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

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

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

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

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

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.

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

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

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

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

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

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

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

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

(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

(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-

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.
References
1 Montecucchi PC, De Castiglione R, Piani S, Gozzini L
& Erspamer VR (1981) Amino acid composition and
sequence of dermorphin, a novel opiate-like peptide
from the skin of Phyllomedusa sauvagei. Int J Peptide
Protein Res 17, 275–283.
2 Amiche M, Delfour A & Nicolas P (1998) Opioid pep-
tides from frog skin. In D-Amino Acids in Sequences of

and anti-4, by injection of VIH II, now known as
D-Trp4 VIH [43].
Primary antisera Secondary antisera
Name Hapten Peptide recognized Animal
HIC ICCr
Fluorochrome Colour Gold particle diameter
Anti-4 VIH II Both VIH gp Al 488 Green Not used
r-anti-
L Dec-L VIH r Al 568 Red 10 nm
gp-anti-
DW4 Dec-DW4 D-Trp4 VIH gp Al 488 Green 20 nm
gp-anti- pQ
L pQL CHH gp Al 488 Green 20 nm
rb-anti- pQ
D pQDD-Phe3 CHH rb Al 568 Red 10 nm
Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al.
4800 FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS
7 Buczek O, Bulaj G & Olivera BM (2005) Conotoxins
and the posttranslational modification of secreted gene
products. Cell Mol Life Sci 62, 3067–3079.
8 Kreil G (1994) Conversion of L- to D-amino acids: a
posttranslational reaction. Science 266, 996–997.
9 Mor A, Amiche M & Nicolas P (1992) Enter a new
post-translational modification: D-amino acids in gene-
encoded peptides. Trends Biochem Sci 17, 481–485.
10 Ollivaux C & Soyez D (2000) Dynamics of biosynthe-
sis and release of crustacean hyperglycemic hormone
isoforms in the X-organ-sinus gland complex of the
crayfish Orconectes limosus. Eur J Biochem 267, 5106–
5114.

eds), pp. 19. Science Publishers, Enfield (NH) USA,
Plymouth, UK.
18 Yasuda A, Yasuda Y, Fujita T & Naya Y (1994) Char-
acterization of crustacean hyperglycemic hormone from
the crayfish (Procambarus clarkii): multiplicity of molec-
ular forms by stereoinversion and diverse functions.
Gen Comp Endocrinol 95, 387–398.
19 Serrano L, Charmantier G, Soyez D, Grousset E &
Spanings-Pierrot C (2003) Putative involvement of crus-
tacean hyperglycemic hormone isoforms in the neuroen-
docrine mediation of osmoregulation in the crayfish
Astacus leptodactylus. J Exp Biol 206, 979–988.
20 Chan SM, Gu PL, Chu KH & Tobe SS (2003) Crusta-
cean neuropeptide genes of the CHH ⁄ MIH ⁄ GIH
family: implications from molecular studies. Gen Comp
Endocrinol 134, 214–219.
21 Soyez D, Van Herp F, Rossier J, Le Caer JP, Tensen
CP & Lafont R (1994) Evidence for a conformational
polymorphism of invertebrate neurohormones.
D-amino-acid residue in crustacean hyperglycemic
hormone. J Biol Chem 269, 18295–18298.
22 Soyez D, Lecaer JP, Noel PY & Rossier J (1991)
Primary structure of two isoforms of the vitellogenesis
inhibiting hormone from the lobster Homarus americ-
anus. Neuropeptides 20, 25–32.
23 De Kleijn DPV, Sleutels FJGT, Martens GJM & Van
Herp F (1994) Cloning and expression of mRNA
encoding prepro-gonad-inhibiting hormone (GIH) in the
lobster Homarus americanus. FEBS Lett 353, 255–258.
24 Van Herp F & Soyez D (1997) Reproductive biology of

