of the tryptophan at the fourth position
of the sequence and, consequently, they were named
VIH and d-Trp4 VIH.
This assumption was further confirmed by immuno-
assays realized with the antisera directed against the
N-terminal decapeptides of VIH with all l-residues [rat
(r)-anti-l] or with the tryptophan residue in d-configu-
ration (gp-anti-dW4). Indeed, ELISA using affinity-
purified antisera with the different heptapeptides
demonstrated a strict specificity of these antisera for
the antigen peptide, with cross-reactivity for the other
heptapeptides being lower than 1% (not shown).
Accordingly, when ELISA was performed on fraction
aliquots from RP-HPLC of sinus gland extract (as in
Fig. 1), no signal was obtained using these antisera
with fractions corresponding to CHHs A and CHHs
B, whereas r-anti-l gave a strong signal with VIH I
only and gp-anti-dW4 with VIH II exclusively, which
confirmed unambiguously the presence of the d-Trp in
VIH II (not shown).
Characterization of cell types in relation to CHH

and VIH isomers
To study the distribution of CHHs and VIHs in the
X organ cells, immunohistochemical labelling of
whole-mounts of X organ–sinus gland complexes was
realized using different sets of antibodies. In addition,
immunogold labelling was performed on sinus gland
sections to localize these peptides at the subcellular
level, within secretory granules.
Localization of VIH and
D-Trp4 VIH in X organ
neuroendocrine cells
Confocal analyses of whole-mounts of X organ–sinus
gland complexes of the lobster H. americanus were per-
formed after double immunofluorescent labelling using
purified antisera r-anti-l and gp-anti-dW4. Different
cell types were observed: the larger neuroendocrine cell
bodies (70 ± 7 lm diameter soma) were strongly
labelled with gp-anti-dW4 (green cells; Fig. 3A), with
the labelling being cytoplasmic and granular; only
some of these cells, with a smaller diameter
(56 ± 7 lm) were also stained with r-anti-l, the yel-
low ⁄ orange colour, variable in a same organ, attesting
to labelling with both antisera. For the sake of clarity,
both types are subsequently referred to as d-VIH cells.
Smaller VIH-producing cells (31 ± 7 lm diameter
soma) were grouped in a distinct region. Their peri-
karya were immunoreactive with r-anti-l exclusively
(red cells called l-VIH cells). A total of 14 d-VIH cells
(nine green and five yellow cell bodies) and 19 l-VIH
cells (red soma) were counted per X organ. Most

Hep-L
Hep-DA3
0.01AU
220 nm
Hep-DF5
34
36
Acetonitrile (%)
32
Retention time (min)
26
30
34
22 38
Hep-DW4
36
Hep-L
Hep-DF5
Hep-
DA3
Hep-
DS2
0.01AU
220 nm
Acetonitrile (%)
32
34
Retention time (min)
26
30

34
22
38
A
B
Fig. 2. (A) RP-HPLC profile of VIH I digest (ten sinus gland equiva-
lents). Only the part of the chromatogram where fragments elute is
shown. The nature of the ultraviolet absorbance peaks was
assessed by comparison with retention times of standards (arrows)
(i.e. heptapeptides Hep-
L, Hep-DA3 and Hep-DF5) coupled with
MALDI-TOF mass analysis. (B) RP-HPLC profile of VIH II digest
(ten sinus gland equivalents). Compared with the previous analysis
shown in Fig. 2A, the synthetic heptapeptides Hep-
DS2 and Hep-
DW4 were added to the standard mixture.
C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells
FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4793
C
50 µm
A
50 µm
B
50 µm
R
LG
SG
XO
ME
MI

