Proteolytic processing in the secretory pathway of Aspergillus niger

A number of filamentous fungi are saprophytes and they secrete a wide spectrum of enzymes to degrade their complex substrates. Many secreted proteins enter the secretory pathway as proproteins and need some form of proteolytic processing before they obtain their mature active state. As described in this thesis we have cloned the genes encoding two proteases involved in such proteolytic processing events occurring in the secretory pathway of Aspergillus niger . In addition, we have characterized both proteases. We have cloned kexB and characterized the kexin maturase. KexB matures by hydrolysation of propeptides from proproteins and plays an important role in the processing of proteins produced in the secretory pathway of A. niger . We found that A. niger strains with a disrupted kexB gene have a hyper-branching morphology at pH 6 (Chapter 3). This morphology is pH dependent, because at pH 4 there are no apparent morphological differences between the kexB disrupted strain and wild-type A. niger . KexB is a modular protein (Chapter 2). In addition to a subtilisin­like protease domain, which in yeast functions in the late Golgi, KexB contains a transmembrane domain and putative cytoplasmic Goigi retention signal. To enable a biochemical characterization of KexB a truncated soluble secreted form of the protease was constructed, by removal of the C­terminal transmembrane domain and Goigi retention signal. (Chapter 3). Using fluorogenic substrates we were able to show that this soluble form of KexB can efficiently process both dibasic and monobasic amino acid cleavage sites. This is in remarkable contrast to Kex2, the KexB homo log of Saccharomyces cerevisiae, which only cleaves dibasic amino acid cleavage sites efficiently. Furthermore we have demonstrated that KexB is responsible for the in vivo processing of endopolygalacturonase I , and is a likely candidate for processing of endopolygalacturonase 11. Both enzymes are naturally secreted by A. niger and enter the secretory pathway as proproteins. We have also cloned dapB and characterized A. niger DapB, a possible ortholog of S. cerevisiae DPAP A (Chapter 4). DPAP A is a Goigilocated exoprotease, which plays a role in the processing and activation of secreted proteins. DP AP A is a type IV dipeptidyl aminopeptidase (DPP IV). Phylogenetic analysis showed that DapB is part of a novel subfamily of DPP IV proteases in filamentous fungi with a transmembrane domain. DPPIV proteases remove dipeptides at the N-terminus with an alanine or proline residue at the penultimate position. We were able to confirm the DPP IV­like activity of DapB by overexpression of the protease, resulting in 5 to 7 times higher DPPIV like activity on artificial substrates in cell extracts from DapB transformants. Although we could predict the presence of a transmembrane domain, we were not able to determine the location of the mature protease. Currently we do not know whether DapB plays a role in protein maturation. DapB is a proline specific protease. Currently not much is known about the role of proline residues in the protection and regulation (delay of activation) of enzymes produced . by filamentous fungi. However in other organisms proline is often found at the termini of secreted enzymes to protect against exoprotease activity. To make a study into the role of proline in protein degradation and regulation feasible we have started with the identification and characterization of proline specific proteases In addition to the A. niger kexB and dapB genes, we have cloned pepP and characterized the proline specific dipeptidase (PepP) of Aspergilllus nidulans (Chapter 5). Publicly available EST sequences of A. nidulans led to the identification and cloning of the prolidase encoding gene of this fungus. A prolidase overexpressing A. nidulans strain was constructed. The protease purified from such an overexpressing strain was used for a biochemical characterization of the protein. The protein is a manganese2+ dependent metallo protease and in vitro cleavage studies showed that it only hydrolyses dipeptides with aC-terminal proline residue. DapB, the A. niger DPP IV protease releases such dipeptides from the N­terminus of a polypeptide chain. In summary, we have cloned the kexB gene and characterized the A. niger endoprotease KexB. KexB removes the propeptide of proteins traveling through the secretory pathway. In addition, we have cloned dapB and characterized the A. niger exoprotease DapB, a possible ortholog of S. cerevisiae DP AP A. DP AP A also plays a role in the maturation of proteins. Furthermore, the A. nidulans prolidase gene has been cloned and PepP characterized. PepP is a proline specific dipeptidase, its substrates are dipeptides like those released by DapB. With our research we have gained insight into proteolytic processing in the secretory pathway of Aspergillus niger , and more specifically we have obtained detailed knowledge about propeptide processing of proproteins destined for secretion.