The complete primary structure of pilin fromHaemophilus influenzae type b strain Eagan

Adherence ofHaemophilus influenzae type b (Hib) to human oropharyngeal cells is mediated by pili which are proteinaceous filaments that extend outward from the bacterial cell surface. Pili from Hib strain Eagan were purified, and the primary structure of the major subunit, pilin, was determined. Sequencing of overlapping peptides showed the mature protein to be comprised of 196 amino acids and to have an M_r of 21,152. The amino terminal sequence was found to be homologous with the sequence previously reported for Hib strain M43 and also to have significant homology to pilins of other gram-negative pathogenic bacteria. Furthermore, Hib pilin had two cysteinyl residues in the amino terminal portion of the protein which were separated by 40 residues (positions 21 and 61); a motif found in other bacterial pilins. The data show that Hib pilin has structural features common to other bacterial pilins.     

The Agrobacterium tumefaciens T pilus composed of cyclic T pilin is highly resilient to extreme environments

Agrobacterium tumefaciens T pili are long semi-rigid, flexuous filaments of 10 nm diameter that are primarily composed of T pilin cyclized protein subunits. The cyclic character of T pilin apparently confers a high level of structural stability on the T pilus. Purified T pili subjected to extreme environmental conditions such as acid and alkali, including glycerol remained relatively unaffected morphologically. T pili lost their semi-rigidity when subjected to high temperatures and high pH, and dissociated into donut shaped subunits when exposed to Triton X-100. Sodium dodecyl sulfate increased the uptake of uranyl acetate exposing a 2 nm wide lumen running the length of the T pilus filament.     

Amino acid sequences of pilins from serologically distinct strains ofBacteroides nodosus

Amino acid sequences of pilin from a strain of Bacteroides nodosus from serogroup B (234) and serogroup C (217) were determined. The amino-terminal N-methylphenlalanine residue of both proteins was followed by a hydrophobic sequence of 30 residues closely related to the N-terminal sequence of other pili having an amino-terminal residue of N-methylphenylalanine. These data lend support to the hypothesis that in pilins of this type, the amino-terminal sequence functions as a transport signal necessary for pilin to reach its external environment, as well as promoting intersubunit interactions for muintenance of the structural integrity of the pilus. Two hydrophilic hypervariable regions can be discerned across the pilin sequences, indicating possible locations of antigenic domains.     

The role of porins in neisserial pathogenesis and immunity

Neisseria meningitidis and Neisseria gonorrhoeae are Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia, and the sexually transmitted disease gonorrhea, respectively. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric beta-barrel structure. Neisserial porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells (APCs). Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of APCs, an effect that is Toll-like receptor 2- and MyD88-dependent. The effect of neisserial porins on the immune system also involves interaction with components of the complement cascade. Furthermore, neisserial porins co-localize with mitochondria of target cells, where they appear to modulate apoptosis.     

The diversity of the glycan shield of sarbecoviruses related to SARS-CoV-2

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Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy

The backbone dynamics of a ¹⁵N-labeled recombinant PAK pilin peptide spanning residues 128–144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys¹²⁸-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys¹⁴⁴) were probed by measurements of ¹⁵N NMR relaxation. This PAK(128–144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The ¹⁵N longitudinal (T₁) and transverse (T₂) relaxation rates and the steady-state heteronuclear {¹H}-¹⁵N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25°C ) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the `model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559 and 4559–4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153–162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II -turns spanning residues Asp¹³⁴-Glu-Gln-Phe¹³⁷ and Pro¹³⁹-Lys-Gly-Cys¹⁴², respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.     

Primary structure of the major glycan from human seminal transferrin

Human seminal transferrin (HSmT) is an iron-containing glycoprotein whose structural properties have not been adequately investigated. The carbohydrate content of the purified glycoprotein amount to 6.1%, and monosaccharide analysis revealed the major oligosaccharide moiety to be of the N-glycoside type. The carbohydrate chains were released from the iron-free form by digestion with peptide N-glycosidase F (PNGase F) in the presence of detergents such as SDS and-octylglucoside. After ethanol precipitation and fractionation on Bio-Gel P-6 and Bio-Gel P-2, the oligosaccharide was further purified on Mono-Q and desalted on Bio-Gel P-2. By 600-MHz¹H-NMR spectroscopy, the primary structure of the major N-linked oligosaccharide component was established to be: Abbreviations used HSmT human seminal transferrin - HSrT human serum transferrin - PNGase F peptide-N⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52), commonly known as peptide N-glycosidase F - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - GLC gas-liquid chromatography - FPLC fast liquid protein chromatography - EDTA ethylenediaminetetraacetic acid, disodium salt - PMFS phenylmethylsulfonyl fluoride - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man, Gal galactose - Fuc fucose     

