A hexokinase from the oyster Crassostrea gigas is involved in immune recognition as a pattern recognition receptor

Abstract

Hexokinase (HK) is generally recognized as a crucial enzyme participating in glycolysis. In the present study, a HK (named CgHK) was identified as a potential pattern recognition receptor (PRR) from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgHK was of 1395 bp, encoding a peptide of 464 amino acids with one Hexokinase_1 domain and one Hexokinase_2 domain. The predicted amino acid sequence of CgHK shared 17%–29% similarities with that of other known HKs. The mRNA transcripts of CgHK were constitutively detected in all the examined tissues, with relative high expression level in labial palp and haemocytes. CgHK protein was mainly observed in the cytoplasm of oyster haemocytes. The mRNA expression level of CgHK in haemocytes was significantly up-regulated and peaked at 3 h after Vibrio splendidus (7.64-fold, p < 0.001) and lipopolysaccharide (LPS) (11.86-fold, p < 0.001) stimulations. The recombinant CgHK protein (rCgHK) exhibited Mg2+-dependent adenosine triphosphate (ATP) binding activity in vitro and activity to bind D-(+)-glucose (GLU) and various pathogen-associated molecular pattern (PAMPs) such as LPS and peptidoglycan (PGN) in the absence of Mg2+. It also displayed higher binding activity towards V. splendidus and relatively lower binding activity towards Staphylococcus aureus, Escherichia coli, and Micrococcus luteus. After the mRNA expression of CgHK in haemocytes was knocked down by dsRNA interference, the expression of CgIL17-5 mRNA in haemocytes was considerably down-regulated at 3 h after the stimulation with V. splendidus (0.33-fold, p < 0.001). These results collectively indicated that CgHK was able to recognize various PAMPs and pathogenic bacteria as a PRR apart from being the enzyme to exert ATP binding activity in glycolysis, and activate the anti-bacterial immune response by promoting the expression of pro-inflammatory cytokines CgIL17-5 in oyster haemocytes.