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Introduction: ABO-incompatible (ABOi) kidney transplantation, a well-established procedure, has good long-term results provided pretransplant desensitization that includes immunosuppression and apheresis., Objective: To compare, within the first pretransplant apheresis session given to 29 ABOi kidney-transplant candidates, the effect on isoagglutinin titers (both IgG and IgM isotypes) of three modalities: centrifugation therapeutic plasmapheresis (cTP; n = 10), filtration TP (fTP; n = 9), and double-filtration plasmapheresis (DFPP; n = 10)., Results: The three groups were comparable according to baseline demographics. Treated plasma volumes were similar across the three groups, that is, 4111 ± 403 mL (cTP), 3861 ± 282 mL (fTP), and 3699 ± 820 mL (DFPP): that is, 54 ± 7, 53 ± 7, and 53 ± 10 mL/kg respectively. One session of centrifugation or filtration TP reduced IgG anti-A/anti-B isoagglutinin titer by ~4, whereas one DFPP session reduced it by ~2. One session of cTP reduced IgM anti-A isoagglutinin titer by a little less than 4, whereas fTP and DFPP sessions reduced it by ~3. There were no statistical differences across the three groups regarding isoagglutinin rebound (IgG and IgM). However, isoagglutinin IgG rebound was >4 dilutions for anti-B titers compared with ~2 dilutions for anti-A titers. The median decreases in IgG level were -3.9 g/L (DFPP), -5.9 g/L (cTP), and - 6.06 g/L (fTP) (p = ns). Median fibrinogen depletions were ~ 60% (fTP), 64% (DFPP), and 76% (cTP)., Conclusions: Isoagglutinin depletions within the first apheresis session were similar across cTP, fTP, and DFPP: this was numerically lower for DFPP., (© 2020 Wiley Periodicals LLC.)

Rather than the internal genome nucleic acids, the biomolecules on the surface of the influenza virus itself should be detected for a more exact and rapid point-of-care yes/no decision for influenza virus-induced infectious diseases. This work demonstrates the ultrasensitive electrical detection of the HA1 domain of hemagglutinin (HA), a representative viral surface protein of the influenza virus, using the top-down complementary metal oxide semiconductor (CMOS) processed silicon nanowire (SiNW) field-effect transistor (FET) configuration. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA) was employed as a probe that specifically binds both to the aldehyde self-aligned monolayer on the SiNWs and to HA1 simultaneously. CMP-NANA was serially combined with two kinds of linkers, namely 3-aminopropyltriethoxysilane and glutaraldehyde. The surface functionalization used was verified using the purification of glutathione S-transferase-tagged HA1, contact angle measurement, enzyme-linked immunosorbent assay test, and isoelectric focusing analysis. The proposed functionalized SiNW FET showed high sensitivities of the threshold voltage shift (ΔV T ) ~51 mV/pH and the ΔV T = 112 mV (63 mV/decade) with an ultralow detectable range of 1 fM of target protein HA1.

Plant ribosome-inactivating proteins (RIPs) are a family of toxins that inhibit protein synthesis. In this study, we have isolated a novel type 2 ribosome-inactivating protein (RIP) present in seeds of the Abrus fruticulosus, named of fruticulosin. Fruticulosin, shows characteristics common to other type 2 RIPs, as specificity by galactosides (d-galactose, N-acetyl-d-galactosamine, and d-lactose), mass of approximately 60 kDa and presence of the of disulfide bonds. The N-terminal amino acid sequence (26 residues) of A-chain fruticulosin, determined by Edman degradation, revealed high similarity of the A-chain with those of other type 2 RIPs. The secondary structure of fruticulosin was analysed by circular dichroism, which showed that fruticulosin contains α-helices (22.3%), β-sheets (43.5%), and random coils and corners (34.2%). Furthermore, fruticulosin showed high toxicity in Artemia sp. (3.12 μg/mL), inhibited in vitro protein synthesis by a cell-free system and showed RNA N-glycosidase activity. Fruticulosin presented biological activities such as agglutination and antileishmanial activity on promastigote forms of Leishmania major., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM -1 ) and recombinant protein productivity per unit area-time (g m -2 /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m -2 than at a low plant density of 100 plants m -2 . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Virus-like particles (VLPs) are becoming established as vaccines, in particular for influenza pandemics, increasing the interest in the development of VLPs manufacturing bioprocess. However, for complex VLPs, the analytical tools used for quantification are not yet able to keep up with the bioprocess progress. Currently, quantification for Influenza relies on traditional methods: hemagglutination assay or Single Radial Immunodiffusion. These analytical technologies are time-consuming, cumbersome, and not supportive of efficient downstream process development and monitoring. Hereby we report a label-free tool that uses Biolayer interferometry (BLI) technology applied on an Octet platform to quantify Influenza VLPs at all stages of bioprocess. Human (α2,6-linked sialic acid) and avian (α2,3-linked sialic acid) biotinylated receptors associated with streptavidin biosensors were used, to quantify hemagglutinin content in several mono- and multivalent Influenza VLPs. The applied method was able to quantify hemagglutinin from crude samples up to final bioprocessing VLP product. BLI technology confirmed its value as a high throughput analytical tool with high sensitivity and improved detection limits compared to traditional methods. This simple and fast method allowed for real-time results, which are crucial for in-line monitoring of downstream processing, improving process development, control and optimization., (© 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Glycine max is commonly used in Algeria for treatment of anemia deficiency and osteoporosis, it ranks first in terms of vegetal proteins. The experiment was aimed at characterizing the proteinaceous Glycine max extract and evaluating its antioxidant, biological and hematological potential., Methodology: Extraction of proteinaceous materials from Glycine max plant was undertaken using water and n-hexane as extracting media. The isolation of proteins from the crude materials was done, providing the use of ammonium sulfate. The Glycine max proteins were characterized by UV-visible and FT-IR spectroscopy and analyzed by SEM micrograph and x-ray diffraction (XRD). Rheological parameters G' and G'' were assessed. The isolated proteins were tested for their antioxidant, antimicrobial and hemagglutination activities., Results: There was a gelling effect of the protein extract which can be used as an alternative in principally made vaccines with its microbiological and antifungal activities., Conclusion: The proteinaceous extract from Algerian Glycine max would have a potential use in biomedical application.