MT
H
20 µm
D
20 µm
E
50 µm
F
20 µm
G
50 µm
Fig. 3. Confocal micrographs of double immunolabelled whole mounts of lobster X organ–sinus gland. Images were collected as a focal
series and processed to create 2D projections (single composite images). Central drawing: schematic representation of the lobster eyestalk
nervous structures and the neuroendocrine complex (X organ–sinus gland). R, retina; LG, lamina ganglionaris; ME, medulla externa; MI,
medulla interna; MT, medulla terminalis. (A) General view of X organ labelled with r-anti-
L and gp-anti-DW4 to visualize small L-VIH cells (red)
and larger
D-VIH cells (green or yellow). (B) Axonal arborizations of both cell types observed in the sinus gland (red for L-VIH varicosities and
green for
D-VIH ones). (C) Distribution of CHH cells in X organ showing green cell bodies (L-CHH cells) labelled only with gp-anti-pQL and
orange somata (
D-CHH cells) corresponding to labelling with gp-anti-pQL and rb-anti-pQD antisera. (D) Enlargement of axon terminals in the
sinus gland showing both types of secretory granules corresponding to
L-CHH cells (green) and D-CHH cells (red). (E) Immunolocalization of
D-Trp4 VIH and D-Phe3 CHH in the X organ where three cell types were observed : D-CHH cells (red), D-VIH cells (green) and D-cells produc-
ing both
D-isomers (orange). (F) Enlarged view of the three cell types showing the variations of coloration for D-cells: D-CHH cells (red, thin
arrow),
D-VIH cells (green, short arrow) and D-cells producing both D-isomers (orange, long arrows). (G) Sinus gland axonal arborizations
containing

D-Trp4 VIH (green) and D-Phe3 CHH (red). (H) Enlargement of axon terminals in the sinus gland with labelling as in (G).
Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al.
4794 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS
d- and l-VIH cells appeared segregated, whereas
several somas of the two types were dispersed in the
X organ (Fig. 3A), most likely as a result of arte-
factual displacement of the cell bodies during the prep-
aration of the organ. In the sinus gland, different types
of axonal arborizations containing VIH, d-Trp4 VIH,
or very rarely both, were observed (Fig. 3B).
Ultrastructural observations after immunogold label-
ling of sinus gland sections reveal the presence of sev-
eral types of terminals differing by the morphology,
size and electron density of the secretory granules.
After double labelling with r-anti-l and gp-anti-dW4,
particles were observed on two categories of axon
terminals: those exclusively labelled with r-anti-l
(l-VIH terminals; Fig. 4A) and a few d-VIH terminals
labelled with gp-anti-dW4 (Fig. 4B). No terminals with
double labelling were found. In each terminal, the
secretory vesicles were densely packed and, although
scarce, the gold labelling was strictly restricted to them.
Localization of CHH and
D-Phe3 CHH in X organ
neuroendocrine cells
Whole-mounts of H. americanus eyestalks were
incubated with antisera gp-anti-pQl and rb-anti-pQ d,
recognizing CHH and d-Phe3 CHH, respectively.
Confocal micrographs revealed two distinct cell types:
either green, labelled only with gp-anti-pQl (l-CHH

cells) or orange ⁄ red, labelled with both gp-anti-pQl
and rb-anti-pQd antisera in variable proportions,
(d-CHH cells; Fig. 3C). In a cluster of 34 cells, 13
d-CHH cells and 21 l-CHH cells were localized in dis-
tinct regions of the X organ. The diameter of their cell
bodies was 64 ± 8 lm and 44 ± 7 lm, respectively.
Intensively stained axons could be traced to the sinus
gland where two types of axonal arborizations were
observed, either green or red. Rarely, orange colour-
ation attested the presence of both CHH types
(Fig. 3D).
Accordingly, when sinus gland ultrathin sections were
observed after double labelling by gp-anti-pQl and
rb-anti-pQd, two different categories of terminals were
revealed: l-CHH terminals, labelled with gp-anti-pQl
(Fig. 4C) and d-CHH terminals with a strong rb-anti-
pQd labelling (Fig. 4D). Those terminals were also
rarely labelled with gp-anti-pQl (Fig. 4E).
Distribution of
D-Trp4 VIH and D-Phe3 CHH in X
organ neuroendocrine cells
To address the question whether d-epimers of CHH and
VIH are expressed in the same cells or not, confocal
analysis of whole-mounts of X organ–sinus gland
complexes was performed after double immunofluo-
rescent labelling using specific antisera gp-anti-dW4
and rb-anti-pQd, recognizing d-Trp4 VIH and
d-Phe3 CHH, respectively. Three types of cells could
be distinguished: seven green cells strongly labelled
with the gp-anti-dW4 (56 ± 7 lm diameter soma; pre-

