TY - JOUR

T1 - A crustacean nitric oxide synthase expressed in nerve ganglia, Y-organ, gill and gonad of the tropical land crab, Gecarcinus lateralis

AU - Kim, Hyun-Woo

AU - Batista, Luisa A

AU - Hoppes, Jodi L

AU - Lee, Kara J

AU - Mykles, Donald L

PY - 2004/7

Y1 - 2004/7

N2 - NO signaling is involved in many physiological processes in invertebrates. In crustaceans, it plays a role in the regulation of the nervous system and muscle contraction. Nested reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) PCR generated a full-length cDNA sequence (3982 bp) of land crab NO synthase (Gl-NOS) from molting gland (Y-organ) and thoracic ganglion mRNA. The open reading frame encoded a protein of 1199 amino acids with an estimated mass of 135 624 Da. Gl-NOS had the highest sequence identity with insect NOS. The amino acid sequences for binding heme and tetrahydrobiopterin in the oxygenase domain, binding calmodulin and binding FMN, FAD and NADPH in the reductase domain were highly conserved. Gl-NOS had single amino acid differences in all three highly conserved FAD-binding sequences, which distinguished it from other NOS sequences. RT-PCR showed that the Gl-NOS mRNA was present in testis, ovary, gill, eyestalk neural ganglia, thoracic ganglion and Y-organ. NOS mRNA varied between preparations of Y-organ, thoracic ganglion and gill, while NOS mRNA was at consistently high levels in the ovary, testis and eyestalk ganglia. Immunohistochemistry confirmed that the Gl-NOS protein was expressed in Y-organ, ovary and gill. These results suggest that NOS has functions in addition to neuromodulation in adults, such as regulating or modulating ecdysteroid synthesis in the Y-organ.

AB - NO signaling is involved in many physiological processes in invertebrates. In crustaceans, it plays a role in the regulation of the nervous system and muscle contraction. Nested reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) PCR generated a full-length cDNA sequence (3982 bp) of land crab NO synthase (Gl-NOS) from molting gland (Y-organ) and thoracic ganglion mRNA. The open reading frame encoded a protein of 1199 amino acids with an estimated mass of 135 624 Da. Gl-NOS had the highest sequence identity with insect NOS. The amino acid sequences for binding heme and tetrahydrobiopterin in the oxygenase domain, binding calmodulin and binding FMN, FAD and NADPH in the reductase domain were highly conserved. Gl-NOS had single amino acid differences in all three highly conserved FAD-binding sequences, which distinguished it from other NOS sequences. RT-PCR showed that the Gl-NOS mRNA was present in testis, ovary, gill, eyestalk neural ganglia, thoracic ganglion and Y-organ. NOS mRNA varied between preparations of Y-organ, thoracic ganglion and gill, while NOS mRNA was at consistently high levels in the ovary, testis and eyestalk ganglia. Immunohistochemistry confirmed that the Gl-NOS protein was expressed in Y-organ, ovary and gill. These results suggest that NOS has functions in addition to neuromodulation in adults, such as regulating or modulating ecdysteroid synthesis in the Y-organ.

KW - Amino Acid Sequence

KW - Animals

KW - Base Sequence

KW - Brachyura/genetics

KW - Cluster Analysis

KW - Conserved Sequence/genetics

KW - DNA Primers

KW - Ganglia, Invertebrate/metabolism

KW - Gene Components

KW - Gills/metabolism

KW - Gonads/metabolism

KW - Immunohistochemistry

KW - Molecular Sequence Data

KW - Molting/physiology

KW - Nitric Oxide Synthase/genetics

KW - Nucleic Acid Amplification Techniques

KW - Phylogeny

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Sequence Alignment

KW - Sequence Analysis, DNA

KW - Signal Transduction/physiology

U2 - 10.1242/jeb.01117

DO - 10.1242/jeb.01117

M3 - Article

C2 - 15235013

VL - 207

SP - 2845

EP - 2857

JO - Journal of Experimental Biology

JF - Journal of Experimental Biology

SN - 0022-0949

IS - 16

ER -