Amino acid sequence of calmodulin from Euglena gracilis

Abstract

The complete amino acid sequence of calmodulin from Euglena gracilis was determined by isolation and sequence analyses of peptides derived from calmodulin by digestion with trypsin and Stuphylococcus aureus V8 protease. Euglena calmodulin consists of 148 amino acid residues; it lacks tryptophan and cysteine and contains one tyrosine, three histidine and two N"-trimethyllysine residues/ molecule of the protein. Its N-terminus was blocked with an acetyl group and C-terminal lysine was trimethylated. Euglenu calmodulin is the first calmodulin so far examined in which the C-terminal lysine is trimethylated. [12], and eel, salmon, sea cucumber and fungi (unpublished).We report here the amino acid sequence of Euglena calmodulin and discuss amino acid substitutions. Materials and Methods, Figs S1 -S5 and Tables S1 and S2 appear as a Supplement at the end of this paper. RESULTS AND DISCUSSIONIsolation of tryptic peptides Tryptic peptides derived from Euglena calmodulin were separated by HPLC on a Cosmosil 5ClsP-30O column ( Note. The novcl amino acid sequencc data publishcd hcre have been deposited with the EMBL sequence data bank and are availablc Sl). Heterogeneous peptide fractions such as T7/T6-7/T12 and Tl/T2/T9/Tll were further purified by rechromatography on the same column using different solvent system as described above (Fig. S2). The 12 tryptic peptides were isolated. Amino acid compositions and overall yields of the purified tryptic peptides and whole Euglena calmodulin are listed in Table S1.Isolation of peptides generated by S. aureus V8 protease digestion S. aureus V8 protease peptides were separated in the same manner as for tryptic peptides. Heterogeneous peptide fractions were rechromatographed on a CIS column using a linear gradient of solvent B in solvent A as described in the text for separation of tryptic peptides ( Figs S3 and S4). The 15 peptides were isolated. Amino acid compositions and overall yields of the purificd peptides are listed in Table S2. N-terminal sequence analysisPeptide TI (1 -13) was resistant to Edman degradation, showing the absence of a free ainino-terminal group. TI was treated with acylamino-acid-releasing enzyme (AARE) prior to Edman degradation. Aliquots of the digest were subjected to automated Edman degradation and the remaining digests were scparated by HPLC on a CIS column (Fig. S5 and Fig. 1). The N-terminal acetylated amino acid was identified as alanine (0.8 mol/mol peptide) by amino acid analysis of fraction A1 after hydrolysis. The N-terminal acetyl group was also confirmed by FAB-MS analysis [I71 of the tryptic peptide, TI (1 -13) (data not shown). On the digestion of TI with acylamino-acid-releasing enzyme, minor cleavage of the Thr5 -His6 bond was observed due to intrinsic endopeptidase action of this enzyme. The results of sequence analysis of under accession number P11118.peptide TI are summarized in Fig. 1

Euglena gracilis Genome and Transcriptome: Organelles, Nuclear Genome Assembly Strategies and Initial Features

“…12, this volume) (Porterfield 1997;Streb et al 2002;Hader et al 2005a, b;Daiker et al 2010), and signal transduction (Daiker et al 2010). Calmodulin and protein kinases are involved in gravitaxis (Streb et al 2002).Five types of CaM proteins have been discovered in E. gracilis and include: CaM.1, CaM.2, CaM.3, CaM.4, and CaM.5, with CaM.2 being implicated in gravitaxis (Toda et al 1992;Daiker et al 2010). In general, CaMs consist of ~150 amino acids and the molecule possesses four calcium binding sites (Aykut et al 2013), each motif consists of a 12-residue loop flanked on either side by a 12-residue α-helix (Kawasaki and Kretsinger 1995).…”

Molecular characterization of a calmodulin involved in the signal transduction chain of gravitaxis in Euglena gracilis

“…First strand cDNAs were synthesized from the total RNA using cDNA Synthesis Kit from Fermentas (RevertAid H Minus First Strand cDNA Synthesis Kit #K1632). Two degenerate primers Ca 56 rev and Ca 56 for (Table 1) were designed according to the calmodulin amino acid sequences (P11118) of E. gracilis Z (Toda et al 1992). Target sequences were amplified by a thermostable DNA polymerase with proofreading activity (Pyrobest DNA Polymerase, Takara) under 33 PCR cycles each consisting of a 30 s 98°C denaturizing step, a 30 s 58°C annealing step and a 30 s 72°C synthesizing step.…”

“…This paper focuses on the characterization of several new calmodulins in comparison to an already known calmodulin in E. gracilis (Toda et al 1992).…”

Gravitaxis in Euglena

“…To our surprise we did not find one gene but at least five calmodulin genes . The amino acid sequence coded by one of these genes (CaM.1) was already known from Euglena (Toda et al 1992). We sequenced the genes of all five calmodulins (CaM.1-CaM.5) isolated from E. gracilis.…”

“…In Chlamydomonas a calmodulin was detected in the flagella (Gitelman and Witman 1980). In Euglena the previously known calmodulin (CaM.1) was found underneath the pellicula (Toda et al 1992) where it may be responsible for the cell form and the euglenoid movements which might explain the effects of RNAi of CaM.1 on motility and cell form abnormalities.…”