viously called d-VIH cells), ten red cells immunoreac-
tive with rb-anti-pQd antiserum (60 ± 5 lm diameter
soma; previously called d-CHH cells) and five yel-
low ⁄ orange cells stained with both antisera, simply
called d-cells (65 ± 7 lm diameter soma; Fig. 3E).
Among these latter cells, large differences in colour-
ation were observed as a result of variations in the
relative amounts of both d-isomers (Fig. 3F).
Immunohistochemical staining of axonal arborization
in the neurohemal organ showed the three cell types
with clustered granules immunoreactive either for one
antiserum or for both antisera (Fig. 3G,H).
To test the hypothesis of vesicular co-packaging of
d-epimers of CHH and VIH, double immunogold
labelling with various associations of antibodies
against the different forms was performed on ultrathin
sections for examination by electron microscopy.
Using rb-anti-pQd and gp-anti-dW4, specific d-VIH
and d-CHH terminals were observed (Fig. 4F–I).
Mixed terminals were also detected in other parts of
the sinus gland, demonstrating that d-Trp4 VIH and
d-Phe3 CHH were not only colocalized in the same
terminals (Fig. 4J), but also in same secretory vesicles
(Fig. 4K). These three categories of terminals were
usually found in different regions of the sinus gland,
as described above, but close juxtapositions of differ-
ent terminals were sometimes observed (Fig. 4G).
Discussion
Although the existence of two VIH isoforms with iden-
tical sequence, molecular mass and isoelectric point

has been known for more than 15 years [22], the nat-
ure of the difference between the two peptides had not
yet been elucidated. The demonstration of the presence
of a d-Phe3 residue in CHH A and B from the
H. americanus some years ago [21] opened the possibil-
ity that a d-residue may be present in one of the VIH
isoforms as well. Furthermore, in a previous study,
ELISA experiments using specific antibodies have
suggested the presence of a d-residue in VIH from
H. gammarus [30].
In the present study, we have demonstrated, using a
combination of RP-HPLC, MALDI-TOF MS (peptide
mapping) and immunoassays, that the most hydropho-
bic VIH isoform contains a d-tryptophanyl residue
at position 4, whereas a l-Trp4 is present in the
C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells
FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4795
500 nm200 nm 200 nm
500 nm500 nm
100 nm
500 nm
200 nm
200 nm 200 nm 500 nm
C
A
B
F
D
GH
I

K
J
E
Fig. 4. Sections of double immunogold labelling of axon terminals in the lobster sinus gland. (A, B) Double labelling with r-anti-L (10 nm gold
particles) and gp-anti-
DW4 (20 nm gold particles) antisera. (A) Axon terminal containing secretion granules with only VIH. (B) Axon terminal
containing secretion with
D-Trp4 VIH. (C–E) Double labelling with gp-anti-pQL (20 nm gold particles) and rb-anti-pQD (10 nm gold particles)
antisera. (C) Two neighbouring CHH axon terminals. (D) Axon terminal with only
D-Phe3 CHH. (E) Axon terminal showing some secretion
granules labelled with both antisera. (F–K) Double labelling with rb-anti-pQ
D (10 nm gold particles) and gp-anti-DW4 (20 nm gold particles).
(F) Axon terminal containing
D-Trp4 VIH. (G) Two neighbouring axon terminals, one containing D-Trp4 VIH and the other D-Phe3 CHH. (H, I)
Higher magnifications of
D-Trp4 VIH and D-Phe3 CHH containing granules, respectively. (J) General view of an axon terminal containing both
D-isomers. (K) Enlarged view of an ending with secretion granules labelled with both rb-anti- pQD and gp-anti-DW4 antisera.
Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al.
4796 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS
hydrophilic form. The results obtained therefore estab-
lish that the lobster neuroendocrine system elaborates a
mixture of epimers of two different neurohormones:
CHH, d-Phe3 CHH, VIH and d-Trp4 VIH. The coex-
istence, in a same organism, of several peptides with a
d-residue of different natures and positions has been
documented in venoms of cone snails [33] and of platy-
pus [32], and also in frog skin secretions [34]. Neverthe-
less, no general consensus for the isomerization site,
nor for the nature of the surrounding residues, could
be demonstrated to date, with the prediction of single