Poultry outbreaks caused by H5N8 highly pathogenic avian influenza viruses (HPAIVs) occurred in Japan between December 2014 and January 2015. During the same period; H5N8 HPAIVs were isolated from wild birds and the environment in Japan. The hemagglutinin (HA) genes of these isolates were found to belong to clade 2.3.4.4 and three sub-groups were distinguishable within this clade. All of the Japanese isolates from poultry outbreaks belonged to the same sub-group; whereas wild bird isolates belonged to the other sub-groups. To examine whether the difference in pathogenicity to chickens between isolates of different HA sub-groups of clade 2.3.4.4 could explain why the Japanese poultry outbreaks were only caused by a particular sub-group; pathogenicities of A/chicken/Miyazaki/7/2014 (Miyazaki2014; sub-group C) and A/duck/Chiba/26-372-48/2014 (Chiba2014; sub-group A) to chickens were compared and it was found that the lethality of Miyazaki2014 in chickens was lower than that of Chiba2014; according to the 50% chicken lethal dose. This indicated that differences in pathogenicity may not explain why the Japanese poultry outbreaks only involved group C isolates., (© 2016 The Societies and John Wiley & Sons Australia, Ltd.)

Gallibacterium anatis has the ability to hemagglutinate rabbit erythrocytes; however, no bacterial component has yet been associated with this function. In the present work, a protein of approximately 65 kDa with hemagglutinating activity for glutaraldehyde-fixed chicken erythrocytes was purified by ion interchange chromatography from G. anatis F149(T) secreted proteins. The protein was recognized by a rabbit polyclonal serum against a hemagglutinin from Avibacterium paragallinarum. The 65 kDa purified protein presented identity with a G. anatis filamentous hemagglutinin by mass spectrometric analysis. As well, the bacterial surface of G. anatis was labeled by immune gold assays using a polyclonal serum against the 65-kDa protein. A similar protein was recognized in four other G. anatis strains by immunoblots using the same antiserum. The protein binds sheep or pig biotinylated fibrinogen, suggesting an interaction with basement membrane eukaryotic cells components, and the protein is present in G. anatis biofilms. Overall, the results suggest that the 65 kDa hemagglutinin is a common antigen and a potential virulence factor in G. anatis.

Lectins (hemagglutinins) are defined as sugar-binding proteins or glycoproteins with various biological activities. A 60 kDa dimeric hemagglutinin with a blocked N-terminus was isolated in large yield (190 mg/60 g) from the common edible bean Phaseolus vulgaris cv. Hokkaido large pinto bean. Its hemagglutinating, antifungal, and antitumor activities as well as the effects of carbohydrate and metal ions on its hemagglutinating activity were examined. It inhibited the proliferation of nasopharyngeal carcinoma (CNE2), human breast cancer (MCF7), and hepatoma (HepG2) cells. The IC50 values toward HepG2, MCF7, and CNE2 cells after treatment for 48 h were 8.1, 6.07, and 7.49 μM, respectively, which were relatively low among lectins of different P. vulgaris cultivars. From the pinto beans, a 10888 Da antifungal peptide with similarity to plant defensins as revealed by mass spectroscopic analysis was also isolated with a yield of 3.2 mg of proteins from 60 g of beans. The large defensin was capable of inhibiting mycelial growth in Mycosphaerella arachidicola, Setosphaeria turcica, Bipolaris maydis, and Fusarium oxysporum but not in Valsa mali.

In the present study, we isolated a novel hemagglutinin from an edible legume and explored its growth-inhibitory effect on osteocarcinoma and liver cancer cells. The protein was purified by liquid chromatography techniques which entailed affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono Q, and gel filtration on Superdex 75 with an FPLC system. The hemagglutinating activity of this hemagglutinin was demonstrated to be ion dependent and stable over a wide range of temperature and pH values. Antiproliferative activity was observed in the tumor cell lines MG-63 and HepG2 but not in the normal cell line WRL 68. Osteocarcinoma cells treated with the hemagglutinin underwent obvious cell shrinkage, chromatin condensation, mitochondrial membrane depolarization, and apoptosis. The mRNA expression level of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) were found to be up-regulated to different extents after treatment of this hemagglutinin.

ABO-incompatible kidney transplantation is nowadays a well-established procedure to expand living donor transplantation to blood group incompatible donor/recipient constellations. In the last two decades, transplantation protocols evolved to more specific isohaemagglutinin elimination techniques and established competent antirejection protection protocols without the need of splenectomy. ABOi kidney transplantation associated accommodation despite isohaemagglutinin reappearance, C4d positivity of peritubular capillaries as well as the increased incidence of bleeding complications is currently under intense investigation. However, most recent data show excellent graft survival rates equivalent to ABO-compatible kidney transplantation outcome., (© 2014 Steunstichting ESOT.)

A novel lectin, CLSL, was purified from Chinese leek seeds by ion exchange chromatography on SP Sephadex C-25 and gel filtration chromatography on Sephadex G50. The lectin had a molecular weight of 23.6 kDa and was composed of two identical subunits linked by disulfide bonds, a conclusion based on SDS-PAGE under reducing and nonreducing conditions. CLSL was a glycoprotein with a carbohydrate content of 3.6%. It exerted potent agglutinating activity against rat red blood cells at a concentration of 8.9 μg/mL. Hemagglutination of rat erythrocytes was inhibited by d-fructose, mannitol, and sorbose at the concentration of 20 mM. The hemagglutinating activity of CLSL was maintained at 100 °C for 60 min and under acidic pH conditions but was lost at neutral and alkaline pH conditions. The hemagglutinating activity was stimulated by Ca(2+), Fe(2+), and Cu(2+) but inactivated by Ba(2+) at a concentration of 10 mM. Ba(2+)-mediated inactivation of CLSL was caused by CLSL conformational change induced by barium ions, according to the results of circular dichroism and fluorescence spectroscopy. Deconvolution of the CLSL circular dichroism indicated that it was an α-helical lectin with α-helix and β-fold contents of 35.8% and 8.6%, respectively. CLSL could also selectively inhibit cell proliferation.

The botulinum toxin complex, the causative agent of botulism, passes through the intestinal wall via sugar-chain-dependent cell binding of a haemagglutinin of 33 kDa molecular weight (HA-33). The amino-acid sequence of the C-terminal half of HA-33 of the serotype C strain Yoichi (C-Yoichi) shares only 46% identity with those of the major serotype C strains. Additionally, C-Yoichi HA-33 exhibits a unique sugar-binding specificity. In the present work, C-Yoichi HA-33 was expressed in Escherichia coli and crystallized. Diffraction data were collected at a resolution of 2.2 Å. The crystals belonged to space group R3. The complete detailed protein structure will yield insight into how the unique HA-33 protein recognizes sugar moieties.

