The neuropeptidergic bag cells of the marine mollusc Aplysia californica are involved in the egg-laying behavior of the animal. These neurosecretory cells synthesize an egg-laying hormone (ELH) precursor protein, yielding multiple bioactive peptides, including ELH, several bag cell peptides (BCP) and acidic peptide (AP). While immunohistochemical studies have involved a number of species, homologous peptides have been biochemically characterized in relatively few Aplysiidae species. In this study, a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and electrospray ionization Fourier transform ion cyclotron resonance MS is used to characterize and compare the ELH peptides from related opisthobranch molluscs including Aplysia vaccaria and Phyllaplysia taylori. The peptide profiles of bag cells from these two Aplysiidae species are similar to that of A. californica bag cells. In an effort to characterize further several of these peptides, peptides from multiple groups of cells of each species were extracted, and microbore liquid chromatography was used to separate and isolate them. Several MS-based sequencing approaches are applied to obtain the primary structures of bag cell peptides and ELH. Our studies reveal that (α)-BCPs are 100 % conserved across all species studied. In addition, the complete sequences of (ε)-BCP and ELH of A. vaccaria were determined. They show a high degree of homology to their counterparts in A. californica, with only a few amino acid residue substitutions.

REFERENCES

Brown
R. O.
,
Mayeri
E.
(
1989
).
Positive feedback by autoexcitatory neuropeptides in neuroendocrine bag cells of Aplysia
.
J. Neurosci
9
,
1443
–.
Brown
R. S.
,
Lennon
J. J.
(
1995
).
Sequence-specific fragmentation of matrix-assisted laser-desorbed protein/peptide ions
.
Analyt. Chem
67
,
3990
–.
Chiu
A. Y.
,
Strumwasser
F.
(
1981
).
An immunohistochemical study of the neuropeptidergic bag cells of Aplysia
.
J. Neurosci
1
,
812
–.
Conn
P. J.
,
Kaczmarek
L. K.
(
1989
).
The bag cell neurons of Aplysia
.
Mol. Neurobiol
3
,
238
–.
Cruz
L. J.
,
De santos
P.
,
Zafaralla
G. C.
,
Ramillo
C. A.
,
Zeikus
R.
,
Gray
W. R.
,
Oliveira
B. M.
(
1987
).
Neuropeptides in the fish gut
.
J. Biol. Chem
262
,
15821
–.
Garden
R. W.
,
Moroz
L. L.
,
Moroz
T. P.
,
Shippy
S. A.
,
Sweedler
J. V.
(
1996
).
Excess salt removal with matrix rinsing: direct peptide profiling of neurons from marine invertebrates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
.
J. Mass Spectrom
31
,
1126
–.
Garden
R. W.
,
Shippy
S. A.
,
Li
L.
,
Moroz
T. P.
,
Sweedler
J. V.
(
1998
).
Proteolytic processing of the Aplysia egg-laying hormone prohormone
.
Proc. Natl. Acad. Sci. USA
95
,
3972
–.
Gauthier
J. W.
,
Trautman
T. R.
,
Jacobson
D. B.
(
1991
).
Sustained off-resonance irradiation for collision-activated dissociation involving Fourier transform mass spectrometry. Collision-activated dissociation technique that emulates infrared multiphoton dissociation
.
Anal. Chim. Acta
246
,
211
–.
Kauer
J. A.
,
Fisher
T. E.
,
Kaczmarek
L. K.
(
1987
).
Alphabag cell peptide directly modulates the excitability of the neurons that release it
.
J. Neurosci
7
,
3623
–.
Leung
M. K.
,
Boer
H. H.
,
Van Minnen
J.
,
Lundy
J.
,
Stefano
G. B.
(
1990
).
Evidence for an enkephalinergic system in the nervous system of the pond snail Lymnaea stagnalis
.
Brain Res
531
,
66
–.
Li
L.
,
Moroz
T. P.
,
Garden
R. W.
,
Floyd
P. D.
,
Weiss
K. R.
,
Sweedler
J. V.
(
1998
).
Mass spectrometric survey of interganglionically transported peptides in Aplysia
.
Peptides
19
,
1425
–.
Lloyd
P. E.
(
1989
).
Interganglionic axonal transport of neuropeptides in Aplysia
.
J. Neurosci
9
,
3243
–.
Marshall
A. G.
,
Wang
T.-C.
,
Ricca
T. L.
(
1985
).
Tailored excitation for Fourier transform ion cyclotron resonance mass spectrometry
.
J. Am. Chem. Soc
107
,
7893
–.
Nagle
G. T.
,
Painter
S. D.
,
Blankenship
J. E.
(
1989
).
Post-translational processing in model neuroendocrine systems: precursors and products that coordinate reproductive activity in Aplysia and Lymnaea
.
J. Neurosci. Res
23
,
359
–.
Nagle
G. T.
,
Painter
S. D.
,
Blankenship
J. E.
,
Choate
J. V.
,
Kurosky
A.
(
1988
).
The bag cell egg-laying hormones of Aplysia brasiliana and Aplysia californica are identical
.
Peptides
9
,
867
–.
Nagle
G. T.
,
Painter
S. D.
,
Blankenship
J. E.
,
Dixon
J. D.
,
Kurosky
A.
(
1986
).
Evidence for the expression of three genes encoding homologous atrial gland peptides that cause egg laying in Aplysia
.
J. Biol. Chem
261
,
7853
–.
Nagle
G. T.
,
Painter
S. D.
,
Blankenship
J. E.
,
Kurosky
A.
(
1988
).
Proteolytic processing of egg-laying hormone-related precursors in Aplysia
.
J. Biol. Chem
263
,
9223
–.