d-amino acid presence being relevant exclusively within
a well-defined and restricted peptide family, as illus-
trated by a recent study of the I
1
-conotoxin superfamily
[35]. In the case of the CHH family, the occurrence of
DAACPs appears to be problematic to predict because
CHH variants with a d-residue have been detected only
in Astacidea (rocky lobsters and crayfish), where the
N-terminal sequence is pGlu-Val-Phe-Asp-Glu-Ala
(with the d-residue always being the Phe3), and not in
CHH from other species with close overall sequence
similarity, such as the peneid shrimp Penaeus vannamei
with the sequence Ser-Leu-Phe-Asp-Pro-Ser . [36],
nor even in those species with an identical sequence of
four residues, such as the spiny lobster Jasus lalandii:
Ala-Val-Phe-Asp-Glu-Ser [37]. However, it is worth
noting that the presence of a d-amino acid in CHHs
from these last species may be overlooked because no
specific investigation procedure, such as amino acid
chiral analysis, was ever performed.
Even if the presence of DAACPs begins to be well
documented in various animal groups, this phenome-
non has been rarely studied at the cellular level.
Indeed, cellular aspects have been investigated in the
serous dermal gland of frogs. However, owing to the
peculiar syncytial structure of this organ, the localiza-
tion of dermorphin and its all-l counterpart in the dif-
ferent cell compartments was difficult to analyze [34].
By contrast, in crustaceans, DAACPs are elaborated in

discrete, well-identified neuroendocrine cells located in
an easily accessible organ, with these characteristics
having already allowed detailed studies of their bio-
genesis [10,38–41].
In previous studies, the expression of CHH and VIH
in H. americanus X organ–sinus gland complex was
investigated at the peptide level, but without distinc-
tion of epimers [42]. It was observed by immuno-
histochemistry that some cells were labelled only by
anti-CHH or anti-VIH antisera, whereas several others
were reactive with both antisera, as an indication of
the presence of both hormones in these cells. The
existence of mixed CHH ⁄ VIH cells was later confirmed
at the mRNA level [26]. When, at an early stage of
our study, we considered the cellular distribution of
CHH and VIH isomers in the lobster X organ, one
attractive simple hypothesis was that the mixed
(double labelled) cells observed previously could con-
stitute the synthesis site of the d-epimer of the two
hormones (d-Phe3 CHH and d-Trp4 VIH), whereas
the cells labelled exclusively by anti-CHH or by anti-
VIH would produce only the l-counterpart of each
peptide. To test this initial hypothesis, the organization
of CHH- and VIH- expressing cells, in relation to iso-
mers, was investigated by immunohistochemistry and
immunocytochemistry, using antisera specific to each
epimer.
Confocal examination of in toto preparations has
shown that approximately 33 cells were immunostained
with gp-anti-dW4 or r-anti-l antiserum, which agrees

with the results obtained in a previously study [26],
but diverges from those of other studies [42,43] report-
ing a smaller figure (approximately 20 cells). It may be
that the number of cells expressing VIH at a given
time varies according to the reproductive stage of the
animals, such as the haemolymphatic VIH level [44].
Regarding the CHH-producing cells, our observa-
tions indicate that their number (n = 34 soma were
counted) is close to the number of VIH cells (n = 33),
with a similar ratio between l-CHH cells (n = 21) and
d-CHH cells (n = 13) of approximately 1.6 versus 1.3
for VIH cells. This figure fits well with the ratio of
CHH ⁄ d-Phe3 CHH quantified in the lobster sinus
glands [45; present study], which is not the case of
VIH isomers, as noted earlier. On confocal mountings,
subtle variations in colouration between d-CHH peri-
karya were observed, although this much less pro-
nounced than for VIH cells. This agrees with the
results obtained in the crayfish Orconectes limosus,
showing that l-isomer of CHH is always present in the
different parts of the d-CHH cells, in decreasing
amounts from the cell body to the axon terminal in
the sinus gland, as a result of late and progressive
isomerization of the Phe3 of the CHH during the
migration of the secretion vesicles along the axonal
tract [39,41].
The immunohistochemical and immunocytochemical
results obtained in the present study invalidate our
starting hypothesis proposing that the cells coexpress-
ing VIH and CHH in the lobster X organ, as observed