A 64-kDa hemagglutinin from a Phaseolus vulgaris cultivar, the northeast red bean, was purified by a protocol composed of three chromatographic steps involving affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose and FPLC-gel filtration on Superdex 75. The purified hemagglutinin appeared as a single 32-kDa band in SDS-PAGE indicating its dimeric nature. The N-terminal amino acid sequence of the hemagglutinin resembled the sequences of lectins and hemagglutinins from a number of Phaseolus species. The hemagglutinin manifested moderate thermostability and pH stability. It retained full activity up to 65 °C and in the pH range 2-12. It did not interact with simple sugars such as glucose, mannose and galactose. The hemagglutinin exerted immunostimulatory effects by upregulating the expression of cytokines like interferon-γ and tumor necrosis factor-α. It also exhibited antiproliferative activity on a number of tumor cells including MCF7 (breast cancer), HepG2 (liver cancer), CNE1 and CNE2 (nasopharyngeal cancer) cells, with stronger activity toward MCF7 and CNE1 cells. The hemagglutinin induced phophatidylserine externalization, mitochondrial depolarization and DNA condensation in MCF7 cells, indicating initiation of apoptosis. However, at high hemagglutinin concentrations, severe damage to the MCF7 cells was detected., Competing Interests: The authors confirm that this article content has no conflict of interest.

Lectin from the seeds of Dioclea lasiophylla (DlyL) was purified in a single step by affinity chromatography on a Sephadex® G-50 column. DlyL strongly agglutinated rabbit erythrocytes and was inhibited by monosaccharides (D-mannose and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Similar to other Diocleinae lectins, DlyL has three chains, α, β and γ, with mass of 25,569 ± 2, 12,998 ± 1 and 12,588 ± 1 Da, respectively, and has no disulfide bonds. The hemagglutinating activity of DlyL was optimal in pH 8.0, stable at a temperature of 70 °C and decreased in EDTA solution, indicating that lectin activity is dependent on divalent metals. DlyL exhibited low toxicity on Artemia sp. nauplii, but this effect was dependent on the concentration of lectin in solution. DlyL immobilized on cyanogen bromide-activated Sepharose® 4B bound 0.917 mg of ovalbumin per cycle, showing the ability to become a tool for glycoproteomics studies.

The current commercial system for influenza vaccine production depends on the culture of virus in embryonated eggs--a strategy that is both costly and poorly scalable. Consequently, a sudden pandemic event with a demand for millions of vaccine doses in a short time could readily overwhelm the available world production capacity. In this communication, we present a process that uses Escherichia coli for scalable production of recombinant vaccine candidates against influenza. A monomeric and a dimeric fragment of hemagglutinin of the influenza A H1N1/2009 virus were successfully expressed in a BL21 (DE3) pLysS variety of C41 E. coli. We present results from batch processes where induction is made with isopropyl thiogalactoside and from fed-batch experiments where expression is induced using lactose/glucose pulses. Concentrations in the range of 1.188-0.605 g/L of recombinant protein were observed in 2-L stirred tank bioreactors. The genetic construct included an N-terminal histidine tag sequence that facilitated recovery, purification, and proper refolding of the vaccine candidate by affinity chromatography in columns loaded with Ni(+2) . The proteins produced by this strategy selectively and specifically recognizes antibodies from patients diagnosed as positive to influenza A H1N1/2009. Overall protein recovery yields between 30.0 and 34.7% were typically observed. Based on these yields, a production of 4.6 × 10(3) doses L(-3) day(-1) is feasible., (© 2013 American Institute of Chemical Engineers.)

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor-binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N-glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) based glycoanalysis (N-glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N-glycosylation pattern of Madin-Darby canine kidney (MDCK) cell-derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum-free growth has only a minor impact on the HA N-glycosylation pattern. Only relative abundances of N-glycan structures are affected. In contrast, host cell adaptation to serum-free suspension growth resulted in significant changes in the HA N-glycosylation pattern regarding the presence of specific N-glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β-propiolactone inactivation resulted-at best-only in minor changes in the relative N-glycan structure abundances of the HA N-glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N-glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum-free suspension growth significantly influenced HA N-glycosylation regarding both, the type of attached glycan structures as well as their abundances., (Copyright © 2013 Wiley Periodicals, Inc.)

The plant Jatropha curcas (Euphorbiaceae) is an important source of biofuel from the inedible oil present in its toxic seeds. The toxicity arises from the presence of curcin, a ribosome-inactivating protein showing haemagglutination activity. In this communication, the purification, crystallization and preliminary X-ray characterization are reported of a small protein isolated from J. curcas seeds with a molecular mass of ~10 kDa that agglutinates rabbit erythrocytes. The protein was crystallized using the hanging-drop vapour-diffusion method and also by the microbatch method in 72-well HLA plates, using PEG 8000 as the precipitant in both conditions. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P2(1)2(1)2(1). The crystals diffracted to 2.8 Å resolution at 103 K.