Crustacean hyperglycemic and vitellogenesis-inhibiting
hormones in the lobster Homarus gammarus. FEBS J
273, 2151–2160.
31 Kamatani Y, Minakata H, Kenny P, Iwashita T,
Watanabe K, Funase K, Sun XP, Yongsiri A, Kim
KH, Novales-Li P et al. (1989) Achatin-I, an endo-
genous neuroexcitatory tetrapeptide from Achatina
fulica Fe
´
russac containing a D-amino acid residue.
Biochem Biophys Res Commun 160, 1015–1020.
C. Ollivaux et al. Peptidyl isomerization in neuroendocrine cells
FEBS Journal 276 (2009) 4790–4802 ª 2009 The Authors Journal compilation ª 2009 FEBS 4801
32 Torres AM, Menz I, Alewood PF, Bansal P, Lahnstein
J, Gallagher CH & Kuchel PW (2002) D-amino acid
residue in the C-type natriuretic peptide from the
venom of the mammal, Ornithorhynchus anatinus, the
Australian platypus. FEBS Lett 524, 172–176.
33 Buczek O, Wei D, Babon JJ, Yang X, Fiedler B, Chen
P, Yoshikami D, Olivera BM, Bulaj G & Norton RS
(2007) Structure and sodium channel activity of an
excitatory I1-superfamily conotoxin. Biochemistry 46,
9929–9940.
34 Auvynet C, Seddiki N, Dunia I, Nicolas P, Amiche M
& Lacombe C (2006) Post-translational amino acid
racemization in the frog skin peptide deltorphin I in the
secretion granules of cutaneous serous glands. Eur J
Cell Biol 85, 25–34.
35 Buczek O, Jimenez EC, Yoshikami D, Imperial JS,
Watkins M, Morrison A & Olivera BM (2008)

42 Kallen J & Meusy JJ (1989) Do the neurohormones
VIH (vitellogenesis inhibiting hormone) and CHH
(crustacean hyperglycemic hormone) of the crustacean
have the same precursor? Immunolocalization of VIH
and CHH in the X-organ sinus gland complex of the
lobster Homarus americanus Invert Reprod Dev 16,
43–52.
43 Laverdure AM, Breuzet M, Soyez D & Becker J (1992)
Detection of the mRNA encoding vitellogenesis inhibit-
ing hormone in neurosecretory cells of the X-organ in
Homarus americanus by in situ hybridization. Gen Comp
Endocrinol 87, 443–450.
44 De Kleijn DPV, Janssen KPC, Waddy SL, Hegeman R,
Lai WY, Martens GJM & VanHerp F (1998) Expres-
sion of the crustacean hyperglycaemic hormones and
the gonad-inhibiting hormone during the reproductive
cycle of the female American lobster Homarus americ-
anus. J Endocrinol 156, 291–298.
45 Meusy JJ & Soyez D (1991) Immunological relationships
between neuropeptides from the sinus gland of the lob-
ster Homarus americanus, with special references to the
vitellogenesis inhibiting hormone and crustacean hyper-
glycemic hormone. Gen Comp Endocrinol 81
, 410–418.
46 Azzouna A, Philippe M, Jarry T, Greve P & Martin G
(2003) Localization of crustacean hyperglycemic and
vitellogenesis inhibiting hormones in separate cell types
in the protocerebrum of the woodlouse Armadillidium
vulgare (Crustacea, Isopoda). Gen Comp Endocrinol 131,
134–142.

(1992) The FMRFamide-related decapeptide of Mytilus
contains a D-amino acid residue. Comp Biochem Physiol
C 102, 91–95.
53 Morishita F, Nakanishi Y, Kaku S, Furukawa Y, Ohta
S, Hirata T, Ohtani M, Fujisawa Y, Muneoka Y &
Matsushima O (1997) A novel D-amino-acid-containing
peptide isolated from Aplysia heart. Biochem Biophys
Res Commun 240 (2), 354–358.
Peptidyl isomerization in neuroendocrine cells C. Ollivaux et al.
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