Cloning, Expression and Isoform Classification of a Minor Oleosin in Sesame Oil Bodies

Abstract

The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosins are present in the oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. In sesame oil bodies, besides the two ubiquitous oleosin isoforms (17 and 15 kDa), an additional minor oleosin (15.5 kDa) was revealed on Tricine SDS-PAGE. A full-length cDNA fragment was cloned, sequenced and deduced to be a putative oleosin of 15,446 Da. The gene was constructed in a fusion or non-fusion vector and then over-expressed with different efficiency in Escherichia coli. All three oleosins purified from sesame oil bodies were subjected to immunoassaying using antibodies raised against the over-expressed oleosin. The results confirmed that this gene encodes the sesame 15.5 kDa oleosin. Sequence comparisons with other known oleosins revealed that sesame 15.5 kDa oleosin does not represent a new oleosin isoform class but may have been derived through gene duplication and truncation of sesame 17 kDa oleosin, and possesses the minimal structure of the high Mr oleosin isoform. A conserved amphipathic alpha-helix is predicted in sesame 15.5 kDa oleosin, which may imply a potential biological function associated with this isoform.

Gene Family of Oleosin Isoforms and Their Structural Stabilization in Sesame Seed Oil Bodies

“…18) We set out to clone the other two sesame oleosin genes. With the sesame 15.5-kDa Holeosin probe, a full-length cDNA clone (accession no …”

“…U43930) was 32 Plabeled and used as a probe to screen for sesame 17-or 15-kDa oleosin genes in the cDNA library as described elsewhere. 18) In the screening with the sesame 15.5-kDa oleosin probe, plaques with weak rather than strong signals were collected. We then excised the pBluescript phagemid from the Uni-ZAP XR vector in vivo from the plaques with weak signals on hybridization by the manufacturer's instructions.…”

Integral Proteins in Plant Oil Bodies

“…Right after the kick-off studies on maize oleosins, homologous oleosin isoforms were subsequently identified in oil bodies of rapeseed (Brassica napus), soybean (Glycine max L.), carrot (Daucus carota), sunflower (Helianthus annuus), Arabidopsis thaliana, and cotton (Gossypium hirsutum) with their corresponding cDNA fragments cloned [27][28][29][30][31][32][33][34]. It seems that oleosin isoforms are universally present in oil bodies of angiosperm seeds including both monocotyledonous and dicotyledonous species [35][36][37][38][39]. Furthermore, oleosin isoforms found as the major proteins (approximately 80-90%) in seed oil bodies were demonstrated to shield the whole surface of the organelles in the company of phospholipids [40].…”

Neutral Lipid Bodies in Prokaryotes: Recent Insights into Structure, Formation, and Relationship to Eukaryotic Lipid Depots

“…Due to their secondary structure, oleosins form a meshwork-like organization on the lipid body surface. Oleosins are composed of two amphiphilic regions at the N and C termini, whereas the central part represents an expanded conserved domain composed of hydrophobic amino acids, which directly contacts the lipid matrix (30,78). The secondary structure of this hydrophobic domain was predicted to be an antiparallel ␤-strain in solvent (63), but more recent experiments performed on native oleosins suggest that it presents mostly as an ␣-helix (78).…”