The isolation of a dimeric 29.8-kDa lectin (LFL) from dried Lactarius flavidulus fruit bodies is reported herein. The chromatographic procedure utilized comprised anion-exchange chromatography on DEAE-cellulose, cation-exchange chromatography on CM-cellulose, anion-exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of LFL was inhibited by a variety of simple sugars, such as lactose, p-nitrophenyl α-D-glucopyranoside, p-nitrophenyl β-D-glucopyranoside and inositol, and by the polysaccharide inulin. The activity of LFL was stable up to 40°C. There was a precipitous drop in activity when the temperature was elevated to 50°C. Hemagglutinating activity was retained when LFL was exposed to 6.25-12.5 mM HCl and NaOH. The activity was potently inhibited by Fe(2+) and Fe(3+) ions, and slightly inhibited by Al(3+) and Mn(2+) ions. LFL suppressed the proliferation of hepatoma (HepG2) and leukemic (L1210) cells with an IC(50) of 8.90 and 6.81 μM, respectively. It inhibited the activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC(50) of 5.68 μM. However, LFL did not exhibit antifungal activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A thermostable novel lectin with a molecular weight of 30.4 kDa was isolated from dried fruiting bodies of Agaricus arvensis. It was a dimer made up of two 15.2 kDa subunits. The lectin was unadsorbed on DEAE-cellulose in 10 mM phosphate buffer (pH 7.5), subsequently adsorbed on CM-cellulose in 10 mM NaAc buffer (pH 4.6) and then on SP-Sepharose in 10 mM NaAc buffer (pH 4.6), and finally purified by fast protein liquid chromatography-gel filtration on Superdex 75. The hemagglutinating activity of the lectin was stable at temperatures up to 90 °C. The activity was preserved in concentrations of NaOH solution up to 50 mM, but was sensitive to HCl and declined to 12.5% in 12.5 mM HCl. The activity was unaffected by Ca(2+), Mn(2+), Zn(2+) and Mg(2+) ions, but was activated by Al(3+) and Fe(3+) ions. Among the carbohydrates tested, only inulin could inhibit the hemagglutinating activity of the lectin. It did not exhibit anti-HIV reverse transcriptase activity. Proliferation of HepG2 and MCF7 tumor cells was inhibited by the lectin with an IC(50) of 1.64 and 0.82 μM, respectively. The lectin was devoid of antifungal activity. The lectin has a remarkable thermostablity and a unique N-terminal amino acid sequence, TYAVLNFVYG. The present report is the first report on a lectin from wild mushroom Agaricus arvensis., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A lectin was purified from fruit bodies of the milk mushroom Lactarius pergamenus (Fr.) Fr. by a combination of ethanol precipitation, affinity chromatography on copolymer of polyvinyl alcohol and human blood B-group-specific substance, and ion-exchange chromatography on DEAE-Toyopearl. The lectin yield was 3 mg/kg of fresh mushrooms. Considerable loss of primary activity was observed during its purification, which, presumably, could be explained by disintegration of the lectin molecule, which consisted of six subunits, first to two molecules of three subunits, and then to individual subunits. There was a reverse tendency to aggregation during concentration of lectin solutions. Similar processes can take place in nature because of considerable individual variations of the lectin activity during growth of mushroom fruit bodies. The lectin weakly interacts with DGalNAc, while DGalβ1-3DGalNAc and DGalβ1-3DGlcNAc are the most probable candidates for ligands, with which the L. pergamenus lectin interacts at disaccharides level. The purified lectin may find application in histochemical research.

Background: Tick carrier proteins are able to bind, transport, and store host-blood heme, and thus they function also as antioxidants. Nevertheless, the role of carrier proteins in ticks is not fully understood. Some of them are found also in tick males which do not feed on hosts to such an extent such as females (there are differences in male feeding in different tick species) and thus they are not dealing with such an excess of heme; some of the carrier proteins were found in salivary glands where the processing of blood and thus release of heme does not occur. Besides, the carrier proteins bind relatively low amounts of heme (in one case only two molecules of heme per protein) compared to their sizes (above 200 kDa). The main aim of this study is the biochemical characterization of a carrier protein from the ornate sheep tick Dermacentor marginatus, hemelipoglycoprotein, with emphasis on its size in native conditions, its glycosylation and identification of its modifying glycans, and examining its carbohydrate-binding specificity., Results: Hemelipoglycoprotein from D. marginatus plasma was purified in native state by immunoprecipitation and denatured using electroelution from SDS-PAGE separated plasma. The protein (290 kDa) contains two subunits with molecular weights 100 and 95 kDa. It is glycosylated by high-mannose and complex N-glycans HexNAc(2)Hex(9), HexNAc(2)Hex(10), HexNAc(4)Hex(7), and HexNAc(4)Hex(8). The purified protein is able to agglutinate red blood cells and has galactose- and mannose-binding specificity. The protein is recognized by antibodies directed against plasma proteins with hemagglutination activity and against fibrinogen-related lectin Dorin M from the tick Ornithodoros moubata. It forms high-molecular weight complexes with putative fibrinogen-related proteins and other unknown proteins under native conditions in tick plasma. Feeding does not increase its amounts in male plasma. The hemelipoglycoprotein was detected also in hemocytes, salivary glands, and gut. In salivary glands, the protein was present in both glycosylated and nonglycosylated forms., Conclusion: A 290 kDa hemelipoglycoprotein from the tick Dermacentor marginatus, was characterized. The protein has two subunits with 95 and 100 kDa, and bears high-mannose and complex N-linked glycans. In hemolymph, it is present in complexes with putative fibrinogen-related proteins. This, together with its carbohydrate-binding activity, suggests its possible involvement in tick innate immunity. In fed female salivary glands, it was found also in a form corresponding to the deglycosylated protein.

A 22.4-kDa dimeric hemagglutinin was isolated from tubers of Colocasia esculenta cv. 'Small Taro' by employing a purification protocol that involved ion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono Q, and FPLC-gel filtration on Superdex 75. The hemagglutinin was isolated from the fraction of the taro extract adsorbed on Q-Sepharose and subsequently adsorbed on Mono Q. The major absorbance peak from the Superdex 75 column constituted purified hemagglutinin. Its hemagglutinating activity could not be inhibited by simple sugars, and was stable after exposure for 30 minutes to temperatures up to 40 degrees C and to ambient pH in the range of pH 2 to pH 13. The activity decreased progressively when the ambient temperature was raised from 40 degrees C to 100 degrees C. Negligible activity was detected at 100 degrees C. The activity plummeted, with about 40% and 10% remaining, 4 minutes and 20 minutes after exposure to 100 degrees C, respectively. About half of the activity remained at pH 0 and pH 1 whereas the activity was completely abolished at pH 14. The hemagglutinin exhibited slight anti-tumor activity toward hepatoma HepG2 cells, and weak mitogenic activity toward murine splenocytes. It induced expression of the cytokines interleukin-1 beta, interleukin-2, interferon-gamma and tumor necrosis factor-alpha. However, it was devoid of anti-fungal activity toward a number of fungal species.

A dimeric 64-kDa hemagglutinin was isolated with a high yield from dried Phaseolus vulgaris cultivar "French bean number 35" seeds using a chromatographic protocol that involved Blue-Sepharose, Q-Sepharose, and Superdex 75. The yield was exceptionally high (1.1g hemagglutinin per 100g seed), which is around 10-85 times higher than other Phaseolus cultivars. Its N-terminal sequence resembled those of other Phaseolus hemagglutinins. The hemagglutinating activity of the hemagglutinin was stable in the pH range 6-8, and in the temperature range 0 degrees C-50 degrees C. It inhibited HIV-1 reverse transcriptase with an IC50 of 2microM. It suppressed mycelial growth in Valsa mali with an IC50 of 10microM. It inhibited proliferation of hepatoma HepG2 cells and breast cancer MCF-7 cells with an IC50 of 100 and 2microM, respectively. It had no antiproliferative effect on normal embryonic liver WRL68 cells., (Copyright 2009 Elsevier GmbH. All rights reserved.)

