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. 2024 Jan 26;14(1):2224.
doi: 10.1038/s41598-024-51950-7.

Cloning, characterization, and spatio-temporal expression patterns of HdhSPARC and its responses to multiple stressors

Md Abu Hanif  1 , Shaharior Hossen  1 , Cheol Young Choi  2 , Kang Hee Kho  3
Affiliations

Cloning, characterization, and spatio-temporal expression patterns of HdhSPARC and its responses to multiple stressors

Md Abu Hanif et al. Sci Rep. .

Abstract

SPARC is an extracellular Ca2+-binding, secreted glycoprotein that plays a dynamic role in the growth and development of organisms. This study aimed to describe the isolation, characterization, and expression analysis of HdhSPARC in Pacific abalone (Haliotis discus hannai) to infer its potential functional role. The isolated HdhSPARC was 1633 bp long, encoding a polypeptide of 284 amino acid residues. Structurally, the SPARC protein in abalone is comprised of three biological domains. However, the structure of this protein varied between vertebrates and invertebrates, as suggested by their distinct clustering patterns in phylogenetic analysis. In early development, HdhSPARC was variably expressed, and higher expression was found in veliger larvae. Moreover, HdhSPARC was highly expressed in juvenile abalone with rapid growth compared to their slower-growing counterparts. Among the testicular development stages, the growth stage exhibited higher HdhSPARC expression. HdhSPARC was also upregulated during muscle remodeling and shell biomineralization, as well as in response to different stressors such as heat shock, LPS, and H2O2 exposure. However, this gene was downregulated in Cd-exposed abalone. The present study first comprehensively characterized the HdhSPARC gene, and its spatio-temporal expressions were analyzed along with its responses to various stressors.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Multiple sequence alignment of HdhSPARC from deduced amino acid sequences of H. discus hannai (OM937904.1), H. discus discus (BAK22657.1), Pinctada fucata (AND99565.1) Patella vulgata (CCJ09602.1), Salmo salar (ACH70833.1), Xenopus tropicalis (AAT01218.1), Rattus norvegicus (NP_036788.1), and Homo sapiens (AAA60570.1). The blue letters indicate hydrophobic residues, red letters signify positively charged residues, magenta letters denote negatively charged residues, green letters represent polar residues, pink letters are for cysteine residues, orange letters for glycine residues, yellow letters for proline residues, cyan letters indicate aromatic residues, and white letters are for the unconserved residues. The green line indicates the signal peptide, and the red boxes indicate the conserved cysteine residues. The red line indicates the SPARC domain.
Figure 2
Figure 2
Structure of the HdhSPARC protein. (A) three functional domains detected in HdhSPARC, (B) three-dimensional structure with the conserved domain.
Figure 3
Figure 3
Phylogenetic tree of SPARC constructed via the maximum likelihood method after ClustalW alignment based on amino acid residues of different vertebrate and invertebrate species. The red circle in the tree indicate bootstrap probability. The green cluster indicates Mammalia, yellow for Amphibia, light green for Actinopterygii, blue for Gastropoda, and violet for Branchiopoda. The following protein sequences with their accession id were used to construct the phylogenetic tree: Homo sapiens (AAA60570.1), Pongo abelii (NP_001127042.1), Bos taurus (ABQ12988.1), Sus scrofa (AAX83050.1), Orcinus orca (XP_004280373.1), Pleurodeles waltl (AWQ28573.1), Rhinatrema bivittatum (AWQ28578.1), Typhlonectes compressicauda (AWQ28577.1), Xenopus laevis (CAA44350.1), Xenopus tropicalis (AAT01218.1), Oryzias latipes (AAT01217.1), Scophthalmus maximus (AGW25370.1), Epinephelus coioides (ACJ66296.1), Oncorhynchus mykiss (AAC99813.1), Salmo salar (ACH70833.1), H. discus discus (BAK22657.1), H. discus hannai (OM937904.1), Patella vulgata (CCJ09602.1), Pinctada fucata (BAK22656.1), Mytilus edulis (CAG2244558.1), Hyalella azteca (XP_018022874.1), Artemia franciscana (BAB20042.1), Daphnia magna (XP_045029097.1), Blattella germanica (CZQ50751.1), and Nilaparvata lugens (UPH52990.1).
Figure 4
Figure 4
Development stage specific mRNA expression of HdhSPARC in Pacific abalone. (a) embryonic and larval developmental (FE, fertilized egg; 2-CL, 2-cell, 4CL, 4-cell, MOR, morula, BLS, blastula, TRO, trochophore; VLG, veliger; JUV, juvenile), (b) testicular development (PS, proliferative stage; GS, growth stage; SS, spawning stage; RS, resting stage). (nsp > 0.05; *p < 0.05; ***p < 0.001).
Figure 5
Figure 5
Expression of HdhSPARC in mantle and muscle tissue of Pacific abalones exhibiting different growth patterns. SG, stunted growth; MG, minimum growth; NG, normal (average) growth; RG, rapid growth. (nsp > 0.05; *p < 0.05; ***p < 0.001).
Figure 6
Figure 6
Expression of HdhSPARC during muscle remodeling and shell biomineralization and injured Pacific abalone. D, day; W, week. (nsp > 0.05; *p < 0.05; ***p < 0.001).
Figure 7
Figure 7
Expression of HdhSPARC in stressed abalone. (a) heat stressed, (b) LPS-exposed Pacific abalone. (nsp > 0.05; *p < 0.05; ***p < 0.001).
Figure 8
Figure 8
Expression of HdhSPARC in stressed abalone. (a) Cd-exposed, (b) H2O2-exposed Pacific abalone. (nsp > 0.05; *p < 0.05; ***p < 0.001).
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