Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Structural role of the T94I rhodopsin mutation in congenital stationary night blindness

Congenital stationary night blindness (CSNB) is an inherited and non‐progressive retinal dysfunction. Here, we present the crystal structure of CSNB‐causing T94I^2.61 rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin‐II state by establishing a direct van der Waals contact with K296^7.43, the site of retinal attachment. This is in stark contrast to the light‐activated state of the CSNB‐causing G90D^2.57 mutation, where the charged mutation forms a salt bridge with K296^7.43. To find the common denominator between these two functional modifications, we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D^2.57 and the hydrophobic T94I^2.61 mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113^3.28. We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.     

Surface structure and properties of plant seed oil bodies

Storage triacylglycerols (TAG) in plant seeds are present in small discrete intracellular organelles called oil bodies. An oil body has a matrix of TAG, which is surrounded by phospholipids (PL) and alkaline proteins, termed oleosins. Oil bodies isolated from mature maize (Zea mays) embryos maintained their discreteness, but coalesced after treatment with trypsin but not with phospholipase A2 or C. Phospholipase A2 or C exerted its activity on oil bodies only after the exposed portion of oleosins had been removed by trypsin. Attempts were made to reconstitute oil bodies from their constituents. TAG, either extracted from oil bodies or of a 1:2 molar mixture of triolein and trilinolein, in a dilute buffer were sonicated to produce droplets of sizes similar to those of oil bodies; these droplets were unstable and coalesced rapidly. Addition of oil body PL or dioleoyl phosphatidylcholine, with or without charged stearylamine/stearic acid, or oleosins, to the medium before sonication provided limited stabilization effects to the TAG droplets. High stability was achieved only when the TAG were sonicated with both oil body PL (or dioleoyl phosphatidylcholine) and oleosins of proportions similar to or higher than those in the native oil bodies. These stabilized droplets were similar to the isolated oil bodies in chemical properties, and can be considered as reconstituted oil bodies. Reconstituted oil bodies were also produced from TAG of a 1:2 molar mixture of triolein and trilinolein, dioleoyl phosphatidylcholine, and oleosins from rice (Oryza sativa), wheat (Triticum aestivum), rapeseed (Brassica napus), soybean (Glycine max), or jojoba (Simmondsia chinensis). It is concluded that both oleosins and PL are required to stabilize the oil bodies and that oleosins prevent oil bodies from coalescing by providing steric hindrance. A structural model of an oil body is presented. The current findings on seed oil bodies could be extended to the intracellular storage lipid particles present in diverse organisms.     

Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem

A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively. The recombinant cystatins were found to be thermally stable up to 60 degrees C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.     

Roles of Rho-associated kinase and myosin light chain kinase in morphological and migratory defects of focal adhesion kinase-null cells

Fibroblasts derived from focal adhesion kinase (FAK)-null mouse embryos have a reduced migration rate and an increase in the number and size of peripherally localized adhesions (Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., and Yamamoto, T. (1995) Nature 377, 539-544). In this study, we have found that Y27632, a specific inhibitor for Rho-associated kinase (Rho-kinase), dramatically reversed the round cell morphology of FAK(-/-) cells to a spread fibroblast-like shape in 30 min and significantly enhanced their motility. The effects of Y27632 on the FAK(-/-) cell morphology and motility were concomitant with reorganization of the actin cytoskeleton and redistribution of focal adhesions. Conversely, the expression of the constitutively active Rho-kinase in FAK(+/+) cells led to round cell shape and inhibition of cell motility. Furthermore, coincident with the formation of cortical actin filaments, myosin light chain (MLC), Ser-19-phosphorylated MLC, and MLC kinase mainly accumulated at the FAK(-/-) cell periphery. We found that the disruption of actin filaments by cytochalasin D prevented the peripheral accumulation of MLC kinase and that inhibition of myosin-mediated contractility by 2,3-butanedione monoxime induced FAK(-/-) cells to spread. Taken together, our results suggest that Rho-kinase may mediate the formation of cortical actomyosin filaments at the FAK(-/-) cell periphery, which further recruits MLC kinase to the cell periphery and generates a non-polar contractile force surrounding the cell, leading to cell rounding and decreased motility.     

