Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
Identification of β tubulin IVb as a pattern recognition receptor with opsonic activity
Graphical abstract
Introduction
Tubulin, an abundant protein present in all eukaryotic cells, belongs to one of several members of a small family of globular proteins. It was initially identified as the target of colchicine, an alkaloid that is capable of arresting mitosis in mammals (Borisy and Taylor, 1967; Weisenberg et al., 1968). The most common members of the tubulin family are α-tubulin and β-tubulin. Nowadays, in humans 10 α-tubulin and 9 β-tubulin genes have been identified in the tubulin gene family (Findeisen et al., 2014). Both α-tubulin and β-tubulin are the proteins that make up microtubules, major intracellular structures that are involved in the control of fundamental processes such as cell division, polarity of growth, intracellular trafficking and communications (Nogales, 2001; Libusová and Dráber, 2006). It has also become clear that tubulin isotypes are involved in multiple cellular functions without being incorporated into microtubule structures. For example, tubulins have been shown to regulate the permeability of the mitochondrial outer membrane voltage-dependent anion channel (Rostovtseva et al., 2018).
Although regarded as a house-keeping protein, tubulin seems to play some “luxury” roles. In the plant Arabidopsis thaliana, both α-tubulin and β-tubulin have been shown involved in defense responses via acting as lipopolysaccharide (LPS)-interacting proteins (Vilakazi et al., 2017). Similarly, tubulin has also been shown involved in pathogen recognition in the snail Biomphalaria glabrata (Tetreau et al., 2017). In addition, tubulins are apparently affected in both vertebrate (alligator) and invertebrate (shrimp and crab) by challenge with LPS (Merchant et al., 2009) or with bacterium such as Vibrio anguillarum (Zhang et al., 2010) and Spiroplasma eriocheiris (Meng et al., 2014). Recently, we have isolated a protein from zebrafish embryos by lipoteichoic acid (LTA)-conjugated Sepharose CL-4B affinity chromatography, and identified it as β-tubulin IVb (Ni et al., 2019). Overall, these suggest that tubulins play an important role in immune responses. However, detailed study on the mode of action of tubulins in defense remains largely lacking to date. Moreover, there is little convincing experimental evidence of functional commitment for specific tubulin isotypes in animals. The present study is thus undertaken to explore the immunological function of β-tubulin IVb in zebrafish Danio rerio. It demonstrates for the first time that β-tubulin IVb can function as a pattern recognition receptor with opsonic activity.
Section snippets
Fish and culture
All the zebrafish used in the experiments were treated in accordance with the guidelines of the Laboratory Animal Administration Law of China with the permit number SD2007695 approved by the Ethics Committee of the Laboratory Animal Administration of Shandong province. Healthy wild-type zebrafish were purchased from a fish dealer and maintained at 27 ± 1 °C. They were fed with brine shrimp twice per day.
Gene cloning
Total RNAs were isolated from D. rerio by RNAiso plus (Takara, Japan) according to the
Structures and characteristics of TBB4b
The β-tubulin IVb cDNA (GenBank accession number: NM_198809) obtained was 1617 bp long, which had an open-reading frame (ORF) of 1338 bp coding for a protein of 445 amino acids with calculated MW of about 49.79 kDa and pI of 4.79 (Supplementary S1). The deduced protein possessed a Tubulin domain positioned at residues 47–244 and a Tubulin_C domain positioned at residues 246–383 (Fig. 1A). Sequence alignment showed that β-tubulin IVb was highly conserved among various species ranging from coral
Discussion
Several lines of evidence suggest that tubulins play important role in immune responses of both plants and animals (Vilakazi et al., 2017; Tetreau et al., 2017; Merchant et al., 2009; Zhang et al., 2010; Meng et al., 2014), but no study has been performed thus far to investigate the mode of action of tubulins in immune defense. Additionally, little convincing experimental evidence of functional commitment for specific tubulin isotypes is available in organisms. In this study, we show first that
Declaration of competing interest
The authors declare no financial or commercial conflict of interest.
Acknowledgements
This work was supported by the Ministry of Science and Technology of the People's Republic of China [grant numbers 2018YFD0900505] and in part by Fundamental Research Funds for the Central Universities [grant numbers 201841013].
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