in previous studies [26,42], were actually producing the
d-isomers of both hormones. Indeed, the lobster neu-
roendocrine system is more complex than expected
because, in addition to cells containing exclusively
l-isomers of CHH or VIH, three types of cells were
found, producing: (a) d-Phe3 CHH (d-CHH cells), (b)
d-Trp4 VIH (d-VIH cells) or (c) a mixture of both
C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells
FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4797
peptides (d-cells). Overall, five cell types producing
CHH and VIH could be identified in the lobster X
organ (Fig. 5). Nevertheless, it should be noted that
d-CHH cells, d-VIH cells and d-cells may correspond
to one single cell type producing a mixture of
d-Trp4 VIH and d-Phe3 CHH in different proportions
depending upon its physiological stage. Indeed, in situ
hybridization in combination with immunohistochem-
istry revealed that strong immunostaining of CHH and
VIH may coincide with a weak or null mRNA label-
ling and vice versa [26]. The existence of a sixth type
of cell, producing a mixture of l-epimers of CHH and
VIH had to been considered. It was researched by
double labellings of successive ultrathin sinus glands
sections (not shown), although this proved to be in
vain. Similarly, no terminals exhibited simultaneous
labelling for CHH and d-Trp4 VIH or VIH and
d-Phe3 CHH (not shown).
At present, it is not possible to assign a functional
significance to the colocalization of the CHH and VIH
epimers, especially because cellular colocalization of

different neurohormones of the CHH family is not a
general rule in the crustaceans. By contrast to the situ-
ation in the lobster, VIH and CHH are synthesized
and released by different cellular types in woodlouse
[46] and in Norway lobster [47]. Similarly, moult-
inhibiting hormone and CHH are localized in distinct
neurosecretory pathways in several crabs [48], whereas
colocalization of both peptides has been reported in
prawns [49].
The presence, in the same cells and even in the same
secretory granules, of peptides displaying a d-residue
of different nature (Phe or Trp) and at different posi-
tion (third or fourth) raises the question of the nature
and characteristics of the enzyme(s) responsible for this
modification. Indeed, only a few peptide isomerases,
exhibiting very different substrate specificity, have been
isolated so far, from the funnel-web spider A. aperta
[50], the frog P. sauvagei [15] and from platypus
venom [12,16]. Only structures of the first two are
known, which appear to be totally unrelated because
the frog enzyme presents similarities with the N-termi-
nal H-domain of human IgG-Fc binding protein and
the spider isomerase appears to belong to the serine
protease family. Obviously, the large structural and
functional divergences between these enzymes impede
any speculation about the characteristics of the puta-
tive lobster isomerase(s). Nevertheless, the occurrence,
in lobster X organ cells, of two distinct enzymes with
different substrate specificity and a variable expression
pattern appears as a likely working hypothesis.

Major challenges remaining for the future are the
identification of the putative peptide isomerase(s) in
crustaceans from these specialized cells from the X
organ–sinus gland complex of the American lobster as
well as the characterization of the receptors of CHH
and VIH epimers, aiming to provide insights on the
functional significance of the intriguing PTM that is
l-tod-aminoacyl isomerization.
Experimental procedures
Animals and peptide purification
H. americanus, weighing 300–500 g, were obtained from a
commercial supplier (Metro, Bobigny, France). To reduce
Fig. 5. General diagram of precursor pro-
cessing of VIH and CHH isomers in relation
to the different cell types in X organ–sinus
gland complex. CPRP, CHH precursor-
related peptide.
a
Amidation can be pre-,
co- or post-cleavage of CPRP.
b
Cylization of
CHH N-terminus is optional (N-terminus
unblocked CHH can be released) and, simi-
lar to isomerization, it occurs after CPRP
cleavage.
c
By contrast to CHH, VIH is not
N-terminal cyclized.
L-CHH and L-VIH cells

