Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase

Abstract: In contrast to the predominantly participate, Ca²⁺/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca²⁺ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca²⁺/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC₅₀= 90 μ M ) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.     

Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.     

The subcellular distribution of carotenoids in light-grown Verticillium agaricinum

Hyphae of light-grown V. agaricinum (Link) Corda contain many lipid bodies. The hypae were disrupted and the membranes immediately pelleted by a 48 000 g spin for 30 min. The major part (90%) of the carotenoids was found in a lipid layer on top of the supernatant. Carotenoids extracted from the lipid layer in diethyl ether had an absorption maximum at 488 nm. On the basis of spectra in different organic solvents the main component was tentatively. Thin layer chromatography of extracted and saponified carotenoids revealed the presence of one major and one minor component.
The 48 000 g pellet contained 80% of the NAD⁺-dependent malate dehydrogenase (marker for mitrochondria), 25% of the antimycin-resistant NADH-cytochrome c reductase (marker for mitochodria and endoplasmic reticulum) and 10% of the proteins and the carotenoids. The latter were different from the carotenoids in the lipid layer in that the absorption maximum was at 471 nm in diethyl ether.
Further fractionation of the 48 000 g pellet showed the presence of carotenoids in both mitochondria and endoplasmic reticulum (ER) all with an absorption maximum at 471 nm. On a thin layer plate these carotenoids migrated in one band coinciding with the minor band of the lipid layer. On a continous Percoll gradient two main areas of mitochodrial activity (1.068 and 1.063 g ml⁻¹) were clearly separated from the ER (1.059 g ml⁻¹). The carotenoid content was about 0.6 and 0.7–1.0 μg (mg protein)⁻¹ for mitochondria and ER, respectively, equivalent to 1–2 carotenoid molecules per 1000 lipid molecules. The possible role of these membrane-bound carotenoid molecules is discussed.     

Temporal Analysis of Changes in Neuronal c-fos mRNA Levels Induced by Depletion of Endoplasmic Reticulum Calcium Stores: Effect of Clamping Cytoplasmic Calcium Activity at Resting Levels

Abstract: Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca²⁺-ATPase. Tg-induced rise in [Ca²⁺]_i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca²⁺]_i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c- fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c- fos mRNA levels (∼10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c- fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c- fos . Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.     

CA2+ BINDING PROTEIN CALRETICULIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): BIOCHEMICAL CHARACTERIZATION, DIFFERENTIAL EXPRESSION DURING SEXUAL REPRODUCTION, AND PHYLOGENETIC ANALYSIS

The occurrence of calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was investigated in the unicellular green alga Chlamydomonas reinhardtii Dangeard. The biochemical characterization of a diethylaminoethyl purified extract highlighted the presence, on SDS-PAGE, of a 55-kDa protein that stained blue with the Stains All dye, a diagnostic feature of acidic Ca²⁺ binding proteins. Immunoblot analyses revealed a strong cross-reaction of the Chlamydomonas reinhardtii protein with antibodies to plant calreticulins and the endoplasmic reticulum retention signal HDEL. Furthermore, the 55-kDa protein bound [⁴⁵Ca²⁺] and had an acidic isoelectric point (pI = 4.9) but was neither glycosylated nor phosphorylated. N-terminal sequencing revealed strong amino acid sequence similarity to calreticulin from other sources. The presence of calreticulin in Chlamydomonas reinhardtii suggested that an endoplasmic reticulum Ca²⁺ buffering mechanism was present in this unicellular chlorophyte. The data suggest an early origin and high conservation of endoplasmic-reticulum-mediated Ca²⁺ functions in eukaryotes, whereby specific posttranslational modifications of the proteinhave been specifically acquired in different lineages of photosynthetic eukaryotes. Moreover, northern and western blot analysis experiments showed a regulation of calreticulin expression during Chlamydomonas sexual reproduction with a high abundance of calreticulin mRNA and protein in reproductive cells.     

Phenotype modulation in primary cultures of arterial smooth-muscle cells

Abstract. The effects of prostaglandin E₁ (PGE₁) on the phenotypic state of enzymatically isolated arterial smooth-muscle cells in primary culture were studied by transmission electron microscopy, thymidine autoradiography, and cell counting. Early in culture (day 0–2), PGE₁, stimulated conversion of the cells from contractile (less euchromatic nucleus and cytoplasm dominated by myofilament bundles) to synthetic state (more euchromatic nucleus and cytoplasm dominated by cisternae of rough endoplasmic reticulum and a large Golgi complex). The rate of entrance of the cells into DNA synthesis and mitosis was also increased at this time. Later on (day 3–6), when the majority of the cells had entered synthetic state, PGE₁ inhibited DNA synthesis and cellular proliferation. These observations indicate that the effect of prostaglandins on arterial smooth muscle is dual in nature and dependent on the state of differentiation of the cells.     

