Creatine Kinase Is A Bacteriostatic Factor With A Lectin-like Activity

Creatine kinase (CK), also known as creatine phosphokinase or phosphocreatine kinase belonging to the family of ATP: guanidine phospho-transferases, is an enzyme (EC 2.7.3.2) catalyzing the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), consuming phosphocreatine and generating creatine. CK has been shown to be present in various species ranging from sponge to vertebrates. It plays a central role in energy homeostasis in the tissues. Clinically, changes in cellular CK concentrations and activities have been linked to a wide range of disease states. Recently, CK has been shown to be an acute phase protein in the humoral fluid of amphioxus B. belcheri. However, immune and functional characterization of this protein remains lacking. In this paper, we report expression and functional analysis of CK gene in amphioxus B. belcheri tsingtauense.Quantitative real-time PCR was employed to quantify BbCK expression in B. belcheri challenged with LPS. Challenge with LPS was able to affect BbCK expression in a dose-dependent fashion. It was found that challenge with 0.1μg/ml LPS had little effect on the expression of BbCK; when challenged with 1 , 5 and 10μg/ml LPS, BbCK expression was significantly up-regulated, peaked at 16 , 8 and 4 h individually. These data indicate that BbCK expression is inducible by the challenge with LPS, agreeing with the observation that CK is an acute phase protein in B. belcheri.Next we sought to examine if the refolded recombinant BbCK has any bacteriostatic activity. It was found that the refolded BbCK exerted no inhibitory effect on the growth of S. aureus, while the growth of E. coli was significantly inhibited by the pre-incubation with the recombinant BbCK, and the inhibition was in a dose-dependent manner. It is highly likely that CK in the humoral fluids is capable of recognizing invading pathogens, binding to them and eventually impairing their propagation, thereby protecting the host from pathogenic attack.The mode of action by which BbCK is involved in the immunity is currently underway. It is of interest to note that the bacteriostatic activity of the recombinant BbCK against E. coli was apparently inhibited by the pre-incubation with the sugars including N-acetyl-D-mannosamine, N-acetyl-D-glucosamine, L-fucose, D-mannose, D-fructose and D-glucose. These data suggest that CK is a bacteriostatic factor with a lectin-like activity, capable of specifically inhibiting the growth of Gram-negative bacteria like E. coli, and therefore may be involved in the host's immune defense against invading pathogens.