A lectin and a galactoxyloglucan were characterized from Mucuna sloanei seed cotyledons. The galactoxyloglucan, isolated by water extraction and ethanol precipitation, had Glc:Xyl:Gal proportions in a molar ratio of 1.8:1.7:1.0 and a molar mass (M(w)) of 1.6x10(6)g mol(-1). The lectin (sloanin), isolated from the same seed by affinity chromatography on cross-linked Adenanthera pavonina galactomannan, gave two protein bands by SDS-PAGE (36 and 34 kDa) and one peak by gel filtration (63.6 kDa). Its N-terminal sequence indicated approximately 69% identity with soybean agglutinin to leguminous lectins. Circular dichroism (CD) spectra established that sloanin predominantly contains beta-sheet structures. Sloanin has approximately 5.5% carbohydrate and displayed hemagglutinating activity against rabbit and enzyme treated human erythrocytes, inhibited only by D-Gal containing sugars. The interaction between sloanin and storage cell-wall galactoxyloglucan was tested by affinity chromatography and fluorescence spectroscopy.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that is one of the causative agents of chronic adult periodontal disease. Among the potential virulence factors of P. gingivalis are the hemagglutinins. Recombinant Hemagglutinin B (rHagB) from P. gingivalis has been shown to activate the immune system by inducing specific antibodies that protect against experimental periodontal bone loss following P. gingivalis infection. Since different microbial products can stimulate dendritic cells (DC) through Toll-like receptors (TLRs), subsequently leading to T cell activation and antibody production, we wanted to investigate the immunostimulatory effect of rHagB on DC and the role of TLR signaling in this process. Using an endotoxin free rHagB preparation, our results show that stimulation of murine bone marrow-derived DC with rHagB leads to upregulation of the costimulatory molecules CD86 and CD40, activation of p38 and ERK MAP kinases, transcription factors NF-kappaB, CREB and IRF-3 and the production of IL-6, TNF-alpha, IL-12p40 and to a lesser extent IL-10 and IFN-beta. This activation process was absolutely dependent on TLR4 and CD14. While upregulation of CD86 was independent of the adaptor molecule MyD88, CD40 upregulation and optimal cytokine (IL-6, TNF-alpha, IL-12p40, IL-10 and IFN-beta) production required both MyD88 and TRIF molecules. These results are of importance since they are the first to provide insights into the interaction of rHagB with DC and TLRs. The information from this study will aid in the design of effective vaccines strategies against chronic adult periodontal disease.

There are only a few reports on agglutinins from ascomycete and medicinal fungi. An HA (haemagglutinin), with an N-terminal amino acid sequence different from those of known lectins, was isolated in the present study from dried fruiting bodies of the medicinal ascomycete fungus Cordyceps militaris. The purification protocol consisted of affinity chromatography, ion-exchange chromatography and gel filtration. The haemagglutinating activity of the HA could not be inhibited by simple sugars or heparin, and was stable over the pH range 2-13 and up to 60 degrees C. Chemical modification of tryptophan and tyrosine residues had no effect. The HA exhibited some antiproliferative activity towards hepatoma (HepG2) cells and inhibited HIV-1 reverse transcriptase (IC50=10 microM). However, it did not exhibit antifungal activity, mitogenic activity towards splenocytes, nitric oxide-inducing activity towards macrophages or RNase activity. The results of the present study add to the meagre information pertaining to agglutinins from ascomycete and medicinal mushrooms. It is revealed in this study that C. militaris HA differs from other ascomycete mushroom HAs in a variety of biochemical characteristics.

Background: Epidemics caused by highly pathogenic avian influenza virus (HPAIV) are a continuing threat to human health and to the world's economy. The development of approaches, which help to understand the significance of structural changes resulting from the alarming mutational propensity for human-to-human transmission of HPAIV, is of particularly interest. Here we compare informational and structural properties of the hemagglutinin (HA) of H5N1 virus and human influenza virus subtypes, which are important for the receptor/virus interaction., Results: Presented results revealed that HA proteins encode highly conserved information that differ between influenza virus subtypes H5N1, H1N1, H3N2, H7N7 and defined an HA domain which may modulate interaction with receptor. We also found that about one third of H5N1 viruses which are isolated during the 2006/07 influenza outbreak in Egypt possibly evolve towards receptor usage similar to that of seasonal H1N1., Conclusion: The presented results may help to better understand the interaction of influenza virus with its receptor(s) and to identify new therapeutic targets for drug development.

The haemagglutinin (HA) content is an important specification of influenza vaccines. Recently, a reversed-phase high performance liquid chromatography (RP-HPLC) method for quantification of HA in PER.C6 cell culture-based whole virus vaccines has been reported, having a high sensitivity, precision, broad range, and high sample throughput [Kapteyn JC, Drissi Saidi M, Dijkstra R, Kars C, Tjon CMS-K, Weverling GJ et al. Haemagglutinin quantification and identification of influenza A&B strains propagated in PER.C6 cells: a novel RP-HPLC method. Vaccine 2006;24:3137-44]. This RP-HPLC assay is based on measuring the peak area of HA1, the hydrophilic subunit of HA, which turned out to be proportional to the amount of HA analyzed. Here, we present data demonstrating that this RP-HPLC method is also highly suitable for HA quantification of active and BPL- or formaldehyde-inactivated egg-based and MDCK cell-based whole virus samples, including egg allantoic harvest, and in final (monovalent) subunit vaccines, including those for pandemic H5N1 strains and for virosomal vaccines. In addition, the RP-HPLC assay was demonstrated to be a very powerful tool in the early stages of seasonal influenza vaccine production, when homologous serial radial immunodiffusion (SRID) reagents are not yet available, enabling fast and reliable viral growth studies in eggs in order to select the best growing virus strains or reassortants for the production of the seasonal trivalent influenza vaccine. Because of its high sensitivity, the RP-HPLC assay has shown its enormous value in supporting small scale MDCK-based (H5N1) influenza virus production models. Finally, the observed differences between HA1 molecules from various HA subtypes in UV absorbance, FLD response, and in the actual retention times in RP-HPLC are discussed in relation to the primary structure of the HA1 molecules studied.

