Identification of averantin as an aflatoxin B1 precursor: placement in the biosynthetic pathway.

A new blocked mutant of Aspergillus parasiticus produces no detectable aflatoxin B1, but accumulates several polyhydroxyanthraquinones. One of these pigments was identified as averantin. This is the first report of its formation by A. parasiticus. Radiotracer studies with [14C]averantin showed that 15.3% of label from averantin was incorporated into aflatoxin B1. This incorporation was blocked by dichlorvos. With radiotracers and other mutants, averantin was placed after norsolorinic acid and before averufin in the biosynthetic pathway in which the general steps are norsolorinic acid leads to averantin leads to averufin leads to versiconal hemiacetal acetate leads to versicolorin A leads to sterigmatocystin leads to aflatoxin B1.     

Averufanin is an aflatoxin B1 precursor between averantin and averufin in the biosynthetic pathway.

Wild-type Aspergillus parasiticus produces, in addition to the colorless aflatoxins, a number of pigmented secondary metabolites. Examination of these pigments demonstrated that a major component was an anthraquinone, averufanin. Radiolabeling studies with [14C]averufanin showed that 23% of the label was incorporated into aflatoxin B1 by the wild type and that 31% of the label was incorporated into O-methylsterigmatocystin by a non-aflatoxin-producing isolate. In similar studies with blocked mutants of A. parasiticus the 14C label from averufanin was accumulated in averufin (72%) and versicolorin A (54%) but not averantin. The results demonstrate that averufanin is a biosynthetic precursor of aflatoxin B1 between averantin and averufin.     

N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.     

Averufanin is an aflatoxin B1 precursor between averantin and averufin in the biosynthetic pathway

Wild-type Aspergillus parasiticus produces, in addition to the colorless aflatoxins, a number of pigmented secondary metabolites. Examination of these pigments demonstrated that a major component was an anthraquinone, averufanin. Radiolabeling studies with [14C]averufanin showed that 23% of the label was incorporated into aflatoxin B1 by the wild type and that 31% of the label was incorporated into O-methylsterigmatocystin by a non-aflatoxin-producing isolate. In similar studies with blocked mutants of A. parasiticus the 14C label from averufanin was accumulated in averufin (72%) and versicolorin A (54%) but not averantin. The results demonstrate that averufanin is a biosynthetic precursor of aflatoxin B1 between averantin and averufin.     

The Alzheimer amyloid precursor protein. Identification of a stable intermediate in the biosynthetic/degradative pathway

The amyloid forming beta-peptide of Alzheimer's disease is synthesized as part of a larger integral membrane precursor protein (beta APP) of which three alternatively spliced versions of 695, 751, and 770 amino acids have been described. A fourth beta APP form of 563 amino acids does not contain the beta-peptide region. Recent experiments using transient expression in HeLa cells (Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L., and Beyreuther, K. (1989) Cell 57, 115-126) indicate that the beta APP undergoes several posttranslational modifications including the cleavage and secretion of a large portion of its extracellular domain. The nature and fate of the fragment that remains cell-associated following this cleavage has not heretofore been described. The metabolism of this fragment may have particular significance in Alzheimer's disease since it must contain at least part of the beta-peptide. To study the metabolic fate of this fragment, we have established cell lines overexpressing the 695- and 751-amino acid versions of beta APP. Pulse-chase studies show that this system is similar to the HeLa cell system in that both proteins are synthesized first as membrane-bound proteins of approximately 98 and 108 kDa carrying asparagine-linked sugar side chains and are subsequently processed into higher molecular mass forms by the attachment of sulfate, phosphate, and further sugar groups including sialic acid, adding approximately 20 kDa in apparent molecular mass. The mature form of beta APP is cleaved and rapidly secreted, leaving an 11.5-kDa fragment with the transmembrane region and the cytoplasmic domain behind in the cell. This fragment is stable with a half-life of at least 4 h.     

Ascorbate as a biosynthetic precursor in plants

BACKGROUND AND AIMS: l-Ascorbate (vitamin C) has well-documented roles in many aspects of redox control and anti-oxidant activity in plant cells. This Botanical Briefing highlights recent developments in another aspect of l-ascorbate metabolism: its function as a precursor for specific processes in the biosynthesis of organic acids. SCOPE: The Briefing provides a summary of recent advances in our understanding of l-ascorbate metabolism, covering biosynthesis, translocation and functional aspects. The role of l-ascorbate as a biosynthetic precursor in the formation of oxalic acid, l-threonic acid and l-tartaric acid is described, and progress in elaborating the mechanisms of the formation of these acids is reviewed. The potential conflict between the two roles of l-ascorbate in plant cells, functional and biosynthetic, is highlighted. CONCLUSIONS: Recent advances in the understanding of l-ascorbate catabolism and the formation of oxalic and l-tartaric acids provide compelling evidence for a major role of l-ascorbate in plant metabolism. Combined experimental approaches, using classic biochemical and emerging 'omics' technologies, have provided recent insight to previously under-investigated areas.     

