Evidence for two-stage binding by the 175-kD erythrocyte binding antigen of Plasmodium falciparum.

Plasmodium falciparum malaria merozoites invade human erythrocytes bearing sialic acid in a multistage process involving the sialic acid-dependent binding of a malaria molecule, the 175-kD erythrocyte binding antigen (EBA-175). We show here that after the initial interaction of EBA-175 with its sialic acid-containing erythrocyte determinant, endogenous proteases can cleave EBA-175 to 65-kD fragment(s), whose binding to erythrocytes is sialic acid independent. A 65-kD fragment was immunoprecipitated by antibodies against peptides between residues 354 and 1061 but not beyond residue 1062. Binding experiments utilizing combinations of native protein, expression-PCR-synthesized EBA-175 polypeptides, peptide synthesis, and antibodies, demonstrated that sialic acid-independent binding could be further mapped to a small (about 40-amino acid) homologous part of the dimorphic allelic region of EBA-175, residues 898-938 (Camp strain numbering). These data support a two-step binding hypothesis and are discussed in relation to the formation of a junction between the merozoite and the erythrocyte, and the finding that after the interaction of some viruses with specific cellular receptors, they undergo conformational changes or cleavage permitting membrane fusion with the host cell.

Purification and preliminary characterisation of praelongin phospholipases, antiplatelet agents from the snake venom of Acanthophis praelongus.

Three praelongin phospholipases were chromatographically purified from the snake venom of Acanthophis praelongus. The purity and homogeneity of the praelongins were assessed by RP-HPLC, HPCE and mass spectrometry. The purified enzymes, praelongins 2bIII, 2cII and 2cIV were found to have phospholipase A2 activities with specific activities of 31.4 +/- 0.4, 326.1 +/- 10.2 and 362.5 +/- 12.0 U/mg, respectively. Mass spectrometry studies showed the molecular mass of praelongin 2bIII to be 12,782.9 +/- 2.6 and praelongins 2cII and 2cIV to have very similar molecular mass values, 12,971.4 +/- 4.5 and 12,971.9 +/- 3.6, respectively. However, platelet aggregation studies showed the praelongins to display different IC50 values, 180 microM for praelongin 2cII and 55 microM for praelongin 2cIV; praelongin 2bIII was found to be a more potent antiplatelet agent, having an IC50 of 0.65 microM. Praelongins 2bIII, 2cIV and 2cII were found to have pI values of 10.3 +/- 0.3, 9.6 +/- 0.6 and 9.4 +/- 0.6 as determined by HPCE. The antiplatelet potencies do not correspond to their in vitro phospholipase catalytic potencies, but appear to be related to the enzyme isoelectric points.

Analysis of swainsonine and its early metabolic precursors in cultures of Metarhizium anisopliae.

The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 microg ml(-1). Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition.

ProDDO: a database of disordered proteins from the Protein Data Bank (PDB).

ProDDO represents a 'pre-screened' database that denotes disorder (or possible disorder) in proteins from the PDB.

O-Mannosylation of Pichia pastoris cellular and recombinant proteins.

O-linked saccharides were released from a major cell wall glycoprotein and from cellular mannan-protein complexes obtained from Pichia pastoris cells. Analysis by a variety of chromatographic methods and exoglycosidase digestions revealed the presence of mannose and (alpha1-2)-linked dimer, trimer and tetramer saccharides of mannose. The recombinant kringle 1-4 domain of human plasminogen expressed in P. pastoris was subjected to hydrazinolysis of both O- and N-linked saccharides. Only a very small quantity of N-linked oligosaccharides was present on the Asn289 consensus site. The major products were O-linked (alpha1-2)-linked mannans, containing dimeric, trimeric, tetrameric and pentameric oligosaccharides with the major amount of the total saccharides being distributed approximately equally between the dimer and trimer components. These results show that short O-linked saccharides of mannose containing (alpha1-2) glycosidic linkages are present in P. pastoris cells and expressed proteins.