The lectin from the marine sponge Axinella corrugata (ACL-I) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-I is a hexameric glycoprotein with a Mr of 82.3 kDa estimated by SDS-PAGE and 78.5 kDa by FPLC on Superose 12 HR column. The pI of lectin is 6.3 and ACL-I is constituted of 13.9 kDa similar subunits some of them linked by disulphide bridges. This lectin agglutinates native rabbit, goat and dog erythrocytes and in less extent human erythrocytes. The hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but it is strongly inhibited by carbohydrates containing N-acetyl groups. ACL-I is stable up to 70 degrees C for 30 min, with optimum pH between 7 and 8, and it is also resistant to enzymatic proteolysis in vitro. In the presence of reducing or denaturant agents, the lectin activity decreases. ACL-I displays chemotactic effect on rat neutrophil in vitro which is inhibited by N-acetyl-d-glucosamine.

A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25-26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5+/-0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as alpha-casein (K(m): 23.0 microM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-beta-Nam (K(m): 55.24 microM, k(cat): 0.92 s(-1)). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40-50 degrees C) over a pH range of 5.5-6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.

The hemagglutinin from the seeds of Moringa oleifera (MoL) agglutinates human as well as rabbit erythrocytes; the affinity for the latter is almost 250 times more than that for the former. MoL was inhibited by glycoproteins namely thyroglobulin, fetuin and holotransferin indicating the complex sugar specificity of the lectin. The protein is a homodimer with molecular mass of 14kDa, subunits (7.1kDa) linked by the disulfide bond(s). The secondary structure elements of MoL are alpha-helix, 28%; beta-sheet, 23%; turn 20% and unordered 28%. While the activity and secondary structure were not affected at extreme pH and high temperature, they were drastically affected in presence of dithiothreitol at and above pH 7.0, indicating that disulfide linkages hold the active conformation of the protein.

A purification protocol is described herein for concurrent isolation of two defense proteins including a 6-kDa defensin-like antifungal peptide and a 60-kDa dimeric hemagglutinin from seeds of the French bean (Phaseolus vulgaris). It involved ion-exchange chromatography on SP-Sepharose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Q-Sepharose, and gel filtration on Superdex Peptide (for defensin-like antifungal peptide) or Superdex 200 (for hemagglutinin). Both antifungal and hemagglutinating activities were adsorbed on SP-Sepharose and then on Affi-gel blue gel. Hemagglutinin was subsequently unadsorbed and defensin-like antifungal peptide adsorbed on Q-Sepharose. The antifungal activity of the antifungal peptide was stable in the temperature range of 0-90 degrees C for 20 min, in the pH range of 4-10, and after exposure to trypsin (1 mg/ml) at 37 degrees C for 1 h. The hemagglutinin was stable from 10 to 80 degrees C, from pH 1 to 12, and after treatment with trypsin at 37 degrees C for 2 h. It inhibited [methyl-(3)H]thymidine incorporation into breast cancer (MCF-7), leukemia (L1210), hepatoma (HepG2) and human embryonic liver (WRL68) cells with an IC50 of 6.6, 7, 13 and 15 microM, respectively, and elicited maximal mitogenic response from mouse splenocytes at 1 microM concentration. It curtailed HIV-1 reverse transcriptase activity with an IC50 of 1.9 microM, but was devoid of antifungal activity.

It is suggested that Haemophilus paragallinarum requires at least three haemagglutinins for adhesion during infection. This paper reports the partial purification and characterization of the HA-L haemagglutinin from H. paragallinarum strain 46-C3, a heat sensitive, trypsin sensitive haemagglutinin that has been shown to be the serovar specific haemagglutinin in this organism. Using the pl and molecular mass obtained, it was shown that this protein shares similarities with other types of adhesins found in Gram-negative bacteria. The haemagglutination assay conditions were optimized at pH 7.5 at 37 degrees C. It was also shown that activity is enhanced by the addition of Ca2+ and Mn2+ ions.

Cultured Histomonas meleagridis cells were readily agglutinated in vitro by horse serum at concentrations as low as 5%, although clumping was more rapid and prominent at 15% or higher. For observation of clumping, the cultured organisms were washed twice in Hanks balanced solution (HBSS) by centrifugation (1,000 x g for 15 min) and filtered through glass wool. The test sera were added and the mixture incubated in a Petri plate or 24-well culture plates at r.t. for 15-30 min. Formation of clumps was time- and concentration-dependent. Gentle agitation hindered agglutination at low serum concentration and accelerated agglutination at higher concentrations. The agglutinating factor (AF) was detected in several batches of serum from different sources, regardless of whether sera were heat-treated to inactivate complement. Histomonads were not clumped by either fetal horse or bovine serum (5-30%). Neither chicken nor turkey serum agglutinated histomonads to the extent seen with horse serum. Immune turkey serum lysed histomonads, hindering observation of clumping. Complement inactivation of immune serum slightly reduced lysis. AF in horse serum was precipitated with 25-40% ammonium sulfate and was active when cleaned by dialysis and reconstituted in HBSS. Clumping by serum facilitated the cleaning of histomonads for other studies where pure suspensions were needed.

Real-time reverse transcription polymerase chain reaction (RRT-PCR) is commonly used for the rapid detection, as well as to determine the subtype, of avian influenza viruses (AIVs). There are 16 known serologically distinct hemagglutinin (HA) subtypes of AIV described. Currently, determination of the subtypes of AIVs by RRT-PCR tests has been limited to the H5 and H7 subtypes. In this study, RRT-PCR assays were developed in simplex formats for rapid detection of AIV subtypes H6, H9, and H11. The primers and probes for RRT-PCR were designed from nucleotide sequences of the HA genes, which were either downloaded from GenBank (for H6 and H9) or sequenced for this study. The specificity and sensitivity of the RRT-PCR assays were determined based on the detection of the virus from a proficiency panel consisting of 15 different HA subtypes of AIVs and from serial dilutions of target viral RNA. The subtype-specific RRT-PCR assays were used to detect the virus in cloacal and oropharyngeal swabs of experimental chickens inoculated with H6, H9, and H11 AIVs, and the test results were compared with validated RRT-PCR assays based on the amplification of AI matrix (MA) gene. A high correlation of the matrix test and the specific H6, H9, and H11 by the RRT-PCR assays was observed; kappa coefficients for the agreement of test results in cloacal and oropharyngeal swabs combined were 0.927, 0.962, and 0.981, respectively.

