Proteome of Seed OB

Exhaustive protein compositions from OB from castor beans (R. communis) [3], A. thaliana [21], and B. napus [4, 37] were recently determined. Proteins from purified OB were often precipitated overnight in cold 100% acetone (—20°C) and subsequently solubilized in electrophoresis or IEF sample medium. Proteins were separated by SDS-PAGE or 2-DGE. Analyses of trypsin peptides obtained after in-gel digestion were performed using MALDI or LC-MS/MS devices. For protein identification, acquired mass spectra were searched against the NCBI nonredundant database or against more specific ESTs database. Major proteins were identified as oleosins, but some other minor proteins were also recognized.

Structural Proteins. Oleosins are alkaline proteins of low molecular mass (15-26 kDa). They all possess three structural domains [7]. Each oleosin molecule has a highly conserved central hydrophobic domain of 68-74 amino acid residues, which is the longest hydrophobic sequence found in any organism. This domain was proposed to be either an anti-parallel a-helix penetrating into the TG matrix or a long stretch of ^-strand structure running underneath the surface of OB. The loop in the anti-parallel ^-strands has a "proline knot" consisting of three proline and one serine residues. An amphipathic domain of 40-60 amino acids, the secondary structure of which is unknown, is present at the N-terminus and likely associated with the OB surface. An amphipathic a-helical domain is situated at the C-terminus. The positively charged residues face the negatively charged lipids on the boundary phos-pholipids layer, whereas the negatively charged residues are exposed to the surface. Together, the N-terminal, the central, and the a-helical portions anchor the oleosin stably on the surface of the OB. Two distinct oleosin isoforms were first described in maize (16-18 kDa [38]), soybean (18-24 kDa [38]), sesame (15-17 kDa [39]), and castor bean (14-16 kDa [3]), only one oleosin was recovered in almond OB [40], and numerous cDNA and genomic sequences encoding for these proteins have been reported from various species. Sixteen oleosin genes have been characterized in the

A. thaliana genome, with five genes specifically expressed in maturing seeds [8]; five genes of the coffee oleosin family have been very recently described, with CcOLE-1 and CcOLE-2 being more highly expressed [41]. Regarding the proteomic analysis of highly purified seed OB, two oleosins were identified in castor bean OB [3], five in A. thaliana [21], and three to eight in B. napus, depending on database used for protein identification [4, 37].

Caleosin gene sequences have been found in a wide range of plants and oil-accumulating fungi [9] composing a multigene family in the case of A. thaliana [9, 42], B. napus [43], or barley [44]. Caleosins contain an N-terminal region with a single calcium-binding EF-hand, a central hydrophobic region with a potential membrane anchor, and a C-terminal region with conserved protein kinase phosphorylation sites [13]. The presence of the hydrophobic region suggested an oleosin-like association with OB. Immunological detection and proteomic analyses suggested that caleosin was unique in seed OB from sesame, sunflower, soybean, peanut [45], rapeseed [4, 45], and A. thaliana [21]. Caleosins could have a potential role in lipid trafficking, membrane expansion, and OB formation during seed development, as hypothesized by Liu et al. [44]. Recent works proved the involvement of AtClo1 in the degradation of storage lipid in OB [46]. According to Hanano et al. [47], AtClo1 could be involved in the biosynthesis of phytooxylipins and capable of catalyzing hydroperoxide-dependent mono-oxygenation reactions that are characteristic of peroxygenases.

Enzymes. Besides abundant oleosins and caleosin, proteins named steroleosins for their sterol-binding capacity have been more recently described in OB of sesame, A. thaliana, and rapeseed [4, 21, 37, 48, 49]. These proteins possess a hydrophobic anchoring segment followed by a soluble domain homologous to sterol-binding dehy-drogenases/reductases with an active site comprised between a NADPH-binding and a sterol-binding subdomain [48]. Sterol-coupling dehydrogenase activity was demonstrated in purified OB from sesame or A. thaliana seeds as well as in the overexpressed soluble domain of steroleosin [48, 50]. According to enzymatic activity assays and sequence homology to hydroxysteroid dehydrogenases, steroleosins are classified as 11P- and 17^-hydroxysteroid dehydrogenases and belong to a very large group of NAD(P)-dependent oxidoreductases, the short-chain dehydrogenase/reductase family. Moreover, A. thaliana steroleosin was described to catalyze NADPH-dependent 17P-ketosteroid reduction [50]. The question of the physiological substrates and functions of steroleosins in plants remain to be elucidated even if Lin and co-workers [48, 49] hypothesized their involvement in plant signal transduction regulated by various sterols. In a recent article, Katavic et al. [4] have reported in B. napus the presence of (a) three isoforms of 11P-hydroxysteroid dehydrogenase, (b) a new short-chain dehydrogenase/reductase, and (c) a myrosinase-like protein showing similarity with the lipase hydrolase family of enzymes with GDSL motif.

Lipase activities have been early characterized in seeds from rape, mustard, or corn and were reported to be associated to OB [51-53]. Enzyme activities are only detectable upon germination and increase concomitantly with TG breakdown. However, until now, only few genes encoding these enzymes have been cloned and characterized [3, 54, 55]. Eastmond [3] recently reported a relatively amphipathic nature of OB-associated lipase from castor bean. He suggested that the N-terminus region of protein, devoid of "proline knot" and less hydrophobic than oleosins central region, might play a role in anchorage to OB.

Valencia-Turcotte and Rodriguez-Sotres [56] demonstrated the presence of a dia-cyl glycerol acyl transferase activity in purified maize OB. Finally, an almond 9-hydroperoxide lyase associated with OB has recently been reported [57] with the targeting of the protein verified through GFP fusions transiently expressed in tobacco protoplasts.

Minor Proteins. Other proteins such as an aquaporin and a putative GPI-anchored protein [21] have been detected in purified OB from A. thaliana upon silver staining, which proves that these proteins existed only in very low abundance. The presence and function of aquaporin remain to be confirmed: this protein is proposed to isolate OB from water or, on the contrary, to facilitate the intake of water and uptake of glycerol during lipolysis and germination. GPI-anchored proteins are often hardly detected in classical proteomics analyses. Some are not recovered from 2D gels and must be analyzed by 1D SDS-PAGE. Moreover, trypsin digestion of these proteins yields few or no peptides for LC-MS/MS analysis due to their high level of glycosylation. Some GPI-anchored proteins could be involved in lipid transfer or associated with membrane lipid microdomains.

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