Leishmania major: identification of developmentally regulated proteins in procyclic and metacyclic promastigotes

The differentiation from procyclic to metacyclic promastigotes (metacyclogenesis) has been correlated with an increased infectivity in a number of Leishmania species. We compared the proteomes of procyclic and metacyclic promastigotes of L. major. Lysates from either life cycle stage were resolved by 2D-PAGE, followed by Coomassie brilliant blue staining. Spots were analyzed by MALDI-TOF MS. 25 protein spots were found to be differentially expressed during metacyclogenesis. We found that proteins involved in protein synthesis were less abundant in metacyclic promastigotes, while proteins involved in motility, including paraflagellar rod protein 1D, α-tubulin and β-tubulin were more abundant. Also, two mitochondrial enzymes (succinyl-CoA synthetase β subunit and cytochrome c oxidase subunit IV) were differentially expressed in both life cycle stages. Down-regulation of proteins related to synthetic pathway in metacyclic promastigotes is consistent with the arrested growth in this life cycle stage, while up-regulation of proteins related to motility in metacyclic promastigotes is in agreement with the high motility observed in this stage.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Brain region-specific N-glycosylation and lipid rafts association of the rat mu opioid receptor

The mu opioid receptor (MOR) in the rat and mouse caudate putamen (CPu) and thalamus was demonstrated as diffuse and broad bands by Western blot with a polyclonal antibody against a C-terminal peptide of MOR, which were absent in the cerebellum and brains of MOR-knockout mice. The electrophoretic mobility of MOR differed in the two brain regions with median relative molecular masses (Mr’s) of 75 kDa (CPu) vs. 66 kDa (thalamus) for the rat, and 74 kDa (CPu) vs. 63 kDa (thalamus) for the mouse, which was due to its differential N-glycosylation. Rat MOR in CPu was found mainly associated with low-density cholesterol- and ganglioside M1 (GM1)-enriched membrane subdomains (lipid rafts), while the MOR in the thalamus was present in rafts and non-rafts without preference. Cholesterol reduction by methyl-β-cyclodextrin decreased DAGMO-induced [³⁵S]GTPγS binding in rat CPu membranes to a greater extent than in the thalamus membranes.     

Immunocytochemical analysis for intracellular dynamics of C1GalT associated with molecular chaperone, Cosmc

The core 1 structure Galβ1-3GalNAcα1-Ser/Thr (T antigen), the major constituent of O-glycan core structure, is synthesized by cooperation of core 1 synthase (C1GalT) and its specific molecular chaperone, Cosmc. The chaperone function of Cosmc has been well investigated biochemically. In this study, we established monoclonal antibodies specifically recognizing either C1GalT or Cosmc, respectively, and investigated the sub-cellular localization of each protein to elucidate how they cooperate to synthesize the core 1 structure.A sequential immunocytochemical analysis of the human colon cancer cell line, LSB, demonstrated different localization of two proteins. C1GalT was localized in Golgi apparatus, while Cosmc was localized in endoplasmic reticulum. In contrast, the LSC cells, which do not have core 1 synthase activity due to a missense mutation in the Cosmc gene, did not express the C1GalT protein. Although the treatment with a proteasome inhibitor, lactacystin, of LSC cells resulted in the increased expression of C1GalT protein, the distribution of C1GalT was not in Golgi apparatus as seen in LSB cells. On the contrary, overexpression of Cosmc but not C1GalT lead to precise localization of C1GalT protein, which distributed in Golgi apparatus and recovered the core 1 synthase activity in LSC cells. These results suggest that the intracellular dynamics of C1GalT is controlled by its specific molecular chaperon, Cosmc, in association with core 1 synthase activity.     

Ferritin acts as a target site for the snowdrop lectin （GNA） in the midgut of the cotton leafworm Spodoptera littoralis

The snowdrop lectin GNA （Galanthus nivalis agglutinin） has been shown to possess insecticidal activity to a range of economically important insect pests. However, the precise mechanism of insecticidal action of GNA against insects remains unknown. In this investigation, we attempted to purify and identify receptor（s） responsible for binding of GNA in the larval midgut of a major lepidopteran pest （the cotton leafworm, Spodoptera littoralis） to better understand its mode of action. Therefore, cytoplasmic as well as membrane proteins from 800 larval midguts were chromatographed on a column with immobilized GNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proteins eluted from the GNA column followed by sequencing of the GNA-binding proteins and BLAST analyses revealed that the N-terminal sequences of a 24 kDa polypeptide purified from the cytoplasmic and membrane protein fraction revealed sequence similarity to sequences encoding heavy chain homologs of ferritin from Manduca sexta （76% sequence identity）, Calpodes ethlius （80% sequence identity） and Bombyx mori （61% sequence identity）. Furthermore, the N-terminal sequence of a 31 kDa polypeptide from the membrane protein fraction showed sequence similarity to a light chain homolog of ferritin from Manduca sexta （88% sequence identity）.     