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.Abbreviations DAA days after anthesis - IgG immunoglobulin G - M_r apparent molecular mass - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis     

Comparison of Type 2 Inositol 1,4,5-Trisphosphate Receptor Distribution and Subcellular Ca2+ Release Sites that Support Ca2+ Waves in Cultured Astrocytes

Abstract: We have examined the mechanisms that underlie Ca²⁺ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca²⁺ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca²⁺ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca²⁺ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP₃R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP₃Rs with Ca²⁺ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP₃Rs (InsP₃R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca²⁺ responses and InsP₃R2 immunofluorescence were compared in the same cell, domains of elevated Ca²⁺ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP₃R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca²⁺ release sites that support regenerative wave propagation include increased levels of InsP₃R2 expression.     

Several compartments of Saccharomyces cerevisiae are equipped with Ca2+-ATPase(s)

Abstract Sucrose density fractionation of yeast membranes revealed two major and two minor peaks of ⁴⁵Ca²⁺ transport activity which all co-migrate with marker enzymes of the endoplasmic reticulum, Golgi and membranes associated with these compartments as well as with ATPase activity measured when all other known ATPase are inhibited. Co-migration of ⁴⁵Ca²⁺ transport and ATPase activities was also found after removal of plasma membranes by concanavalin A treatment. SDS-PAGE at pH 6.3 shows the Ca²⁺-dependent formation of acyl phosphate polypeptides of about 110 and 200 kDa. It is concluded that several compartments or sub-compartments of yeast are equipped with Ca²⁺-ATPase(s). It is proposed that these compartments are derived from the protein secretory apparatus of yeast.     

Identification and purification of the calcium‐regulated Ca2+‐ATPase from the endoplasmic reticulum of a higher plant mechanoreceptor organ

Erythrosin b, a potent inhibitor of the Ca²⁺‐ATPases and the Ca²⁺‐release channel (BCC1) in mechanosensitive tissue of Bryonia dioica Jacq., effectively suppresses a tendril's reaction to touch, suggesting that Ca²⁺‐transporters are involved in signal transduction in this organ. The Ca²⁺‐ATPase located in the endoplasmic reticulum (ER) represents a multiregulated enzyme that is stimulated by calmodulin (CaM), KCl and lysophospholipids. Limited proteolysis of ER‐membranes by trypsin results in an irreversible activation of the Ca²⁺‐ATPase and loss of the CaM sensitivity, presumably through removal of an autoinhibitory domain where CaM binds. Mild trypsination mimics the effects of CaM on V_max and the affinity for Ca²⁺ and ATP. Irrespective of a trypsin treatment, the enzyme can be additionally stimulated by KCl and lysolipids, indicating that the sites of interaction for these effectors are not located in the domain removed by the protease. CaM‐stimulated ATPase activity was purified from microsomal and ER fractions using a combination of CaM‐affinity and anion‐exchange chromatography. The isolated polypeptide was enzymatically active, showed a calcium‐dependent mobility‐shift in SDS‐PAGE from 109 kDa in the absence of Ca²⁺ to 104 kDa in the presence of 10 m M CaCl₂ and could be radiolabeled with [³⁵S]‐CaM. The characteristics of the purified enzyme remained closely similar to those of the ER‐bound Ca²⁺‐transporting activity, including the enzymatic data, CaM stimulation, and the sensitivity towards a range of inhibitors.     

ER retention may play a role in sorting of the nuclear pore membrane protein POM121

Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 micro m(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15 degrees C. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Endoplasmic reticulum Ca(2+) homeostasis and neuronal death

The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca²⁺ signalling by either amplifying Ca²⁺ entry through voltage-gated Ca²⁺ channels by Ca²⁺-induced Ca²⁺ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca²⁺ release (IICR). The ER Ca²⁺ store emerges as a single interconnected pool, thus allowing for a long-range Ca²⁺ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca²⁺ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca²⁺ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca²⁺ handling may be involved in pathogenesis of various, neurodegenerative diseases including brain ischemia and Alzheimer dementia.     

Endoplasmic reticulum in developing seeds of Vicia faba

The changes in endoplasmic reticulum (ER) morphology during seed development have been followed using a thick section electron microscope technique. The tissues were stained with a zinc iodineosmium tetroxide complex which preferentially accumulated in the lumen between double membranes. Sections up to 2 m in thickness were examined in a high voltage electron microscope (HVEM) with tilt facility to produce stereo pairs. The micrographs from HVEM showed an increase in the extent of interconnecting tubular and cisternal ER during the protein deposition phase of seed maturation with subsequent degeneration of the cisternae to a reticular form during the final seed maturation phase. No evidence of cisternal ER vesicles was found, instead our work suggests that such structures are artefacts of thin sectioning with the so-called vesicles representing the interconnection of cisternal and tubular ER. The results are discussed with reference to the transport of storage protein from its site of synthesis, the rough cisternal ER, to that of accumulation, the vacuolar protein bodies.Abbreviations ER endoplasmic reticulum - HVEM high voltage electron microscopy     

Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: A protective role

Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage.     