Alteration in N-acetylglucosaminyltransferase activities and glycan structure in tissue and bile glycoproteins from extrahepatic bile duct carcinoma

The activities of three N-acetylglucosaminyltransferases (GnT)-III, IV and V, as well as the structural alterations of N-glycans on the glycoproteins in cancer tissues and bile specimens from 28 cases of extrahepatic bile duct carcinoma (EBDC) were compared with those from 18 cases of benign biliary duct diseases (BBDD). GnT activities were determined with fluorescence-labeled substrate using a HPLC method. It was found that GnT-III and GnT-V activities in EBDC were increased to 3.14 and 15.96 times respectively of the mean BBDD values, but GnT-IV remained unchanged. The activity of GnT-V was correlated with the grade of differentiation and TMN stage of EBDC. The up-regulation of GnT-III resulted in the increased bisecting-GlcNAc on the N-glycans of glycoproteins in cancer tissues and a 201 kDa bile glycoprotein when analyzed with HRP-labeled E₄-PHA. The increased GnT-V activity led to the elevation of the β1,6GlcNAc branch (or antennary number) on the N-glycans in cancer tissue glycoproteins and 201, 163, 122 kDa proteins in the bile as probed with HRP-labeled DSA. These findings suggest that the alteration in GnT activities may be involved in the malignant transformation and development of EBDC, resulting in the aberrant glycosylation of some tissue and bile proteins. The latter was expected to be used in the clinical diagnosis and prognosis evaluation in EBDC patients. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stability

Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N‐linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high‐molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high‐affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N‐linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N‐linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex.     

Kinetic analysis of ligand interaction with the gonococcal transferrin-iron acquisition system

The transferrin iron acquisition system of Neisseria gonorrhoeae consists of two dissimilar transferrin binding proteins (Tbp) A and B. TbpA is a TonB dependent transporter while TbpB is a lipoprotein that makes iron acquisition from transferrin (Tf) more efficient. In an attempt to further define the individual roles of these receptors in the process of Tf-iron acquisition, the kinetics of the receptor proteins in regards to ligand association and dissociation were evaluated. Tf association with TbpB was rapid as compared to TbpA. Tf dissociation from the wild-type receptor occurred in a biphasic manner; an initial rapid release was followed by a slower dissociation over time. Both TbpA and TbpB demonstrated a two-phase release pattern; however, TbpA required both TonB and TbpB for efficient Tf dissociation from the cell surface. The roles of TbpA and TbpB in Tf dissociation were further examined, utilizing previously created HA fusion proteins. Using a Tf-utilization deficient TbpA-HA mutant, we concluded that the slower rate of ligand dissociation demonstrated by the wild-type transporter was a function of successful iron internalization. Insertion into the C-terminus of TbpB decreased the rate of Tf dissociation, while insertion into the N-terminus had no effect on this process. From these studies, we propose that TbpA and TbpB function synergistically during the process of Tf iron acquisition and that TbpB makes the process of Tf-iron acquisition more efficient at least in part by affecting association and dissociation of Tf from the cell surface.     