Analysis of equilibrium dissociation and unfolding in denaturants of soybean agglutinin and two of its derivatives

The equilibrium denaturation of tetrameric soybean agglutinin (SBA) in urea and guanidine hydrochloride (GdnHCl) has been examined by steady-state fluorescence and size-exclusion chromatography. The denaturation of SBA reveals two distinct and separable transitions: dissociation (native tetramer↔tertiary monomer) and unfolding (tertiary monomer↔unfolded monomer). The urea denaturation curves of N-dimethyl and acetyl derivatives of SBA are also similar to unmodified lectin but the midpoints, [D]_1/2, are shifted to lower denaturant concentrations. The free energy of stabilization of tertiary structure (ΔG_u,aq) of SBA is estimated to be 4.5–4.6 kcal mol⁻¹, which shows a decrease by 10–15% for both N-dimethyl SBA and acetyl-SBA. The free energy term (ΔG_{d, aq}) for the relative stability of the quaternary structure of SBA and its derivatives shows that the decrease in stability relative to SBA occurs by <10% for N-dimethyl SBA while for acetyl-SBA, this occurs by 30%. However, the m values depicting the dependence of free energy on denaturant concentration for SBA and its derivatives are similar for dissociation as well as unfolding, which suggest similar denaturation pathways of unmodified and modified SBA.     

Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey

Casein glycomacropeptide (CMP) is a 64‐ amino acid peptide found in cheese whey, which is released after κ‐casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N‐acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171–180, 2017     

Monomer/dimer equilibrium of the AB-type lectin from mistletoe enables combination of toxin/agglutinin activities in one protein: analysis of native and citraconylated proteins by ultracentrifugation/gel filtration and cell biological consequences of dimer destabilization

The biological activity of a lectin is influenced by its quaternary structure. Viscumin is special among the family members of toxic AB-type plant lectins, because it triggers mitogenicity, toxicity, and agglutination. Its activity profile is dependent on the concentration, motivating a thorough inspection of the status of quaternary structure. Over a broad range of protein concentrations (0.01-25 mg/mL), viscumin occurs as a dimer. At high concentrations, the solutions exhibited nonideality, self-association, and polydispersity in sedimentation equilibrium and velocity experiments caused by irreversible aggregation. Calculation of viscumin's overall shape based on sedimentation velocity data resulted in an elongated dimer form resembling that of crystallized agglutinin. Appearance of monomers was restricted to concentrations in the submicrogram/mL level, as demonstrated by fast protein liquid chromatography gel-filtration analysis. To shift the equilibrium to the monomer for comparative cell biological assays, we performed chemical modification under conditions protecting the lectin activity. Citraconylation was effective to destabilize the dimer. Binding studies by fluorescence-activated cell scan analysis revealed a reduction in cell association upon modification and a tendency for increased sensitivity towards haptenic inhibitors at microg/mL concentrations. Nonetheless, growth inhibition continued to be potent for the ricin-like monomer despite reduced extent of binding. Occurrence of a concentration-dependent monomer/dimer equilibrium appears to achieve the same objectives as the development of two separate protein entities in Ricinus communis, an alternative strategy to emergence of a monomeric toxin, and cell cross-linking dimeric agglutinin.     

Brefeldin A-regulated retrograde transport into the endoplasmic reticulum of internalised wheat germ agglutinin

The effects of the fungal metabolite brefeldin A (BFA) on the endocytic routes of internalised wheat germ agglutinin (WGA) were studied in human HepG2 hepatoma cells, drawing particular attention to the application times in relation to the membrane dynamics occurring at the trans Golgi face during endocytosis. As shown in previous studies, transport of internalised WGA into the Golgi apparatus can be classified in three stages being characterised by predominance of vesicular endosomes (stage I), formation of an extended endocytic trans Golgi network (stage II) and uptake of WGA into the stacked Golgi cisternae (stage III). BFA treatment of the cells led to rapid tubular-reticular transformations of the Golgi stacks. Retrograde transport and further destinations of internalised WGA depended on the time of BFA application. When BFA was administered during stages I or II, WGA was localised within the BFA-induced tubules and networks, but never was found within the endoplasmic reticulum. By contrast, BFA treatment during stage III led to a redistribution of internalised WGA into cisternae of the endoplasmic reticulum. These results show that BFA administered according to a precise time schedule can be used as a regulatory agent that allows to control retrograde traffic of internalised molecules into the endoplasmic reticulum.     

Immobilized Marasmius oreades agglutinin: use for binding and isolation of glycoproteins containing the xenotransplantation or human type B epitopes

The type B-specific lectin from the mushroom Marasmius oreades was immobilized onto Sepharose 4B. The immobilized lectin bound murine laminin and bovine thyroglobulin, glycoproteins that contain the Galalpha1,3Galbeta1,4GlcNAc epitope. This epitope is responsible for hyperacute rejection of xenotransplants from lower mammals to humans, Old World monkeys, or apes. The immobilized lectin also bound a fraction of serum proteins from type B human serum but little or none from type A or O(H) serum. The major protein bound from human B serum was a portion of the alpha2-macroglobulin. Treatment of this fraction with N-glycosidase F resulted in decreased molecular weight of bands associated with alpha2-macroglobulin and loss of their M. oreades lectin reactivity, whereas on treatment with coffee bean alpha-galactosidase, this bound fraction also lost reactivity with M. oreades lectin but became reactive with Ulex europaeus I lectin, suggesting the presence of L-fucosyl-alpha1,2-terminated structures. The presence of blood group epitopes on alpha2-macroglobulin has been detected previously by immunological methods, but this is the first isolation and characterization of the specifically glycosylated fraction of this serum protein. The immobilized lectin also bound a number of proteins from pig, rabbit, and rat serum that were distinct in electrophoretic mobility from the human B-serum components and presumably contain the xenotransplantation epitope among their glycan structures. This report further demonstrates the utility of immobilized lectins in isolating and characterizing glycan structures of naturally occurring glycoproteins.