Universal influenza B vaccine based on the maturational cleavage site of the hemagglutinin precursor

Conventional influenza vaccines can prevent infection, but their efficacy depends on the degree of antigenic "match" between the strains used for vaccine preparation and those circulating in the population. A universal influenza vaccine based on invariant regions of the virus, able to provide broadly cross-reactive protection, without requiring continuous manufacturing update, would solve a major medical need. Since the temporal and geographical dominance of the influenza virus type and/or subtype (A/H3, A/H1, or B) cannot yet be predicted, a universal vaccine, like the vaccines currently in use, should include both type A and type B influenza virus components. However, while encouraging preclinical data are available for influenza A virus, no candidate universal vaccine is available for influenza B virus. We show here that a peptide conjugate vaccine, based on the highly conserved maturational cleavage site of the HA(0) precursor of the influenza B virus hemagglutinin, can elicit a protective immune response against lethal challenge with viruses belonging to either one of the representative, non-antigenically cross-reactive influenza B virus lineages. We demonstrate that protection by the HA(0) vaccine is mediated by antibodies, probably through effector mechanisms, and that a major part of the protective response targets the most conserved region of HA(0), the P1 residue of the scissile bond and the fusion peptide domain. In addition, we present preliminary evidence that the approach can be extended to influenza A virus, although the equivalent HA(0) conjugate is not as efficacious as for influenza B virus.     

In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa

Bacterial peptidoglycan is the cell wall component responsible for maintaining cell integrity against osmotic pressure. Biosynthesis of the cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is catalyzed by the Mur enzymes. Genomic analysis of the three regions encoding Mur proteins was achieved. We have cloned and over-expressed the murA, -B, -D, -E and -F genes of Pseudomonas aeruginosa in pET expression system by adding a His-Tag to the C-termini of the proteins. Mur proteins were purified to homogeneity by a single chromatographic step on affinity nickel columns. Protein identities were verified through N-terminal sequencing. Enzyme activity was proved by the identification of the pathway's final product.     

Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin.

Influenza virus A/seal/Mass/1/80 (H7N7) mutants were obtained; the hemagglutinins (HAs) of the mutants were not activated by trypsin, as in the wild-type virus, but by thermolysin. The mutants grew efficiently under multiple replication cycle conditions and formed plaques in chicken embryo cells only when thermolysin was added to the culture medium. They exhibited hemolytic activity and induced protective immunity in chickens after an asymptomatic course of infection. Nucleotide sequencing of the HA gene and direct amino acid sequencing showed that insertion of a single leucine into the fusion peptide of the HA2 chain close to the cleavage site and a shift of the cleavage site toward the C terminus by one amino acid were responsible for the changes in the biological properties of the thermolysin activation mutants. Revertants could be obtained when trypsin or trypsin-like endoproteases were present in the virus-producing system.     

Hinokiresinol is not a precursor of agatharesinol in the norlignan biosynthetic pathway in Japanese cedar

The biosynthetic relationship between the two norlignans agatharesinol and trans-hinokiresinol was investigated. Fresh sapwood sticks of Cryptomeria japonica were fed with stable isotope-labeled compounds, namely p-coumaryl alcohol-[9,9-²H], p-coumaryl alcohol-[9-¹⁸O] and trans-hinokiresinol-[1-²H], and then incubated under high-humidity for approximately 20 days, during which the two norlignans were produced simultaneously. While trans-hinokiresinol was strongly deuterium-labeled after feeding with p-coumaryl alcohol-[9,9-²H], agatharesinol was only lightly labeled after feeding with either p-coumaryl alcohol-[9,9-²H] or -[9-¹⁸O]. These results suggest that p-coumaryl alcohol, which is a precursor of hinokiresinol, is not involved in the biosynthesis of agatharesinol. Therefore, the norlignan carbon skeleton of agatharesinol must be framed from different types of phenylpropanoid monomers compared to those utilized by the trans-hinokiresinol pathway. The biosynthesis of these two norlignans seems to branch at an early stage, i.e., before the framing of the norlignan carbon skeleton. Furthermore, agatharesinol was not labeled with deuterium after feeding with ²H-labeled trans-hinokiresinol, which has the simplest norlignan structure. This result strongly supports the suggestion that the conversion of trans-hinokiresinol to agatharesinol is not part of the biosynthesis of norlignans and that early branching occurs instead.     

Immunogenic structure of the influenza virus hemagglutinin

We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenic determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of its pieces.     

Autocatalytic activation of influenza hemagglutinin

Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.     

On the origin of influenza A hemagglutinin

Recent advances in phylogenetic methods have produced some reassessments of the ages of the most recent common ancestor of hemagglutinin proteins in known strains of influenza A. This paper applies Bayesian phylogenetic analysis implemented in BEAST to date the nodes on the influenza A hemagglutinin tree. The most recent common ancestor (MRCA) of influenza A hemagglutinin proteins is located with 95% confidence between 517 and 1497 of the Common Era (AD), with the center of the probability distribution at 1056 AD. The implications of this revised dating for both historical and current epidemiology are discussed. Influenza A can be seen as an emerging disease of mediaeval and early modern times.     