Lectins play important role in innate immunity of animals. The affinity of the natural haemagglutinin of the giant freshwater prawn Macrobrachium rosenbergii towards vertebrate erythrocytes and its level with relation to sex, size and moult stages were studied. The strongest agglutinating titres in haemolymph of prawns were marked against guinea pig, chicken, Clarias batrachus, and rabbit erythrocytes, and the weakest towards cattle, dog, horse and goat erythrocytes. A moderately agglutinating titre was evident in duck and human erythrocytes. The haemolymph of adult, male or intermoult stage prawns weighing more than 100 g had the highest haemagglutinating activity as compared to their respective counterparts with varied responses observed towards various erythrocytes.

A rhamnose-binding glycoprotein (lectin), named SML, was isolated from the eggs of Spanish mackerel (Scomberomorous niphonius) by affinity and ion-exchange chromatographies. SML was composed of a non-covalently linked homodimer. The SML subunit was composed of 201 amino acid residues with two tandemly repeated domains, and contained 8 half-Cys residues in each domain, which is highly homologous to the N-terminal lectin domain of calcium-independent alpha-latrotoxin receptor in mammalian brains. Each domain has the same disulfide bonding pattern; Cys10-Cys40, Cys20-Cys99, Cys54-Cys86 and Cys67-Cys73 were located in the N-terminal domain, and Cys108-Cys138, Cys117-Cys195, Cys152-Cys182 and Cys163-Cys169 were in the C-terminal domain. SML was N-glycosylated at Asn168 in the C-terminal domain. The structure of the sugar chain was determined to be NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-6-(NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc-Asn.

A 67-kDa hemagglutinin composed of two identical subunits was purified from Phaseolus vulgaris cv. 'Dark Red Kidney Bean'. It was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. The hemagglutinin was highly purified after the two aforementioned chromatographic steps as revealed by a single peak in gel filtration on Superdex 75 and a single band in SDS-PAGE. The hemagglutinating activity was stable between 25 degrees C and 70 degrees C, and between pH 4 and pH 11, and in the presence of a variety of divalent metal chlorides at 500 mM concentration. The activity was reduced by 50% at 80 degrees C, and also when the pH was lowered to 3 or elevated to 12. The activity was reduced by 75% in the presence of 250 mM KCl or NaCl. A variety of sugars tested failed to inhibit the hemagglutinating activity of the hemagglutinin. Although the hemagglutinin exhibited mitogenic activity toward murine splenocytes, it had no effect on the activity of HIV-1 reverse transcriptase or mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It exerted an antiproliferative activity on leukemia L1210 cells.

We previously identified a strong haemagglutination activity in the freshwater unicellular green alga, Chlorella pyrenoidosa. Here, we sought to purify and characterize the haemagglutinin associated with this activity. Ammonium sulfate precipitation, gel filtration on sephacryl S-200 and DEAE-Sepharose ion-exchange chromatography were used to purify the haemagglutinin, which was designated CPH (Chlorella pyrenoidosa haemagglutinin). The molecular weight of CPH was estimated as 58 kDa by SDS-PAGE and 60 kDa by gel filtration of the native protein, indicating that this haemagglutinin exists as a monomer. The haemagglutinin activity of CPH was inhibited by glycoproteins, especially yeast mannan, but not by monosaccharides or disaccharides, indicating that CPH is carbohydrate-specific. In addition to the composition of CPH shown to be rich in glycine and acidic amino acids, heamagglutinating activity of CPH was insensitive to variations in pH or the presence of divalent cations, and atomic force microscopy revealed that the protein is rod-shaped. These results indicate that the characteristics of CPH are consistent with its identification as a haemagglutinin, and suggest that CPH may be a viable candidate for applications in a variety of biomedical fields.

A nonfimbrial mannose-sensitive hemagglutinin (MSH) with adhesive properties produced by Salmonella enterica serovar Enteritidis was characterized. The MSH was characterized as glycoprotein and consisted of three noncovalently bound subunits of M(r) 28, 33 and 40 kDa determined by SDS-PAGE. The hemagglutinin was heat-stable and resistant to alkaline (high) or acid (low) pH, however, it was inhibited by proteolytic enzymes, by EDTA and by sodium periodate. Mouse antiserum raised against MSH reacted with the 28 kDa band in immunoblotting, and also inhibited hemagglutination and bacterial adherence to HeLa cells. Electron microscope examinations showed that MSH is not a fimbriae-like structure. MSH and anti-MSH IgG competitively inhibited bacterial adherence to HeLa cells. The immunofluorescence test, using MSH on HeLa cells and specific anti-MSH IgG, supported the view that MSH contributes to adherence of the organism. These results indicate that MSH is a nonfimbrial putative adhesive factor that may mediate the adherence of Salmonella enteritidis to eucaryotic cells.

Bordetella pertussis is the causative agent of whooping cough, a major childhood pathogen; acellular vaccines consisting of purified B. pertussis antigens such as filamentous haemagglutinin (FHA) are commonly used to prevent pertussis. Despite the importance of FHA in B. pertussis pathogenesis and its inclusion in most acellular vaccines, the functional importance of individual domains in the induction of protective immunity is largely unknown. In this study, we have purified a recombinant FHA protein from Escherichia coli consisting of a 42 kDa maltose binding domain of E. coli and the 43 kDa type I immunodominant domain of FHA. The fusion protein (Mal85) was purified from E. coli cell lysates via affinity chromatography with an amylose column. Mal85 was then delivered to BALB/c mice intranasally encapsulated in liposomes, formulated with Protollin(TM) or in conjunction with an immunostimulatory CpG oligonucleotide. Mice were also vaccinated intraperitoneally with alum-adsorbed Mal85. Sera from all treatment groups showed strong IgG responses to Mal85 and recognized native FHA. Specific salivary IgA was induced in mice vaccinated with Mal85 in liposomes, Protollin(TM) and delivered with CpG. Vaccination with Mal85 encapsulated in liposomes or formulated with Protollin(TM) provided protection against aerosol challenge with B. pertussis in BALB/c mice. These data indicate that the type I domain of FHA is a protective antigen in mice and may serve as a candidate for inclusion in new acellular pertussis vaccines.

