TY - JOUR
T1 - Characterization of Aplysia Alb-1, a candidate water-borne protein pheromone released during egg laying
AU - Cummins, Scott F.
AU - Degnan, Bernard M.
AU - Nagle, Gregg T.
N1 - Funding Information:
Supported by NSF grant IBN-0314377 to G.T.N. and UQ fellowship to S.F.C. We acknowledge the assistance of the UTMB Protein Chemistry Lab. The nucleotide sequence of Alb-1 (previously designated clone Alb-149 [3] ) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession no. DQ924535 .
PY - 2008/2
Y1 - 2008/2
N2 - Pheromones are known to be important to the innate behavior of marine animals. Attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, temptin and seductin, which are released from the albumen gland during egg laying. Other pheromones are predicted to act in concert with these pheromones, but their identities are unknown. To identify additional pheromone candidates, we employed differential library screening of an albumen gland cDNA library, RT-PCR, recombinant protein expression, rhinophore contraction bioassays and immunocytochemistry. Alb-1 is expressed in the Aplysia californica albumen gland and encodes a novel protein that does not share significant sequence identity with any proteins in the database. RT-PCR analysis detected Alb-1 transcripts in the albumen gland, exocrine atrial gland and ovotestis. The Alb-1 precursor has a signal peptide sequence followed by a predicted 101-residue protein sequence containing eight cysteine residues. Recombinant protein expression, RP-HPLC, microsequence analysis and MALDI mass spectrometry analyses demonstrated that mature recombinant Alb-1 was processed at a paired basic residue site to generate an N-terminal and C-terminal protein fragment; this was consistent with immunoblot observations on purified albumen gland extracts. In rhinophore contraction (twitch) bioassays, the recombinant N-terminal protein induced rhinophore contractions whereas the C-terminal protein did not. An antibody generated to the N-terminal protein was used for immunocytochemical and immunoblot analyses and demonstrated that this protein is present in albumen gland secretory cells, egg cordons and egg eluates. Overall, the data suggest that Alb-1 may be processed in the albumen gland and that the Alb-11-56 protein released during egg laying may serve a pheromonal function in concert with attractin, enticin, temptin and seductin.
AB - Pheromones are known to be important to the innate behavior of marine animals. Attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, temptin and seductin, which are released from the albumen gland during egg laying. Other pheromones are predicted to act in concert with these pheromones, but their identities are unknown. To identify additional pheromone candidates, we employed differential library screening of an albumen gland cDNA library, RT-PCR, recombinant protein expression, rhinophore contraction bioassays and immunocytochemistry. Alb-1 is expressed in the Aplysia californica albumen gland and encodes a novel protein that does not share significant sequence identity with any proteins in the database. RT-PCR analysis detected Alb-1 transcripts in the albumen gland, exocrine atrial gland and ovotestis. The Alb-1 precursor has a signal peptide sequence followed by a predicted 101-residue protein sequence containing eight cysteine residues. Recombinant protein expression, RP-HPLC, microsequence analysis and MALDI mass spectrometry analyses demonstrated that mature recombinant Alb-1 was processed at a paired basic residue site to generate an N-terminal and C-terminal protein fragment; this was consistent with immunoblot observations on purified albumen gland extracts. In rhinophore contraction (twitch) bioassays, the recombinant N-terminal protein induced rhinophore contractions whereas the C-terminal protein did not. An antibody generated to the N-terminal protein was used for immunocytochemical and immunoblot analyses and demonstrated that this protein is present in albumen gland secretory cells, egg cordons and egg eluates. Overall, the data suggest that Alb-1 may be processed in the albumen gland and that the Alb-11-56 protein released during egg laying may serve a pheromonal function in concert with attractin, enticin, temptin and seductin.
KW - Alb-1
KW - Aplysia
KW - Attractin
KW - Mollusk
KW - Protein pheromone
UR - http://www.scopus.com/inward/record.url?scp=39149128738&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=39149128738&partnerID=8YFLogxK
U2 - 10.1016/j.peptides.2007.07.031
DO - 10.1016/j.peptides.2007.07.031
M3 - Article
C2 - 18190999
AN - SCOPUS:39149128738
SN - 0196-9781
VL - 29
SP - 152
EP - 161
JO - Peptides
JF - Peptides
IS - 2
ER -