secrete exclusively CHH and VIH, respec-
tively, whereas
D-CHH and D-VIH cells
release mainly the
D-isomer of the respec-
tive hormone, in addition to a variable
amount of
L-isomer. D cells secrete mainly
the
D-form of both CHH and VIH. Besides
isomerization, the same PTMs occur in
every type of CHH or VIH cell.
Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al.
4798 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS
their stress, lobsters were maintained in the laboratory for
2 weeks before experimentation, in filtered and re-circulat-
ing artificial seawater at 13 °C. In the aquarium, hollow
bricks provided shelters for the animals, which were fed
weekly with mussels. The physiological status of the donors
(sex, moulting and reproductive stage) was not recorded.
The animals were anaesthetized on crushed ice for 30 min
before dissection.
VIH I and II were extracted from 30 H. americanus sinus
glands (sinus gland equivalents, i.e.  1.5 lg or 160 pmol
for VIH I and  2.7 lg or 280 pmol for VIH II). Peptides
were extracted and purified as described previously [10].
HPLC fractions from the elution zone of CHHs and VIHs
were collected manually to achieve optimal resolution
between CHH and VIH isomers, respectively. HPLC frac-
tions containing VIH I and II were dried under vacuum

(Speed-Vac, Savant Instruments Inc., Holbrook, NY,
USA).
Enzymatic cleavage and digest fractionation
Purified and dried VIHs (30 sinus gland equivalents) were
redissolved in 5 lL of acetonitrile ⁄ water (1 : 1, v ⁄ v) and
mixed with 0.4 lg of enzyme (Endoproteinase Asp-N
sequencing grade, EC 3.4.24.33; Roche Diagnostics, Baˆ le,
Switzerland) in 50 lL of phosphate buffer (50 mm, pH 8).
After 22 h at 37 °C under stirring, the reaction was stopped
by adding 5 lL of acetic acid (2 m). Then, the digests of
each VIH were fractionated on a Nucleosil C18 (5 lm,
250 · 2.0 mm; Macherey-Nagel, Du
¨
ren, Germany) con-
nected to the pump system and spectrophotometer. Peptides
were eluted from the column by a gradient of acetonitrile
in water at a flow-rate of 0.2 mLÆmin
)1
. Both solvents con-
tained trifluoroacetic acid (0.1% in water and 0.08% in ace-
tonitrile). HPLC fractions from the elution zone of
synthetic peptides (see below) were collected manually and
dried under vacuum.
Solid-phase peptide synthesis and antisera
production
Heptapeptides (Hep-l, Hep-dS2, Hep-dA3, Hep-dW4 and
Hep-dF5) with the sequence corresponding to the N-termi-
nus of lobster VIH and with all l -residues or a d-residue at
different positions (Table 2) were synthesized using solid-
phase FastMoc chemistry with a 433A Automated Peptide

Synthesizer (Applied Biosystems, Foster City, CA, USA) as
described previously [51]. The homogeneity of the synthetic
peptides preparation was assessed by MALDI-TOF MS
and analytical RP-HPLC.
Decapeptides named Dec-l and Dec-dW4 (Table 2), cor-
responding to the N-terminus of VIH I and II, respectively,
were commercially synthesized (Eurogentec, Angers,
France) and injected, after coupling to keyhole limpet
haemocyanin, into rats (for Dec-l) or guinea pigs (for
Dec-dW4) to generate polyclonal antisera. When necessary,
specific IgG was purified according to a batch procedure
that has been described previously [41].
MS
Positive-ion mass spectra were recorded in reflectron mode
with a single stage reflectron matrix-assisted laser desorp-
tion ⁄ ionization time-of-flight (MALDI-TOF) mass spec-
trometer (Voyager DE RP; Perseptive Biosystems Inc.,
Framingham, MA, USA) as described previously [51].
ELISA
Specificity assays
Direct ELISA was performed to determine the specificity of
antisera. The wells of a plastic microtitre plate (Nunc, Ros-
kilde, Denmark) were coated in triplicate with 100 ng of
synthetic hepta- or decapeptides. In addition to the antisera
r-anti-l (made in rat) and gp-anti-dW4 (produced in guinea
pig), two antisera discriminating CHH isomers (gp-anti-pQl
made in guinea pig and rb-anti- pQd in rabbit) [38] and an
antiserum raised against purified VIH II (i.e. anti-4 pro-
duced in guinea pig and recognizing both VIHs) [45] were
used as primary antisera (Table 3). Secondary antibodies