A protein with antifungal and hemagglutinating activities was isolated from dried flageolet beans (Phaseolus vulgaris cv. 'Flageolet Bean'). The protein was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. The protein demonstrated antifungal activity against Mycophaerella arachidicola with an IC50 of 9.8 microM, but was inactive toward Fusarium oxysporum and Botrytis cinerea. Its hemagglutinating activity could not be inhibited by a variety of the sugars tested. The activity was stable up to 60 degrees C. At 70 degrees C, 75% of the hemagglutinating activity remained while no activity was discernible at and above 100 degrees C. The hemagglutinating activity was stable in the presence of a variety of monovalent, divalent and trivalent chlorides, and also when the ambient pH changed from 3 to 12. It did not exert any mitogenic activity on mouse splenocytes in vitro. Neither did it inhibit HIV-1 reverse transcriptase. It inhibited [3H-methyl]-thymidine incorporation into leukemia L1210 cells with an IC50 of about 4 microM.

To date, arabinose-binding lectins have been reported only from the human opportunistic pathogen Pseudomonas aeruginosa, the plant aggressive pathogen Ralstonia solanacearum, and the sponge Pellina semitubulosa. An arabinose-binding lectin with mitogenic activity toward splenocytes and a high specific hemagglutinating activity was isolated in the present study from a wild discomycete mushroom, Peziza sylvestris. The maximal mitogenic activity was induced by a lectin concentration of 8 microM. The lectin was a single-chained protein with a molecular mass of 20 kDa. Its N-terminal sequence showed only slight resemblance to other mushroom lectins. It was adsorbed on both diethylaminoethyl-cellulose and carboxymethyl-cellulose. Unlike previously reported mushroom lectins, the hemagglutinating activity of the lectin was inhibited by arabinose, but not by a large variety of other carbohydrates. The lectin activity was adversely affected in the presence of 0.05 M NaOH or 0.025 M HCl, and when the ambient temperature was elevated above 35 degrees C.

Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogen Porphyromonas gingivalis and are encoded by rgpA and rgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the alpha-catalytic chain and the beta-adhesin chain, the monomeric soluble Arg-gingipain comprising only the alpha-catalytic chain (RgpA(cat)), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the alpha-catalytic chain (mt-RgPA(cat)), are derived from rgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight. rgpB encodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated that rgpB is involved in the glycosylation process, since inactivation of rgpB results in the loss of not only RgpB and mt-RgpB but also mt-RgpA(cat). This work aims to confirm the role of RgpB in the posttranslational modification of RgpA(cat) and the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivated rgpB strain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream of rgpB had no effect on rgpA-derived enzymes, suggesting that the phenotype of the rgpB mutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpA(cat) from W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpA(cat) from D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived from rgpA and that this process is required for enzyme stabilization.

The temperature-sensitive hemagglutinin (Tsh) is an autotransporter protein secreted by avian-pathogenic Escherichia coli strains that colonize the respiratory tract and lead to airsacculitis, pericarditis, and colisepticemia. It is synthesized as a 140-kDa precursor protein, whose processing results in a 106-kDa passenger domain (Tshs) and a 33-kDa beta-domain (Tsh(beta)). The presence of a conserved 7-amino-acid serine protease motif within Tshs classifies the protein in a subfamily of autotransporters, known as serine protease autotransporters of the Enterobacteriaceae. In this study, we report that purified Tshs is capable of adhering to red blood cells, hemoglobin, and the extracellular matrix proteins fibronectin and collagen IV. We also demonstrate that Tshs exerts proteolytic activity against casein, and we provide experimental evidence demonstrating that serine 259 is essential for the protease function. However, this residue is not required for adherence to substrates, and its replacement by an alanine does not abolish binding activity. In summary, our results demonstrate that Tsh is a bifunctional protein with both adhesive and proteolytic properties.

The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis. Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein. Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies. The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter. The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4. Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M. smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA. We constructed a recombinant M. smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified. The use of fast-growing strains of M. smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis.

Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles. In this study, we investigated the capacity of P. gingivalis vesicles to promote the shedding or cleavage of the lipopolysaccharide (LPS) receptor CD14 from the surface of human U937 macrophage-like cells. SDS-PAGE/Western immunoblotting analysis of gingival crevicular fluid samples from patients affected by moderate or advanced periodontitis revealed the presence of soluble CD14 and CD14 fragments, thus supporting the hypothesis of an in vivo shedding and cleavage of CD14 receptors. Flow cytometry analysis of macrophage-like cells treated with a vesicle-containing culture supernatant of P. gingivalis showed a significant decrease in the binding of anti-human CD14 to the cell surface. However, no accumulation of soluble CD14 or immunoreactive CD14 fragments in the assay supernatant could be demonstrated by ELISA. Treatment of macrophage-like cells with various concentrations of P. gingivalis vesicles substantially suppressed TNF-alpha production triggered by Escherichia coli LPS. This suppressive effect was much less important using heat-treated vesicles or in the presence of leupeptin, a gingipain inhibitor, during the treatment. Recombinant human CD14 receptors were found to be susceptible to proteolytic degradation by P. gingivalis vesicles. A purified Arg-gingipain preparation produced much more degradation than a Lys-gingipain preparation. This study provides evidence that P. gingivalis outer membrane vesicles contribute to the loss of membrane-bound CD14 receptors and that gingipains degrade this LPS receptor. Such a phenomenon, which results in an hyporesponsiveness of macrophages to LPS stimulation, may contribute to an increased capacity of P. gingivalis, and other periodontopathogens, to evade the host immune system mechanisms.

We have developed and analyzed several mutant lines (M6 generation) of pigeonpea (Cajanus cajan (L.) Millsp.) for the content of defensive proteins and antinutritional factors. Inhibitors of proteinase and of amylase, lectins, and raffinose family oligosaccharides were analyzed in mature seeds of different pigeonpea accessions (untreated) and compared with mutant lines. Proteinase inhibitor profiles were similar in terms of number and intensities of activity bands but they differ marginally in the activity units in pigeonpea accessions and mutants. Pigeonpea mutants showed significant differences in amylase inhibitor profiles as well as activity units from those of pigeonpea accessions. Interestingly, two mutants (A6-5-1 and A7-3-2) were identified to have absence of amylase inhibitor isoforms. Hemagglutinating activity and raffinose family oligosaccharides content were found to be significantly higher in mutants than in accessions. It is evident from the results that proteinase inhibitors of pigeonpea are stable while amylase inhibitors, lectins, and raffinose family oligosaccharides show altered expression upon mutagen treatments. These mutants will be ideal candidates for further evaluation.