The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and up-regulates its own transcription.

The unfolded protein response (UPR) involves a complex signalling pathway in which the transcription factor HACA plays a central role. Here we report the cloning and characterisation of the hacA gene and its product from Aspergillus niger. ER (endoplasmic reticulum) stress results in the splicing of an unconventional 20-nt intron from the A. niger hacA mRNA, and is associated with truncation of the 5'-end of the hacA mRNA by 230 nt. In this study the UPR was triggered by over expressing tissue plasminogen activator (t-PA), and by treatment of mycelia with dithiothreitol (DTT) or tunicamycin. Overexpression of the processed form of hacA not only led to the up-regulation of bipA, cypB and pdiA--mimicking the UPR--but also led to the up-regulation of the hacA gene itself. In vitro binding assays confirmed that the HACA protein binds to the promoters of genes encoding ER-localised chaperones and foldases, and to the promoter of the hacA gene itself. Finally, a GFP-HACA fusion was shown to localise in the nucleus.

Production of tissue plasminogen activator (t-PA) in Aspergillus niger.

A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.

The foldase CYPB is a component of the secretory pathway of Aspergillus niger and contains the endoplasmic reticulum retention signal HEEL.

Here we report the isolation and characterization of the cypB gene from Aspergillus niger. The cypB gene encodes a protein with a predicted molecular weight of 20.7 kDa, which shows a high degree of identity to the cyclophilin family of peptidyl prolyl cis-trans isomerases (PPIases) from other eukaryotes. The 5' untranslated region of cypB includes three sequences resembling UPREs (unfolded protein response elements). The expression of cypB is upregulated by tunicamycin and DTT, suggesting that at least one UPRE is functional. The CYPB protein also has a 23-amino acid sequence which serves to target the protein to the endoplasmic reticulum (ER), and the ER retention sequence HEEL. CYPB-(His)(6) was expressed in Escherichia coli; the purified protein is capable of isomerizing a substrate peptide in vitro. This is also the first report to show that C-terminal addition of the sequence HEEL is sufficient to ensure retention of the green fluorescent protein (GFP) within the ER.

The Aspergillus niger cypA gene encodes a cyclophilin that mediates sensitivity to the immunosuppressant cyclosporin A.

Here we report the cloning and characterization of a gene, cypA, from Aspergillus niger that encodes a peptidyl prolyl cis-trans isomerase (PPIase) belonging to the cyclophilin family. Sequencing of both genomic and cDNA clones revealed two ORFs in cypA, one encoding a 19-kDa protein of 174 amino acid residues and the other a 24-kDa protein of 219 amino acid residues, with overall identities of 27-77% to the homologous cyclophilins from prokaryotic and eukaryotic organisms. Expression of the 19-kDa CYPA-(His)(6) in E. coli shows that the purified protein has PPIase activity which is inhibited by cyclosporin A. Northern analysis shows two specific cypA transcripts, the smaller transcript encodes the cytosolic 19-kDa CYPA protein, the larger transcript encodes the putative mitochondrial 24 kDa CYPA protein. The transcript for the cytosolic CYPA is expressed at a higher basal level than that for the mitochondrial protein. The presence of tunicamycin, DTT or cyclosporin A in the medium does not affect the expression level of cypA. Its expression is however slightly induced by heat shock. Growing A. niger mycelium in the presence of cyclosporin A leads to an increase in hyphal branching prior to growth arrest. Overexpression of cypA under the control of its own promoter in A. niger results in increased sensitivity to cyclosporin A, suggesting that cypA encodes the cellular target for cyclosporin A in A. niger.

Crystallization and preliminary X-ray analysis of arabinofuranosidase C from Aspergillus niger strain 3M43.

Crystals of arabinofuranosidase C purified from Aspergillus niger strain 3M43 have been obtained by vapour diffusion. The crystal belongs to the space group P2(1) with cell parameters a = 44.28, b = 71.99, c = 45.27 A and beta = 105.98 degrees with one molecule in the asymmetric unit. The X-ray diffraction pattern of these crystals extends to at least 2.20 A resolution with the use of synchrotron radiation. These crystals are stable on exposure to radiation and are suitable for structure determination.

Characterization of a new antifungal chitin-binding peptide from sugar beet leaves.

The intercellular washing fluid (IWF) from leaves of sugar beet (Beta vulgaris L.) contains a number of proteins exhibiting in vitro antifungal activity against the devastating leaf pathogen Cercospora beticola (Sacc.). Among these, a potent antifungal peptide, designated IWF4, was identified. The 30-amino-acid residue sequence of IWF4 is rich in cysteines (6) and glycines (7) and has a highly basic isoelectric point. IWF4 shows homology to the chitin-binding (hevein) domain of chitin-binding proteins, e.g. class I and IV chitinases. Accordingly, IWF4 has a strong affinity to chitin. Notably, it binds chitin more strongly than the chitin-binding chitinases. A full-length IWF4 cDNA clone was obtained that codes for a preproprotein of 76 amino acids containing an N-terminal putative signal peptide of 21 residues, followed by the mature IWF4 peptide of 30 residues, and an acidic C-terminal extension of 25 residues. IWF4 mRNA is expressed in the aerial parts of the plant only, with a constitutive expression in young and mature leaves and in young flowers. No induced expression of IWF4 protein or mRNA was detected during infection with C. beticola or after treatment with 2,6-dichloroisonicotinic acid, a well-known inducer of resistance in plants.

New antifungal proteins from sugar beet (Beta vulgaris L.) showing homology to non-specific lipid transfer proteins.

Two novel, nearly identical antifungal proteins, IWF1 and IWF2, were isolated from the intercellular washing fluid (IWF) of sugar beet leaves. The proteins were purified to homogeneity and their amino acid sequences were determined. They are basic, monomeric proteins of 91 amino acid residues, 89 of which are identical. Both proteins show strong in vitro antifungal activity against Cercospora beticola, the casual agent of leaf spot disease in sugar beet. Based on primary sequence homology, including the presence of 8 conserved cysteine residues, IWF1 and IWF2 are related to the family of plant non-specific lipid transfer proteins (nsLTPs). Antibodies were raised against IWF2 after conjugation to diphtheria toxoid. The amino acid sequence data was used to generate a polymerase chain reaction (PCR) clone, employed for the isolation of a cDNA clone encoding a closely related isoform IWFA, which differs from IWF1 by two amino acid substitutions only. The induction and subcellular localization of these proteins were studied by western and northern blotting analyses after treatment with 2,6-dichloroisonicotinic acid (INA), a compound capable of inducing resistance against C. beticola, and after fungal infection. The following observations were made: (1) the proteins were present in leaves of non-INA-treated and uninfected control plants, (2) they were only slightly induced by INA treatment and during infection with C. beticola, and (3) they were present both intra- and extracellularly. However, their strong antifungal potentials together with immunohistological investigations, the proteins accumulating in contact with the fungus and in autolysing cells, suggested a role of these proteins in plant defence. Finally, immunohistology revealed a remarkable expression pattern of the IWF1 and IWF2 proteins, or serologically related proteins, in sugar beet styles, in that single or a few scattered papillae and a few cells in the lower transmitting tissue strongly and specifically reacted with the antibody.