Nambu
J. R.
,
Scheller
R. H.
(
1986
).
Egg-laying hormone genes of Aplysia: evolution of the ELH gene family
.
J. Neurosci
6
,
2026
–.
Painter
S. D.
,
Kalman
V.K.
,
Nagle
G.T.
,
Blankenship
J. E.
(
1989
).
Localization of immunoreactive alpha-bag-cell peptide in the central nervous system of Aplysia
.
J. Comp. Neurol
287
,
515
–.
Proux
J. P.
,
Miller
C. A.
,
Li
J. P.
,
Carney
R. L.
,
Girardie
A.
,
Delaage
M.
,
Schooley
D. A.
(
1987
).
Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria
.
Biochem. Biophys. Res. Commun
149
,
180
–.
Ram
J. L.
(
1982
).
Interspecific induction of egg laying in Hawaiian aplysiids
.
Gen. Comp. Endocr
4
,
522
–.
Ram
J. L.
,
Gallardo
C. S.
,
Ram
M. L.
,
Croll
R. P.
(
1998
).
Reproduction-associated immunoreactivity peptides in the nervous systems of prosobranch gastropods
.
Biol. Bull.
,
195
,
308
–.
Redman
R. S.
,
Berry
R. W.
(
1991
).
Temperature-dependent peptidergic feedback: potential role in seasonal egg-laying in Aplysia
.
J. Neurosci
11
,
1780
–.
Rock
M. K.
,
Shope
S. B.
,
Blankenship
J. E.
,
Schlesinger
D. H.
(
1986
).
Effects of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia
.
J. Neurobiol
17
,
273
–.
Rothman
B. S.
,
Mayeri
E.
,
Brown
R. O.
,
Yuan
P.-M.
,
Shively
J. E.
(
1983
).
Primary structure and neuronal effects of alpha-bag cell peptide, a second candidate neurotransmitter encoded by a single gene in bag cell neurons of Aplysia
.
Proc. Natl. Acad. Sci. USA
80
,
5753
–.
Salzet
M.
,
Bulet
P.
,
Van Dorsselaer
A.
,
Malecha
J.
(
1993
).
Isolation, structural characterization and biological function of aL. LIANDOTHERS2973 Egg-laying hormone peptides in the Aplysiidae lysine-conopressin in the central nervous system of the pharyngobdellid leech Erpobdella octoculata
.
Eur. J. Biochem
217
,
897
–.
Salzet
M.
,
Bulet
P.
,
Wattez
C.
,
Malecha
J.
(
1994
).
FMRFamide-related peptides in sex segmental ganglia of the pharyngobdellid leech Erpobdella octoculata
.
Eur. J. Biochem
221
,
269
–.
Sawyer
W. H.
,
Deyrup-Olsen
I.
,
Martin
A. W.
(
1984
).
Immunological and biological characteristics of the vasotocin-like activity in the head ganglia of gastropod molluscs
.
Gen. Comp. Endocr
54
,
97
–.
Scheller
R. H.
,
Jackson
J. F.
,
McAllister
L. B.
,
Rothman
B. S.
,
Mayeri
E.
,
Axel
R.
(
1983
).
A single gene encodes multiple neuropeptides mediating a stereotyped behavior
.
Cell
35
,
7
–.
Scheller
R. H.
,
Jackson
J. F.
,
McAllister
L. B.
,
Schwartz
J. H.
,
Kandel
E. R.
,
Axel
R.
(
1982
).
A family of genes that codes for ELH, a neuropeptide eliciting a stereotyped pattern of behavior in Aplysia
.
Cell
28
,
707
–.
Senko
M. W.
,
Speir
J. P.
,
McLafferty
F. W.
(
1994
).
Collisional activation of large multiply charged ions using Fourier transform mass spectrometry
.
Analyt. Chem
66
,
2801
–.
Sigvardt
K. A.
,
Rothman
B. S.
,
Brown
R. O.
,
Mayeri
E.
(
1986
).
The bag cells of Aplysia as a multitransmitter system: identification of alpha bag cell peptide as a second neurotransmitter
.
J. Neurosci
6
,
803
–.
Smit
A. B.
,
Van Kesteren
R. E.
,
Li
K. W.
,
Van Minnen
J.
,
Spijker
S.
,
Van Heerikhuizen
H.
,
Geraerts
W. P.
(
1998
).
Towards understanding the role of insulin in the brain: lessons from insulin-related signaling systems in the invertebrate brain
.
Progr. Neurobiol
54
,
35
–.
Spengler
B.
(
1997
).
Post-source decay analysis in matrix-assisted laser desorption/ionization mass spectrometry of biomolecules
.
J. Mass Spectrom
32
,
1019
–.
Van Minnen
J.
,
Schallig
H. D. F. H.
,
Ramkema
M. D.
(
1992
).
Identification of putative egg-laying hormone containing neuronal systems in gastropod molluscs
.
Gen. Comp. Endocr
86
,
96
–.
Vreugdenhil
E.
,
Jackson
J. F.
,
Bouwmeester
T.
,
Smit
A. B.
,
Van Minnen
J.
,
Van Heerikhuizen
H.
,
Klootwijk
J.
,
Joosse
J.
(
1988
).
Isolation, characterization and evolutionary aspects of a cDNA clone encoding multiple neuropeptides involved in the stereo-typed egg-laying behavior of the freshwater snail Lymnaea stagnalis
.
J. Neurosci
8
,
4184
–.
Wiens
B. L.
,
Brownell
P. H.
(
1994
).
Neuroendocrine control of egg-laying behavior in the nudibranch, Archidoris montoereynsis
.
J. Comp. Neurol
344
,
619
–.
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