Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase
- 469 Downloads
Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.
KeywordsDevelopment Embryo Glycoconjugates Japanese medaka Neu4 Sialidase
We appreciated the expert technical assistance of Mako Ikeda, Momo Shiozaki, Yusuke Harasaki and Midori Fukuda. This work was supported by Grant-in-Aid for Young Scientist (B) (Grant No. 24780204) from Japan Society for the Promotion of Science (JSPS).
- Hinek A, Bodnaruk TD, Bunda S, Wang Y, Liu K (2008) Neuraminidase-1, a subunit of the cell surface elastin receptor, desialylates and functionally inactivates adjacent receptors interacting with the mitogenic growth factors PDGF-BB and IGF-2. Am J Pathol 173(4):1042–1056PubMedCentralPubMedCrossRefGoogle Scholar
- Perez-Sanchez J, Estensoro I, Redondo MJ, Calduch-Giner JA, Kaushik S, Sitja-Bobadilla A (2013) Mucins as diagnostic and prognostic biomarkers in a fish-parasite model: transcriptional and functional analysis. PLoS One 8(6):e65457Google Scholar
- Zanchetti G, Colombi P, Manzoni M, Anastasia L, Caimi L, Borsani G, Venerando B, Tettamanti G, Preti A, Monti E, Bresciani R (2007) Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures. Biochem J 408(2):211–219PubMedCentralPubMedCrossRefGoogle Scholar