Novel Nile tilapia Neu1 sialidases: Molecular cloning and biochemical characterization of the sialidases Neu1a and Neu1b

Abstract

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.