Creatine kinase in human erythrocytes: A genetic anomaly reveals presence of soluble brain-type isoform

Abstract

For maintaining energy homeostasis, creatine kinase (CK) is present at elevated levels in tissues with high and/or fluctuating energy requirements such as muscle, brain, and epithelia, while there is very few CK, if any, in peripheral blood cells. However, an ectopic expression of brain-type creatine kinase (BCK) has been reported for platelets and leukocytes in an autosomal dominant inherited anomaly named CKBE. Here we investigated CK in erythrocytes of CKBE individuals from eight unrelated families. The data revealed a varying but significant increase of CK activity in CKBE individuals as compared to controls, reaching an almost 800-fold increase in two CKBE individuals which also had increased erythrocyte creatine. Immunoblotting with highly specific antibodies confirmed that the expressed CK isoform is BCK. Cell fractionation evidenced soluble BCK, suggesting cytosolic and not membrane localization of erythrocyte CK as reported earlier. These results are discussed in the context of putative CK energy buffering and transfer functions in red blood cells.

Introduction

Isoforms of creatine kinase (CK) are key players in cellular energy homeostasis by providing together with creatine and phosphocreatine an efficient energy buffering and transfer system [1], [2], [3], [4]. High CK levels are therefore present in tissues and organs with high and/or fluctuating energy demands such as muscle, brain and epithelia, while this enzyme is much less abundant or even undetectable in some other cell types including hepatocytes and human blood cells. However, in erythrocytes of three probands from a family of Italian origin, unusually high CK activity in peripheral blood cells was described by Arnold et al. (1978) [5]. The CK isoform responsible for this activity was identified to be the brain-type CK (BCK) [6]. This ectopic expression of BCK (CKBE, MIM ID 123270) in blood cells is determined by a positive CK activity test in blood cells and normal CK activity in serum. It represents an autosomal dominant inherited anomaly with a frequency of about 1 out of 5000 persons. Most of the probands are in good health and do not show any hematologic disorder [6]. The mutation underlying the CKBE phenotype has been mapped to chromosome 14q32 where the BCK gene is located, but the molecular basis of this anomaly remains unknown [6]. CK in platelets and leukocytes has been analyzed in more detail in a recent study comparing CKBE probands of eight unrelated families or normal control individuals [6]. CK activity in probands was 50- and 7-fold higher in platelets and leukocytes, respectively, as compared to control individuals. Probands also showed significantly higher BCK mRNA levels that correlated with CK protein abundance.

Much less is known about CK in erythrocytes. Earlier data indicated that in contrast to other peripheral blood cells, human erythrocytes contain muscle-type CK (MCK) that is present in the membrane fraction [7], and this has been confirmed by first proteomic approaches [8]. Appreciable CK activities of 6.0 U/mg hemoglobin (Hb) and up to 37.3 U/mg Hb were also reported for rainbow trout and domestic chicken, respectively [9], [10], and in the latter case again MCK was suggested as the principal erythrocyte CK. However, more recent proteomic studies do not identify any CK isoform in erythrocytes [11], [12], [13], [14] and CK mRNA has neither been detected [15]. Thus, the questions remain whether red blood cells still express any CK activity, and if this is the case, which isoform and which putative function this would represent, and in particular how this activity would be affected in CKBE individuals.

In this study, we re-investigated the issue of erythrocyte CK by comparing CKBE and control probands [6]. We detected varying degrees of erythrocyte CK activity, but significantly higher in CKBE probands as compared to control individuals, and could identify this CK as a soluble, cytosolic form of BCK [4].