Characterization and Expression Analysis of Mollusk-like Growth Factor: A Secreted Protein Involved in Pacific Abalone Embryonic and Larval Development

Abstract

Growth factors are mostly secreted proteins that play key roles in an organism's biophysical processes through binding to specific receptors on the cell surface. The mollusk-like growth factor (MLGF) is a novel cell signaling protein in the adenosine deaminase-related growth factor (ADGF) subfamily. In this study, the MLGF gene was cloned and characterized from the digestive gland tissue of Pacific abalone and designated as Hdh-MLGF. The transcribed full-length sequence of Hdh-MLGF was 1829 bp long with a 1566 bp open reading frame (ORF) encoding 521 amino acids. The deduced amino acid sequence contained a putative signal peptide and two conserved adenosine deaminase domains responsible for regulating molecular function. Fluorescence in situ hybridization localized Hdh-MLGF in the submucosa layer of digestive tubules in the digestive gland. The mRNA expression analysis indicated that Hdh-MLGF expression was restricted to the digestive gland in the adult Pacific abalone. However, Hdh-MLGF mRNA expressions were observed in all stages of embryonic and larval development, suggesting Hdh-MLGF might be involved in the Pacific abalone embryonic and larval development. This is the first study describing Hdh-MLGF and its involvement in the Pacific abalone embryonic and larval development.

Keywords: Pacific abalone; catalytic activity; digestive gland; larval development; mollusk-like growth factor.

Figure 1

The Full-length nucleotide and amino acid sequences of Hdh-MLGF (GenBank accession no. ON803449). The numerical numbers at the left and right side of the sequence indicate the position of the nucleotide and amino acid sequence of the Hdh-MLGF gene, respectively. The coding regions starting with the start codon (ATG) and ending with the stop codon (TAA) are shown in red bold letters. The signal peptide for the secreted Hdh-MLGF protein has been indicated by the red box. The adenosine deaminase domain is marked with a red underline and an additional N-terminal adenosine deaminase with a black underline. N-glycosylation sites are denoted with a violet box. Potential protein kinase C (PKC) phosphorylation sites are pointed out with a green box. Predicted casein kinase II phosphorylation sites are marked with a blue box. The N-myristoylation site is boxed in yellow. The putative polyadenylation signal is denoted by a blue colored letter with a plain underline.

Figure 2

Schematic diagram of detected motifs in the Hdh-MLGF protein sequence and other ADGF subfamily members of vertebrates and invertebrates. Detected motifs are explained by different colors. The motif analysis included the Hdh-MLGF amino acid sequence and other growth factors of the following ADGF family members: MLGF of H. discus (ABO26607.1), MDGF of Aplysia californica (AAD13112.1), IDGF-Al of Xenopus laevis (AAY42596.1), IDGF-Bl of Xenopus laevis (AAY42597.1), CECR1 of Danio rerio (AAL40922.1), TSGF-1 precursor of Glossina morsitans (AAD52850.1), TSGF-2 of Glossina morsitans (AAD52851.1), and ADGF precursor of Bombyx mori (NP_001098698.1).

Figure 3

Multiple sequence alignments of mollusk-like growth factor from the deduced amino acid sequences of H. discus hannai (accession no. ON803449), H. discus (ABO26607.1), Aplysia californica (AAD13112.1), Xenopus laevis (AAY42596.1), Bombyx mori (BAF73622.1), Danio rerio (AAL40922.1), and Glossina morsitans (AAD52850.1). Conserved residues are indicated by an asterisk.

Figure 4

Three-dimensional structure and zinc ion binding sites prediction of Hdh-MLGF from the Pacific abalone, H. discus hannai (A) and catalytic activity with binding sites (B).

Figure 5

Functional analysis of the Hdh-MLGF amino acid sequence of Pacific abalone, H. discus hannai. Different color box in the upper side of the figure indicates the confidence score.

Figure 6

Phylogenetic tree constructed by the bootstrap neighbor-joining method after ClustalW alignment based on the amino acid sequences of different ADGF family members. The numbers at the nodes indicate bootstrap probability. Different colors of squares indicate different genes in the growth factor family ADGF, for instance: the orange square box for CECR1 (specific to vertebrate); green square for mollusks specific growth factor MDGF and MLGF; blue square for ADGF; teal square for IDGF (vertebrate and invertebrates separately formed cluster); lime square for ADA and violet square for TSGF. The sequences with their protein IDs used to construct the phylogenetic tree are as follows: MLGF of H. discus hannai (accession no. ON803449), H. discus (ABO26607.1), MDGF of Aplysia californica (AAD13112.1), IDGF of Sarcophaga peregrina (BAA11812.1), IDGF-Al of Xenopus laevis (AAY42596.1), IDGF-Bl of Xenopus laevis (AAY42597.1), CECR1 of Xenopus laevis (AAX10952.1), Gallus (AAX10953.1), Homo sapiens (CAG30303.1), Sus scrofa (AAL40921.1), Danio rerio (AAL40922.1), Branchiostoma lanceolatum (CAH1231807.1), ADA of Lutzomyia longipalpis (AAF78901.1), Aedes aegypti (AAL76033.1), Culex quinquefasciatus (AAK97208.1), TSGF-1 precursor of Glossina morsitans (AAD52850.1), TSGF-2 of Glossina morsitans (AAD52851.1), ADGF of Bombyx mori (BAF73622.1), ADGF precursor of Bombyx mori (NP_001098698.1), ADGF A of Drosophila melanogaster (AAL40913.1), ADGF B of Drosophila melanogaster (AAL40920.1), and ADGF E of Drosophila melanogaster (AAL40910.1).

Figure 7

Expression of Hdh-MLGF/Hdh-β-Actin mRNA in different tissues of Pacific abalone, H. discus hannai. In graph, error bars indicate standard error and different letters above the bar indicate significance difference (p < 0.05).

Figure 8

Tissue specific distribution of Hdh-MLGF and Hdh-β-Actin in the Pacific abalone, H. discus hannai.

Figure 9

Microscopic (confocal laser scanning microscope) observation of Hdh-MLGF in digestive gland tissue of adult Pacific abalone using FISH.

Figure 10

Expression of Hdh-MLGF at different stages of embryonic and larval developmental stages of Pacific abalone, H. discus hannai. In bar graph, error bars indicate standard error and different letters above the bar indicate significance difference (p < 0.05).