(anti-rat IgG, anti-guinea pig IgG and anti-rabbit IgG; all
raised in goat and conjugated to alkaline-phosphatase;
Sigma, Saint Louis, MO, USA) were used at 1 : 2000 dilu-
tion (Table 3). Cross-reactivity of antisera between l- and
d-peptides was calculated as the ratio between absorbance
values obtained with the cross-reacting and the immunogen
peptides.
Analysis of native VIH
Direct ELISA on RP-HPLC fractions from the elution zone
of CHHs and VIHs from lobster sinus glands was per-
formed: 10 lL aliquots of each fraction were pipetted in
triplicate into the wells of a microtitre plate and dried
under vacuum. The immunoassay was performed as
Table 2. N-terminal amino acid sequence of vitellogenesis inhibit-
ing hormone (VIH) and the synthetic peptides used in the present
study.
D-residues are indicated by bold and underlined letters.
Peptide Sequence
VIH ASAWFTNDECPG.
Dec-
L ASAWFTNDEC
Dec-
DW4 ASAWFTNDEC
Hep-
L ASAWFTN
Hep-
DS2 ASAWFTN
Hep-
DA3 ASAWFTN
Hep-

DW4 ASAWFTN
Hep-
DF5 ASAWFTN
C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells
FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4799
described previously [38] with primary antisera and second-
ary IgG as described above.
Immunohistochemical staining and confocal laser
scanning microscopy
After removal of the exoskeleton and muscles, the eyestalks
were immediately fixed overnight at 4 °C in a 8% parafor-
maldehyde solution in 0.01 m NaCl ⁄ P
i
(pH 7.2) and washed
with NaCl ⁄ P
i
. All subsequent incubations for immunofluo-
rescence labelling of whole mounts were performed as
described previously [40]. Primary antisera against different
peptides were applied at appropriate dilutions overnight:
r-anti-l (1 : 300), gp-anti-dW4 (1 : 300), gp-anti-pQl
(1 : 200) and rb-anti-pQd (1 : 400). After washes, the eye-
stalks were incubated overnight with secondary fluorescent
antibodies (Molecular Probes, Eugene, OR, USA) diluted
1 : 1000, goat anti-(rat IgG) conjugated to Alexa568, goat
anti-(guinea pig IgG) ⁄ Alexa488 and goat anti-(rabbit
IgG) ⁄ Alexa568. Analyses were performed on a confocal
laser-scanning microscope (TCS4D confocal imaging system;
Leica, Heidelberg, Germany) with an argon–krypton ion
laser [40].

Immunocytochemistry and electron microscopy
After dissection of the eyestalks, the sinus glands were
removed and dipped in fixative solution (2% paraformalde-
hyde, 2% glutaraldehyde, 0.1% picric acid buffered with
sodium cacodylate, pH 7.4) at 4 °C overnight. A previously
described protocol [41] was employed using different primary
antisera (gp-anti-pQl 1 : 5000, rb-anti-pQd 1 : 10 000,
r-anti-l 1 : 250, gp-anti-dW4 1 : 250) and the adequate
secondary antibodies coupled with gold particles of different
sizes: goat-anti-gp IgG coupled with 10 or 20 nm, goat-anti-
rb IgG coupled with 10 nm, goat-anti-r IgG coupled with
20 nm gold particles. Finally, sections were examined with a
JEOL JEM-100 CX transmission electron microscope (Jeol
Ltd, Tokyo, Japan).
Acknowledgements
We thank Jean-Jacques Montagne (Institut Jacques
Monod) and Joelle Vinh (Ecole Superieure de Phy-
sique et de Chimie Industrielles de la Ville de Paris)
for MALDI-TOF MS analysis. We are grateful to
Richard Schwartzmann for providing technical assis-
tance with the confocal microscopy and Dr Laurence
Dinan for critically reading the manuscript.
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