Size and stability of reconstituted sesame oil bodies

Oil bodies of sesame seeds comprise a triacylglycerol matrix, which is surrounded by a monolayer of phospholipids embedded with unique proteins, mainly structural proteins termed oleosins. Artificial oil bodies were successfully reconstituted with various compositions of triacylglycerols, phospholipids, and oil-body proteins. The sizes of reconstituted oil bodies displayed a normal distribution with an average size proportional to the ratio of triacylglycerols to oil-body proteins. Both thermostability and structural stability of reconstituted oil bodies decreased as their sizes increased, and vice versa. Proteinase K digestion indicated that oleosins anchored both native and reconstituted oil bodies via their central hydrophobic domains. The stability of reconstituted oil bodies, as well as the purified ones from sesame seeds, could be substantially enhanced after their surface proteins were cross-linked by glutaraldehyde or genipin.     

Analysis of the Three Essential Constituents of Oil Bodies in Developing Sesame Seeds

Oil bodies of plant seeds contain a matrix of triacylglycerolssurrounded by a monolayer of phospholipids embedded with alkalineproteins termed oleosins. Triacylglycerols and two oleosin isoformsof 17 and 15 kDa were exclusively accumulated in oil bodiesof developing sesame seeds. During seed development, 17 kDaoleosin emerged later than 15 kDa oleosin, but it was subsequentlyfound to be the most abundant protein in mature oil bodies.Phosphotidylcholine, the major phospholipid in oil bodies, wasamassed in microsomes during the formation of oil bodies. Priorto the formation of these oil bodies, a few oil droplets ofsmaller size were observed both in vivo and in vitro. Theseoil droplets were unstable, presumably due to the lack of sterichindrance shielded by the oleosins. The temporary maintenanceof these droplets as small entities seemed to be achieved byphospholipids, presumably wrapped in ER. Oil bodies assembledin late developing stages possessed a higher ratio of oleosin17 kDa over oleosin 15 kDa and were utilized earlier duringgermination. It seems that the proportion of oleosin 17 kDaon the surface of oil bodies is related to the priority of theirutilization. (Received July 16, 1997; Accepted October 27, 1997)     

Binding orientation and specificity of calmodulin to rat olfactory cyclic nucleotide-gated ion channel

Calmodulin (CaM), the primary intracellular Ca²⁺ receptor, regulates a large number of key enzymes and controls a wide spectrum of important biological responses. Recognition between CaM and its target sequence in rat olfactory cyclic nucleotide-gated ion channel (OLFp) was investigated by circular dichroism (CD), fluorescence, and NMR spectroscopy. Fluorescence data showed the OLFp tightly bound to CaM with a dissociation constant of 12?nM in a 1:1 stoichiometry. Far-UV CD data showed that approximately 60% of OLFp residues formed α-helical structures when associated with CaM. NMR data showed that most of the ¹⁵N–¹H HSQC cross-peaks of the ¹⁵N-labeled CaM not only shifted but also split into two sets of peaks upon association with the OLFp. Our data indicated that the two distinct CaM/OLFp complexes existed simultaneously with stable structures that were not interexchangeable within the NMR time scale. In light of the palindromic sequence of OLFp (FQRIVRLVGVIRDW) for CaM targeting, we proposed that the helical OLFp with C₂ symmetry may bind to CaM in two orientations. This hypothesis is supported by the observation that only one set of ¹⁵N–¹H HSQC cross-peaks of the ¹⁵N-labeled CaM was detected upon association with OLFp-M13 chimeric peptide (OLFMp), a mutated OLFp lacking the palindromic feature. The binding specificity of OLFMp to CaM was restored when the palindromic feature was destroyed. Binding modes of CaM/OLFp and CaM/OLFMp simulated by molecular docking were in accord with their distinct patterns observed in HSQC spectra. Our studies suggest that the palindromic residues in OLFp are crucial for the orientation-specific recognition by CaM.     

Stable oil bodies sheltered by a unique oleosin in lily pollen

Stable oil bodies were purified from mature lily (Lilium longiflorum Thunb.) pollen. The integrity of pollen oil bodies was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Immunodetection revealed that a major protein of 18 kDa was exclusively present in pollen oil bodies and massively accumulated in late stages of pollen maturation. According to mass spectrometric analyses, this oil body protein possessed a tryptic fragment of 13 residues matching that of a theoretical rice oleosin. A complete cDNA fragment encoding this putative oleosin was obtained by PCR cloning with primers derived from its known 13-residue sequence. Sequence analysis as well as immunological non-cross-reactivity suggests that this pollen oleosin represents a distinct class in comparison with oleosins found in seed oil bodies and tapetum. In pollen cells observed by electron microscopy, oil bodies were presumably surrounded by tubular membrane structures, and encapsulated in the vacuoles after germination. It seems that pollen oil bodies are mobilized via a different route from that of glyoxysomal mobilization of seed oil bodies after germination.