Alternative splicing produces transcripts coding for alpha and beta chains of a hetero-dimeric phosphagen kinase

Abstract

Glycocyamine kinase (GK) catalyzes the reversible phosphorylation of glycocyamine (guanidinoacetate), a reaction central to cellular energy homeostasis in certain animals. GK is a member of the phosphagen kinase enzyme family and appears to have evolved from creatine kinase (CK) early in the evolution of multi-cellular animals. Prior work has shown that GK from the polychaete Neanthes ( Nereis) diversicolor exits as a hetero-dimer in vivo and that the two polypeptide chains (termed α and β) are coded for by unique transcripts. In the present study, we demonstrate that the GK from a congener Nereis virens is also hetero-dimeric and is coded for by α and β transcripts, which are virtually identical to the corresponding forms in N. diversicolor. The GK gene from N. diversicolor was amplified by PCR. Sequencing of the PCR products showed that the α and β chains are the result of alternative splicing of the GK primary mRNA transcript. These results also strongly suggest that this gene underwent an early tandem exon duplication event. Full-length cDNAs for N. virens GKα and GKβ were individually ligated into expression vectors and the resulting constructs used to transform Escherichia coli expression hosts. Regardless of expression conditions, minimal GK activity was observed in both GKα and GKβ constructs. Inclusion bodies for both were harvested, unfolded in urea and α chains, β chains and mixtures of α and β chains were refolded by sequential dialysis. Only modest amounts of GK activity were observed when α and β were refolded individually. In contrast, when refolded the α and β mixture yielded highly active hetero-dimers, as validated by size exclusion chromatography, electrophoresis and mass spectrometry, with a specific activity comparable to that of natural GK. The above evidence suggests that there is a preference for hetero-dimer formation in the GKs from these two polychaetes. The evolution of the alternate splicing and an additional exon in these GKs, producing α and β transcripts, can be viewed as a possible compensation for a mutation(s) in the original gene, which most likely coded for a homo-dimeric protein.