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First published online July 2, 2004
Journal of Experimental Biology 207, 2845-2857 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01117

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

Hyun-Woo Kim, Luisa A. Batista, Jodi L. Hoppes, Kara J. Lee and Donald L. Mykles^*

Department of Biology, Colorado State University, Fort Collins, CO 80523, USA

View larger version (88K): [in a new window]   Fig. 1. The nucleotide and deduced amino acid sequences of land crab nitric oxide synthase (Gl-NOS) cDNA. The cDNA (3982 bp) contained a complete open reading frame (ORF) encoding a protein of 1199 amino acids (residue number indicated on the right). Locations and directions of degenerate primers used for nested PCR to obtain the initial cDNA are indicated by bold letters and solid arrowheads with broken lines. Locations and directions of sequence-specific forward primers and a degenerate reverse primer used for semi-nested PCR to obtain more of the 3' ORF are indicated by open arrowheads with broken lines. Solid arrowheads with solid lines indicate locations and directions of nested sequence-specific primers used for 5' and 3' RACE. The poly(A) signal (AATAAA) in the 3' UTR is boxed. GenBank accession #AY552549.

View larger version (113K): [in a new window]   Fig. 2. Comparison of deduced amino acid sequences of NO synthase from land crab, insects, mollusk and human. Land crab (Gecarcinus lateralis) NOS was aligned with NOS sequences from insects (Manduca sexta, Bombyx mori, Rodnius prolixus, Anopheles stephensi and Drosophila melanogaster), mollusk (Aplysia californica) and human (iNOS, nNOS, eNOS) using the ClustalW program (see Materials and methods). Identities in all 10 sequences are highlighted in black. Boxes with broken borders identify highly conserved binding sequences for heme, tetrahydrobiopterin (H4), calmodulin, FMN, FAD and NADPH. Inverted triangles indicate amino acid sequence deviation in the FAD binding motif of Gl-NOS. Regular triangles indicate the two conserved cysteine residues in the zinc tetrathiolate cluster. Accession numbers: Rhodnius prolixus, Q26240; Anopheles stephensi, O61608; Bombyx mori, BAB85836; Drosophila melanogaster, Q27571; Manduca sexta, T30555; Aplysia californica, AF288780; and human iNOS (AAB49041), eNOS (NP000594) and nNOS (NP000611).

View larger version (8K): [in a new window]   Fig. 3. Domain organization of arthropod NOS. The ORFs of Drosophila (dNOS) and land crab NOS (Gl-NOS) are compared. The oxygenase domain contains heme-binding and tetrahydrobiopterin (H4) domains; the reductase domain contains binding domains for FMN, FAD and NADPH. A calmodulin (CaM) binding domain is located between the oxygenase and reductase domains and is involved in dimerization and regulation of catalytic activity (Regulski and Tully, 1995; Stasiv et al., 2001). dNOS has a Gln-rich sequence near the amino terminus.

View larger version (8K): [in a new window]   Fig. 4. Phylogenetic relationships of NO synthases from arthropods, mollusk and mammals. The deduced amino acid sequences of the oxygenase domain were analyzed using ClustalW and Treeview programs. Arthropod NOS sequences form a group divergent from molluscan and mammalian NOS sequences. Within the arthropods, the land crab NOS was diverged from insects. Within Insecta, NOS sequences grouped according to major taxonomic lineages: Lepidoptera (Manduca and Bombyx), Diptera (Drosophila and Anopheles) and Hemiptera (Rhodnius). Accession numbers were the same as those given in the legend for Fig. 2.

View larger version (86K): [in a new window]   Fig. 5. Expression of NO synthase (Gl-NOS) in land crab tissues. Total RNA was DNase-treated, reverse-transcribed and PCR-amplified using sequence-specific primers (see Materials and methods). Shown are inverse images of ethidium bromide-stained agarose gels of the PCR products. (A) First-round PCR generated a product of the expected size (2.1 kb). Gl-NOS was expressed in testis (Tt), gill (Gi), ovary (Ov), eyestalk neural ganglia (EG) and Y-organ (YO). Gl-NOS mRNA was not detected in integument (Ig), thoracic ganglion (TG), digestive gland (DG), heart (Ht) or claw muscle (CM). (B) Nested PCR of the initial PCR generated a product of the expected size (800 bp), which confirmed the identity of the initial product as Gl-NOS. In other experiments, a Gl-NOS product was obtained from thoracic ganglion (data not shown). (C) Elongation factor 2 (EF2) served as internal positive control, as it was constitutively expressed in all tissues. Reactions without template [water (W) lane k] served as a negative control. Positions of DNA size markers are indicated on the left.

View larger version (187K): [in a new window]   Fig. 6. Immunocytochemical localization of NO synthase in land crab Y-organs. Sections of Y-organ were incubated with either a universal anti-NOS rabbit antibody (A, C and D; 1:2000 dilution) or a non-immune rabbit antibody (B; 1:2000 dilution); detection used BCIP/NBT (see Materials and methods). (A) NOS localization in the cytoplasm and nuclei of Y-organ (YO) cells and nuclei in some cells in the adjacent hemolymph space (HS) and connective tissue (CT). (B) Control serial section adjacent to A showing no specific antibody binding in Y-organ tissue. (C) NOS localization in the nuclei of some connective tissue cells. (D) NOS localization in the tendinous cells (TC), which anchor connective tissue to the cuticle (C). BC, branchial chamber. Scale bars, 200 µm.

View larger version (157K): [in a new window]   Fig. 7. Immunocytochemical localization of NO synthase in Y-organ, gill and ovary. Sections were incubated with a universal anti-NOS rabbit antibody (1:1000 dilution; A, B and D) or no primary antibody (C); detection used Vector Red (see Materials and methods). (A) Y-organ (YO). NOS was localized in cytoplasm and nuclei of YO cells. Connective tissue (CT) showed little or no staining. BC, branchial chamber. (B) Gill. The field includes a portion of the central axis (CA) and transverse sections through three lamellae. NOS was localized in the epithelium and pillar cells (arrowheads). The epithelium lining the central axis stained more intensely (arrows). Control sections of Y-organ and gill incubated with either non-immune primary antibody or no primary antibody showed no specific staining (data not shown). Cuticular ridges (CR) keep lamellar surfaces from touching, creating a space for air circulation between gill lamellae. (C) Ovary control (no primary antibody), showing weak non-specific staining in the perinuclear region of oocytes (arrowhead). (D) Ovary. NOS was confined to the perinuclear cytoplasm of oocytes (arrowheads). Scale bars: 400 µm in A, 50 µm in B, and 200 µm in C and D.

View larger version (16K): [in a new window]   Fig. 8. Hypothetical signaling pathway inhibiting ecdysteroidogenesis in the crustacean Y-organ. Molt inhibiting hormone (MIH) binds to a G protein-coupled receptor (G), activating adenylyl cyclase (AC); intracellular Ca2+ rises when protein kinase A (PKA) activates a membrane Ca2+ channel; nitric oxide synthase (NOS) is activated and released from the membrane when Ca2+/calmodulin (CaM) binds, and NOS is dephosphorylated by the Ca2+/calmodulin-dependent phosphatase, calcineurin (CaN); NO activates an NO-sensitive (class I) GC (GC-I). Activation of a cGMP-dependent protein kinase (cGPK) inhibits expression and/or activities of ecdysteroidogenic proteins, resulting in reduced ecdysteroid synthesis. NOS is inactivated by phosphorylation by PKA, protein kinase C (PKC) or other protein kinases.