The role of glycosylation in storage-protein synthesis in developing pea seeds

Intact pea (Pisum sativum L.) cotyledons were incubated with [¹⁴C]glucosamine at several stages of seed development and the resultant radioactive proteins were analysed by gel electrophoresis combined with immunoaffinity chromatography and sucrose gradient fractionation. Glucosamine was incorporated into at least five vicilin polypeptides (approx. molecular weight 70,000; 50,000, two components; 14,000, two components). No incorporation was detected into the subunits of legumin. Tunicamycin at 50 g/ml largely inhibited glucosamine incorporation but had little effect on the incorporation of ¹⁴C-labelled amino acids into cotyledon proteins, including vicilin. The assembly of vicilin polypeptides into full-sized protein oligomers (7–9 S) was also unaffected by tunicamycin. Chromatography on concanavalin A confirmed that glycosylation of cotyledon proteins was inhibited by tunicamycin. It is concluded that glycosylation of most cotyledonary proteins involves lipid-linked sugar intermediates, but that glycosylation itself is not an essential step in the synthesis of vicilin polypeptides nor in their assembly into oligomers.Abbreviations IgG immunoglobulin G - M W_t approximate molecular weight based on electrophoretic mobility relative to that of protein standards - SDS-PAGE Na-dodecyl sulfate-polyacrylamide gel electrophoresis     

The role of natural enemies in controllingIcerya purchasi in South Australia

The obscure occurrence of the cottony-cushion scaleIcerya purchasi Maskell in its native country Australia is generally attributed to its natural enemies. Twelve natural-enemy-exclusion experiments were conducted at monthly intervals to confirm the role of natural enemies. Each experiment had uncaged, open-caged, and caged treatments. The natural enemies were active throughout the year. The percentage of scales surviving to adults in the cages was significantly higher than in the open-caged treatments, except the April (first) cohort. In both open-caged and uncaged treatments, the percentage of scales surviving to adults was similar and matched the changes in the numbers of natural enemies, thus confirming their importance in controllingI. purchasi in South Australia.      

磷脂酰肌醇类脂质在嗜肺军团菌发病机制中作用的研究进展北大核心

嗜肺军团菌(Legionella pneumophila)是一种能引起被称为“军团病”的严重肺炎的致病菌,其利用自身的IVB型分泌系统(type IVB secretion systems)将效应蛋白转运到宿主细胞中,作用于宿主蛋白质和脂质,以形成军团菌在宿主细胞内生长所需的吞噬泡(Legionella-containing vacuole,LCV)。磷酸酰肌醇(phosphatidylinositols,PIs)作为细胞的重要脂质组成,参与细胞信号转导及囊泡转运等过程。而大量的证据表明嗜肺军团菌利用其效应蛋白调控宿主磷酸酰肌醇类脂质代谢及其LCV膜的脂质组成,以促进LCV的成熟。本文主要从军团菌的致病机制、其效应蛋白对磷酸酰肌醇类脂质的代谢调控及对宿主磷脂酰肌醇代谢酶的招募等方面进行了综述分析,期望对进一步理解军团菌调控宿主脂质代谢分子机制和其致病机制提供参考。     

Evaluation of the role of angiogenic factors in the pathogenesis of schistosomiasis

Schistosomiasis is one disease produced by helminths, which affect many people in tropical areas. Granuloma formation is the main mechanism involved in the pathogenesis of this disease. Experimental studies have demonstrated angiogenesis (blood vessels formation from pre-existing vessels) in the initial phase of granuloma formation. In the present work, VEGF (vascular endothelial growth factor) levels were analyzed in sera from people diagnosed with different helminthic infections. Patients with schistosomiasis and filariasis had significantly high VEGF levels in compared with healthy people and patients diagnosed with hookworms. In addition, the effects of angiogenesis inhibition using anti-angiogenic factors (endostatin) were evaluated in a schistosomiasis murine model. A lesion decrease was observed in mice infected with Schistosoma mansoni and treated with endostatin. Finally, mechanisms of angiogenesis induction were studied and observed that cercariae antigens stimulated the angiogenic factors by host alveolar macrophages.     

Petunia hybrida S-proteins: ribonuclease activity and the role of their glycan side chains in self-incompatibility

Summary Self-incompatibility in flowering plants is controlled by the S-gene, encoding stylar S (allele-specific) glycoproteins. In addition to three previously characterized Petunia hybrida S-proteins, we identified by N-terminal sequence analysis another stylar S-protein, co-segregating with the S _b-allele. Purified S-proteins reveal biological activity, as is demonstrated for two of them by the allele-specific inhibition of pollen tube growth in vitro. Moreover, the four isolated S-proteins are ribonucleases (S-RNases). Specific activities vary from 30 (S₁) to 1000 (S₂) units per min per mg protein. We attempted to investigate the functionality of the carbohydrate portion of the S-RNases. Deglycosylation studies with the enzyme peptide-N-glycosidase F (PNGase F) reveals differences in the number of N-linked glycan chains present on the four S-RNases. Variability in the extent of glycosylation accounts for most of the molecular weight differences observed among these proteins. By amino acid sequencing, the positions of two of the three N-glycosylation sites on the S₂-RNase could be located near the N-terminus. Enzymic removal of the glycan side chains has no effect on the RNase activity of native S-RNases. This suggests another role of the glycan moiety in the self-incompatibility mechanism.     