An exposed carboxyl group on sialic acid is essential for gangliosides to inhibit calcium uptake via the sarco/endoplasmic reticulum Ca2+-ATPase: relevance to gangliosidoses

We previously observed that gangliosides GM2, GM1, and GM3 inhibit Ca²⁺-uptake via the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) in neurons and in brain microsomes. We now systematically examine the effect of various gangliosides and their analogs on Ca²⁺-uptake via SERCA and demonstrate that an exposed carboxyl group on the ganglioside sialic acid residue is required for inhibition. Thus, asialo-GM2 and asialo-GM1 have no inhibitory effect, and modifications of the carboxyl group of GM1 and GM2 into a hydroxymethyl residue (CH₂OH), a methyl ester (COOCH₃) or a taurine-conjugated amide (CONHCH₂CH₂SO₃H) drastically diminish their inhibitory activities. We also demonstrate that the saccharides must be attached to a ceramide backbone in order to inhibit SERCA as the ceramide-free ganglioside saccharides only inhibit SERCA to a minimal extent. Finally, we attempted to use the ceramide-free ganglioside saccharides to antagonize the effects of the gangliosides on SERCA; although some reversal was observed, the inhibitory effects of the gangliosides were not completely abolished.     

Localization of calcium ions in wounded characean internodal cells

Ca²⁺ was localized in chemically injured internodal cells of the characean alga Nitella flexilis (L.) Ag. using alizarin red and antimonate precipitation. The presence of Ca²⁺ in the antimonate precipitates was verified by X-ray analysis and EGTA chelation. Callose-containing amorphous wound walls were induced by 0·1 m m chlortetracycline (CTC) and cellulosic fibrillar wound walls were induced by 50 m m CaCl₂. Numerous precipitates were found in the amorphous wound walls and in the adjacent cytoplasm. Precipitates were mainly localized in single membrane-bound cisternae, probably of the endoplasmic reticulum, which accumulate at the wound and become a component of the amorphous wound wall via membrane fusion. In fibrillar wound walls, which do not contain membranous residues, precipitate density was significantly lower and similar to that in the secondary cell wall.
The data suggest that the high Ca²⁺ content of amorphous wound walls is due to incorporation of cytoplasmic Ca²⁺ stores. The possible function of amorphous wound walls in maintaining cellular Ca²⁺ homeostasis is discussed.     

Biochemical and immunological characterization of the plant-derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB–MPR649–684

Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB–MPR_649–684[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB–MPR_649–684 expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB–MPR_649–684 proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N- glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine–X–serine/threonine (Asn-X-Ser/Thr) N- glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR_649–684 moiety. Furthermore, the protein induced mucosal and serum anti-MPR_649–684 antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.     

Functional Conservation of Calreticulin in Euglena gracilis

Calreticulin is the major high capacity, low affinity Ca²⁺ binding protein localized within the endoplasmic reticulum. It functions as a reservoir for triggered release of Ca²⁺ by the endoplasmic reticulum and is thus integral to eukaryotic signal transduction pathways involving Ca²⁺ as a second messenger. The early branching photosynthetic protist Euglena gracilis is shown to possess calreticulin as its major high capacity Ca²⁺ binding protein. The protein was purified, microsequenced and cloned. Like its homologues from higher eukaryotes, calreticulin from Euglena possesses a short signal peptide for endoplasmic reticulum import and the C-terminal retention signal KDEL, indicating that these components of the eukaryotic protein routing apparatus were functional in their present form prior to divergence of the euglenozoan lineage. A gene phytogeny for calreticulin and calnexin sequences in the context of eukaryotic homologues indicates i) that these Ca²⁺ binding endoplasmic reticulum proteins descend from a gene duplication that occurred in the earliest stages of eukaryotic evolution and furthermore iii that Euglenozoa express the calreticulin protein of the kinetoplastid (trypanosomes and their relatives) lineage, rather than that of the eukaryotic chlorophyte which gave rise to Euglena's plastids. Evidence for conservation of endoplasmic reticulum routing and Ca²⁺ binding function of calreticulin from Euglena traces the functional history of Ca²⁺ second messenger signal transduction pathways deep into eukaryotic evolution.