Hierarchical analysis of influenza A hemagglutinin gene sequences.

Five recently sequenced hemagglutinin genes from Influenza A virus strains are studied for similarities in a hierarchical fashion. The sequences are compared for similarity, first on the level of sequence homology, and then on several progressively more general levels. Though the HA1 subsequences contain regions where homology drops to that of a Monte Carlo generated reference value, subsequent tests reveal great similarity due to constraints on the level of amino acid sequence. Other tests detect statistically significant differences between subtypes due to constraints acting below the level of amino acid sequence, such as the 2 degrees structure of the viral RNA, or involving translation of the mRNA. The general applicability of the hierarchical approach to sequence analysis is discussed.     

Pyrimidine biosynthetic pathway of Baccillus subtilis.

Biochemical and genetic data were obtained from a series of 51 Pyr- strains of Bacillus subtilis. The observed enzymatic deficiencies allowed the mutants to be placed into 12 clases, some of which represent defects in more than one of the six known pyrimidine biosynthetic enzymes. Mapping analysis by transformation has shown that all the Pyr- mutations are located in a single small area of the B. subtilis genome. A correlation of the biochemical defects and the genetic data has been made. Those mutations conferring similar enzymatic deficiencies were found to be contiguous on the B. subtilis map. Regulatory aspects of the pyrimidine pathway have also been investigated and are compared to previously reported results from other organisms. Evidence is presented which bears upon the possible physical association of the first three enzymes and the association of at least some of the enzymes of this pathway with particulate elements of the cell. A model for the organization of the enzymes is presented with dihydroorotate dehydrogenase as the central enzyme in a proposed aggregate.     

Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change

The hemagglutinin (HA) spike glycoprotein of influenza virus catalyzes a low pH-induced membrane fusion event which releases the viral genome into the host cell cytoplasm. To study the fusion mechanism in more detail, we have prepared the ectodomain of HA in water-soluble form by treating virus particles with bromelain. Under mildly acidic conditions (pH less than or equal to 5.8), the ectodomain undergoes a conformational change which we found to be biochemically and immunologically equivalent to that in native viral HA. It became sensitive to proteinase K, it exposed new antigenic epitopes in its HA1 chain, and it acquired amphiphilic properties, notably the ability to bind to liposomes. The attachment to liposomes exhibited the same pH dependence and rapid kinetics as the conformational change and was mediated by HA2. The nature of the attachment resembled that of an integral membrane protein except that the bound HA was partially removed by base. As observed for virus fusion, attachment is independent of divalent cations and lipid composition. Temperature was found to be a critical parameter only with dimyristoylphosphatidycholine vesicles where attachment was partially blocked below the major phase transition. These and other results obtained indicated that the low pH-induced conformational change in the isolated ectodomain is equivalent to that occurring in intact viral HA, and that its attachment to liposomes can serve as a model for the initial stages in the HA-induced membrane fusion reaction.     

流感病毒血凝素蛋白裂解机制的研究进展

血凝素(Hemagglutinin,HA)是流感病毒的主要表面抗原之一,诱导机体产生中和抗体,介导病毒囊膜与靶细胞膜融合,从而启动病毒对宿主细胞的感染过程。HA蛋白以前体形式合成,需经宿主蛋白酶水解为HA1、HA2两个亚单位,并以二硫键连接,病毒才获得感染性。研究表明宿主蛋白酶的分布与流感病毒感染后的致病力和组织嗜性有直接关系。潜在的裂解酶及其抑制因子的发现为流感的防治提供了新的思路,成为干预治疗的新潜在靶点。就当前国内外关于流感病毒血凝素的结构与功能、裂解机制及其应用的研究进展进行综述。     

Integrity of a recombinant hemagglutinin protein of an avian influenza virus

An open reading frame representing cDNA from a hemagglutinin (HA) encoding gene of a low pathogenic avian influenza virus (AIV) subtype H10N7 was cloned in the pNMT1-TOPO vector under the control of thiamine response promoter. This construct was designated as pNMT1-HA. The pNMT1-HA construct was transformed into Schizosaccharomyces pombe for expression of HA antigen. The correct expression of recombinant HA protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The level of expression of recombinant HA protein was approximately 0.2% of total soluble protein. Purified yeast-derived recombinant HA protein showed hemagglutination activity. The 2-D and 3-D scanning images of recombinant HA protein were observed with an atomic force microscope (AFM). The structural integrity of the HA protein under AFM and hemagglutination activity provided support that the recombinant HA protein may be suitable for development of AIV subunit vaccine for mass administration to poultry.     

Substrate specificity of the sialic acid biosynthetic pathway.

Unnatural analogues of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogues bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogues with ketone-containing N-acyl groups that varied in the length or steric bulk was chemically synthesized and tested for metabolic conversion to cell surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.