The role of omnivorous crayfish in littoral communities

Large omnivorous predators may play particularly important roles determining the structure of communities because of their broad diets and simultaneous effects on multiple trophic levels. From June 2001 to June 2002 we quantified community structure and ecosystem attributes of six newly establishing freshwater ponds (660 m² each) after populations of omnivorous crayfish (Orconectes virilis) were introduced to three of the ponds. Crayfish preyed heavily on fish eggs in this experiment, which reduced recruitment of young-of-year fish. This effect indirectly enhanced zooplankton biomass in crayfish ponds. Phytoplankton abundance exhibited a more complex pattern and was probably influenced by non-trophic (e.g., bioturbation) effects of crayfish. Peak dissolved oxygen levels were lower in the crayfish ponds indicating that they had lower primary production: respiration ratios. Metaphytic algae were strongly affected by crayfish presence; filamentous greens quickly disappeared and the blue-green Gleotrichia (a less preferred food item) eventually dominated the composition in crayfish ponds. Chara vulgaris and vascular macrophytes established 34% cover in control ponds by June 2002, but were not able to establish in crayfish ponds. Two important periphyton herbivores (tadpoles and gastropods) were absent or significantly reduced in the crayfish ponds, but periphyton differences were temporally variable and not easily explained by a simple trophic cascade (i.e., crayfish—snails and tadpoles—periphyton). Our results indicate that crayfish can have dramatic direct and indirect impacts on littoral pond communities via feeding links with multiple trophic levels (i.e., fish, invertebrates, and plants) and non-trophic activities (bioturbation). Although the effects of omnivorous crayfish on littoral communities can be large, their complex effects do not fit neatly into current theories about trophic interactions or freshwater community structure.     

The role of spider cocoons in controlling desiccation

Summary The abilities of the cocoons of the spiders Mecynogea lemniscata and Argiope aurantia to protect the enclosed egg and spiderling stages from desiccation were investigated in the laboratory under controlled humidities, and in the field under ambient conditions. For M. lemniscata, which has a relatively small clutch (8–30 eggs) and remains in the cocoon for approximately 9–10 months, removal of the cocoon had no effect on water loss from the egg stage, nor did it adversely affect hatching or molting success. Cocoon removal did, however, significantly affect water loss and, consequently, survival in the spiderling stage at all humidities in the laboratory and in the field. The importance of the cocoon for survival is probably related to the unusually long time M. lemniscata spiderlings spend in the cocoon overwintering. For A. aurantia, which has a substantially larger clutch size (300–1400 eggs) and remains in the cocoon for a shorter 6–7 months, cocoon removal had no effect on water loss, egg hatching success, molting success, nor spiderling survival. The lack of an effect suggests that other factors (e.g., relative humidity at the oviposition site, or a large clutch size) may be more important in controlling water loss for A. aurantia.     

The ecological role of chemical stimuli for the zooplankton: predator-induced morphology in Daphnia

Summary Numerous adaptive predator-induced responses occurred when eight clones representing seven Daphnia (Crustacea: Cladocera) species were tested against three common predators: fourth instar larval phantom midge Chaoborus americanus, adult backswimmer Notonecta undulata, and small sunfish Lepomis macrochirus. The predators were confined within small mesh bags, suggesting that the signal for induction is chemical. The induced responses included longer tail spines, longer heads, smaller bodies, increased egg clutches, and decreased lipid reserves. Each Daphnia species responded to each of the three predators in a unique manner. Induced responses in the above characters showed no significant association. The induced morphological changes are generally consistent with current theories of what is an adaptive response for the various sizes of Daphnia exposed to tactile and visual predators. The abundance of induced responses in these experiments suggests that predator-induced responses are a widespread and ecologically important phenomenon of the freshwater zooplankton.