Crenomytilus grayanus 40 kDa calponin-like protein: cDNA cloning, sequence analysis, tissue expression, and post-translational modifications

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Abstract

Calponin-like protein (CaP-40), a third major protein after actin and tropomyosin, has recently been identified by us in the Ca2 +-regulated thin filaments of mussel Crenomytilus grayanus. It contains calponin homology domain, five calponin family repeats and possesses similar biochemical properties as vertebrate smooth muscle calponin. In this paper, we report a full-length cDNA sequence of CaP-40, study its expression pattern on mRNA and protein levels, evaluate CaP-40 post-translational modifications and perform protein-protein interaction analysis.

The full-length sequence of CaP-40 consists of 398 amino acids and has high similarity to calponins among molluscan species. CaP-40 gene is widely expressed in mussel tissues, with the highest expression in adductor and mantle. Comparison of these data with protein content established by mass-spectrometry analysis revealed that the high mRNA content is mirrored by high protein levels for adductor smooth muscles. To provide unbiased insight into the function of CaP-40 and effect of its over-expression in adductor smooth muscle, we built protein-protein interaction network of identified Crenomytilus grayanus proteome. In addition, we showed that CaP-40 is subjected to post-translational N- and C-terminal acetylation at N127, G229 and G349 sites which potentially regulates its function in vivo.

Introduction

Molluscan smooth muscles, similar to that of in other species, contract in response to cholinergic nerve stimulation and relax via the serotonin-mediated mechanism (Twarog, 1976). Cholinergic nerve stimulation increases production of acetylcholine, which leads to release of [Ca2 +] into the cytoplasm and triggers muscle contraction (Szent-Györgyi, 1975). When [Ca2 +] drops down to the resting level (10 7 M) through serotonin nerve stimulation, the muscle relaxes.

However, under some circumstances (e.g. when animal is attacked), molluscan smooth muscle exhibits unique mechanical features: the decrease in [Ca2 +] does not lead to relaxation. Thus, the muscle able to maintain a high level of tension even at the basal [Ca2 +]. This condition is known as the catch state (Rüegg, 1964).

It is well established that molluscan smooth muscle exhibits a myosin-linked regulation: Ca2 + directly binds and activates myosin heads which then able to interact with actin (Lehman and Szent-Gyorgyi, 1975). However, a growing body of evidence indicates the presence of additional actin-linked thin filament system regulating muscle contraction in molluscan smooth muscle. (Lehman, 1981, Lehman, 1983, Nishita et al., 1997, Mendez-Lopez et al., 2012, Dobrzhanskaya et al., 2010, Dobrzhanskaya et al., 2013, Vyatchin et al., 2015). Indeed, molluscan thin filaments are regulated by calcium (Dobrzhanskaya et al., 2010) demonstrating high Ca2 +-sensitivity (Dobrzhanskaya et al., 2013), although the mechanism is still obscure.

Recently, several new proteins associated with molluscan thin filament other than tropomyosin and actin were identified. These are filamin-like protein (Mendez-Lopez et al., 2012), a troponin complex of three subunits (Vyatchin et al., 2015) and calponin-like protein (CaP-40) (Dobrzhanskaya et al., 2013).

CaP-40, a novel 40-kDa actin-binding protein of molluscan smooth muscle, contains CH– and calponin family repeat domains known as a hallmark of calponin family members, has 31% similarity with human and chicken calponin and possesses similar biochemical properties such as inhibitory effect on Mg2 +-ATPase myosin activity and binding to actin filaments (Dobrzhanskaya et al., 2010, Dobrzhanskaya et al., 2013, Sirenko et al., 2013, Sirenko et al., 2016). Surprisingly, association of CaP-40 with thin filaments is dynamic and depends on the isolation conditions: extraction at low temperature (2 °C) leads to selective unbinding of protein from thin filaments (Dobrzhanskaya et al., 2010). Even if this unbinding has no impact on Ca2 +-sensitivity (Dobrzhanskaya et al., 2013) it indicates that CaP-40 is able to change its localization in the cell and be a link between contractile domain and cytoskeletal network, as was reported for mammalian calponin (North et al., 1994). This redistribution might trigger multiple changes in biochemical properties of CaP-40 or, alternatively, the protein may be reversibly modified through post-translational modifications (PTMs) to be able to redistribute. To address these questions, we obtained the full sequence of C. grayanus CaP-40, studied its expression pattern in mussel tissues and analyzed the most accessible sites for PTMs.

Section snippets

Material and methods

All procedures with animals were approved by the Animal Care Committee of A.V. Zhirmunsky Institute of Marine Biology, Far East Branch of the Russian Academy of Sciences (Protocol N 21 from 08.09.2014).

Absence of caldesmon in mussel proteome

Previously we have shown by Western Blot analysis that caldesmon, an abundant protein of Ca2 +-dependent regulation in vertebrate thin filaments (Marston and Lehman, 1985), is absent in molluscan smooth muscle thin filaments (Dobrzhanskaya et al., 2013). Here we confirm this funding by a proteomic approach. We were not able to detect caldesmon not only in adductor smooth muscle (Table 3) but also in mantle and heart tube of the marine bivalve Crenomytilus grayanus (Table S1). Absence of

Similarity of CaP-40 across calponin family members

Calponin-like protein (CaP-40) was recently identified as a protein associated with Ca2 +-regulated thin filaments isolated from smooth muscle of bivalve mollusc Crenomytilus grayanus where its function is still obscure. Based on mass-spectrometry analysis of identified peptides we predicted that it contains one CH-domain and five calponin family repeats (Dobrzhanskaya et al., 2013), which are hallmarks of calponin family members. Full-length sequence analysis confirmed these data (Fig. 1).

Funding

This work was supported by the Russian Science Foundation (No 14-14-00080).

Author contributions

Conceived and designed the experiments: OSM, AD, VP, KK, NS. Performed the experiments: OSM, AD, VP, KK, GU. Analyzed the data: OSM, VP, KK. Contributed reagents/materials/analysis tools: OSM, NS. Wrote the paper: OSM.

Acknowledgments

We thank Lennart Hilbert for reading of the manuscript and comments. We are grateful to Proteomic Technology Platform of the Research Institute of the McGill University Health Centre (Montreal, Canada) and staff (Lorne Taylor and Amy Wang) for the help with mass-spectrometry experiments and comments.

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