A novel calcium-binding protein from Euglena gracilis. Characterisation of a cDNA encoding a 74-kDa acidic-repeat protein targeted across the endoplasmic reticulum.
We have isolated a novel cDNA from Euglena gracilis that encodes a protein composed of 24.9% aspartate with an estimated pI of 3.56, and a deduced molecular mass of 73,542 Da. The first 20 or so amino acids are hydrophobic and resemble a signal sequence. The rest of the polypeptide is composed of a 23-amino-acid repeat. There are 30 repeats, of which 23 are full length. Part of the consensus sequence derived from the repeats has some similarity to the loop of the EF-hand type calcium-binding motif. Evidence is presented that a fusion protein of this novel protein with beta-galactosidase can bind calcium. Northern blotting indicates a single transcript of 2.3 kb (the same size as the cDNA). In-vitro translation of the cDNA gives a protein that migrates on SDS/PAGE with an apparent molecular mass of 120-125 kDa. The protein is processed into a smaller, protease-protected form (110-120 kDa) when translated in the presence of canine pancreatic microsomal vesicles. This suggests that the protein is targeted across the endoplasmic reticulum membrane in vivo, and is the first report of a signal sequence from E. gracilis. We propose that the cDNA obtained encodes a novel calcium-binding protein that is either secreted or resident in the endomembrane system of E. gracilis, and call it the acidic-repeat protein.
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005056:Euglena gracilis / Q000235:genetics* / Q000378:metabolism*; D015723:Gene Library; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics; D011506:Proteins; D015800:Protozoan Proteins; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002135:Calcium-Binding Proteins; C099695:MANF protein, human; D009414:Nerve Growth Factors; D021382:Protein Sorting Signals; D011506:Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C079235:acidic repeat protein, Euglena gracilis; D012313:RNA; D004247:DNA; D001616:beta-Galactosidase; D002118:Calcium