CgAATase with specific expression pattern can be used as a potential surface marker for oyster granulocytes. Issue 87 (April 2019)

• Record Type: Journal Article
• Authors: Dong, Miren; Song, Xiaorui; Wang, Min; Wang, Weilin; Zhang, Peng; Liu, Yu; Li, Meijia; Wang, Lingling; Song, Linsheng
• Is Part Of: Fish & shellfish immunology. Issue 87(2019)
• Page Start: 96
• Page End: 104
• Publication Date: 2019-04
• Subjects: Granulocytes -- Crassostrea gigas -- Surface marker -- Alcohol acyltransferase -- Monoclonal antibody -- Immunomagnetic bead; Fishes -- Immunology -- Periodicals; Shellfish -- Immunology -- Periodicals; Poissons -- Immunologie -- Périodiques; Crustacés -- Immunologie -- Périodiques; 571.9617
• DOI: 10.1016/j.fsi.2019.01.003
• Languages: English
• ISSNs: 1050-4648

Abstract

Abstract: Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas . In the present study, an alcohol acyltransferase (designed as Cg AATase) with specific expression pattern was identified from oyster C. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) of Cg AATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts of Cg AATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level of Cg AATase in hemocytes was significantly up-regulated at 3–12 h and reached the highest level (27.40-fold compared to control group, p < 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll ® density gradient centrifugation. Cg AATase transcripts were dominantly observed in granulocytes, which was 8.26-fold ( p < 0.05) and 2.80-fold ( p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody against Cg AATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead. Cg AATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ± 4.60% of the granulocytes were positive for Cg AATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti- Cg AATase monoclonal antibody, and 97.7 ± 1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative for Cg AATase. The isolated primary granulocytes could maintain cell activity for more than one week in vitro culture that exhibited numerous filopodia. These results collectively suggested that Cg AATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti- Cg AATase monoclonal antibody. Highlights: A new member of AATases family was identified from oyster C. gigas. Cg AATase mRNA was highly expressed in granulocytes and Cg AATase protein was located on their membrane. Cg AATase could be used as a marker for the identification and purification of oyster granulocytes.