Host Cell Proteome of Physcomitrella patens Harbors Proteases and Protease Inhibitors under Bioproduction Conditions.

Host cell proteins are inevitable contaminants of biopharmaceuticals. Here, we performed detailed analyses of the host cell proteome of moss ( Physcomitrella patens) bioreactor supernatants using mass spectrometry and subsequent bioinformatics analysis. Distinguishing between the apparent secretome and intracellular contaminants, a complex extracellular proteolytic network including subtilisin-like proteases, metallo-proteases, and aspartic proteases was identified. Knockout of a subtilisin-like protease affected the overall extracellular proteolytic activity. Besides proteases, also secreted protease-inhibiting proteins such as serpins were identified. Further, we confirmed predicted cleavage sites of 40 endogenous signal peptides employing an N-terminomics approach. The present data provide novel aspects to optimize both product stability of recombinant biopharmaceuticals as well as their maturation along the secretory pathway. Data are available via ProteomeXchange with identifier PXD009517.

Phylogenomic evolutionary surveys of subtilase superfamily genes in fungi.

Subtilases belong to a superfamily of serine proteases which are ubiquitous in fungi and are suspected to have developed distinct functional properties to help fungi adapt to different ecological niches. In this study, we conducted a large-scale phylogenomic survey of subtilase protease genes in 83 whole genome sequenced fungal species in order to identify the evolutionary patterns and subsequent functional divergences of different subtilase families among the main lineages of the fungal kingdom. Our comparative genomic analyses of the subtilase superfamily indicated that extensive gene duplications, losses and functional diversifications have occurred in fungi, and that the four families of subtilase enzymes in fungi, including proteinase K-like, Pyrolisin, kexin and S53, have distinct evolutionary histories which may have facilitated the adaptation of fungi to a broad array of life strategies. Our study provides new insights into the evolution of the subtilase superfamily in fungi and expands our understanding of the evolution of fungi with different lifestyles.

Evolutionary History of Subtilases in Land Plants and Their Involvement in Symbiotic Interactions.

Subtilases, a family of proteases involved in a variety of developmental processes in land plants, are also involved in both mutualistic symbiosis and host-pathogen interactions in different angiosperm lineages. We examined the evolutionary history of subtilase genes across land plants through a phylogenetic analysis integrating amino acid sequence data from full genomes, transcriptomes, and characterized subtilases of 341 species of diverse green algae and land plants along with subtilases from 12 species of other eukaryotes, archaea, and bacteria. Our analysis reconstructs the subtilase gene phylogeny and identifies 11 new gene lineages, six of which have no previously characterized members. Two large, previously unnamed, subtilase gene lineages that diverged before the origin of angiosperms accounted for the majority of subtilases shown to be associated with symbiotic interactions. These lineages expanded through both whole-genome and tandem duplication, with differential neofunctionalization and subfunctionalization creating paralogs associated with different symbioses, including nodulation with nitrogen-fixing bacteria, arbuscular mycorrhizae, and pathogenesis in different plant clades. This study demonstrates for the first time that a key gene family involved in plant-microbe interactions proliferated in size and functional diversity before the explosive radiation of angiosperms.

Subtilases - versatile tools for protein turnover, plant development, and interactions with the environment.

Subtilases (SBTs) constitute a large family of serine peptidases. They are commonly found in Archaea, Bacteria and Eukarya, with many more SBTs in plants as compared to other organisms. The expansion of the SBT family in plants was accompanied by functional diversification, and novel, plant-specific physiological roles were acquired in the course of evolution. In addition to their contribution to general protein turnover, plant SBTs are involved in the development of seeds and fruits, the manipulation of the cell wall, the processing of peptide growth factors, epidermal development and pattern formation, plant responses to their biotic and abiotic environment, and in programmed cell death. Plant SBTs share many properties with their bacterial and mammalian homologs, but the adoption of specific roles in plant physiology is also reflected in the acquisition of unique biochemical and structural features that distinguish SBTs in plants from those in other organisms. In this article we provide an overview of the earlier literature on the discovery of the first SBTs in plants, and highlight recent findings with respect to their physiological relevance, structure and function.

Inferring hypotheses on functional relationships of genes: Analysis of the Arabidopsis thaliana subtilase gene family.

The gene family of subtilisin-like serine proteases (subtilases) in Arabidopsis thaliana comprises 56 members, divided into six distinct subfamilies. Whereas the members of five subfamilies are similar to pyrolysins, two genes share stronger similarity to animal kexins. Mutant screens confirmed 144 T-DNA insertion lines with knockouts for 55 out of the 56 subtilases. Apart from SDD1, none of the confirmed homozygous mutants revealed any obvious visible phenotypic alteration during growth under standard conditions. Apart from this specific case, forward genetics gave us no hints about the function of the individual 54 non-characterized subtilase genes. Therefore, the main objective of our work was to overcome the shortcomings of the forward genetic approach and to infer alternative experimental approaches by using an integrative bioinformatics and biological approach. Computational analyses based on transcriptional co-expression and co-response pattern revealed at least two expression networks, suggesting that functional redundancy may exist among subtilases with limited similarity. Furthermore, two hubs were identified, which may be involved in signalling or may represent higher-order regulatory factors involved in responses to environmental cues. A particular enrichment of co-regulated genes with metabolic functions was observed for four subtilases possibly representing late responsive elements of environmental stress. The kexin homologs show stronger associations with genes of transcriptional regulation context. Based on the analyses presented here and in accordance with previously characterized subtilases, we propose three main functions of subtilases: involvement in (i) control of development, (ii) protein turnover, and (iii) action as downstream components of signalling cascades. Supplemental material is available in the Plant Subtilase Database (PSDB) (http://csbdb.mpimp-golm.mpg.de/psdb.html), as well as from the CSB.DB (http://csbdb.mpimp-golm.mpg.de).

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.

Isolation and characterization of a subtilisin-like serine proteinase secreted by the sap-staining fungus Ophiostoma piceae.

Identification of key enzymes of sapstain fungi which cause wood discoloration is necessary for targeted inhibition strategies. Therefore proteinases involved in the nitrogen pathway have been characterized. The sap-staining fungus Ophiostoma piceae strain 387N produced proteolytic enzymes when grown on wood and protein-supplemented media. Proteolytic activity in culture filtrates was inhibited by PMSF and EDTA. The major protein in culture filtrates was a proteinase with a pI of 5.6 and a molecular weight of 33 kDa. This was the major proteinase produced by O. piceae and it was purified from culture filtrates by hydrophobic interaction chromatography. The proteinase was susceptible to autolytic degradation during chromatographic separations when ammonium sulfate was not present. When azocoll was used as a substrate, the proteolytic activity of the purified proteinase was determined to be optimal at pH 7-9 and 40 degrees C. Similar pH and temperature optima were obtained using succinyl-ala-ala-pro-phe-p-nitroanilide as the substrate. The N-terminal sequence of the protein showed a high degree of homology with fungal alkaline serine proteinases classified as subtilisin class II enzymes. Agreement in inhibition patterns and electrophoretic and catalytic properties suggested the secretion of the same proteinase during growth on wood. Understanding the role of this proteinase during fungal colonization is an important step toward disrupting fungal growth on wood.

Distribution of the Kexin family proteases in pancreatic islets: PACE4C is specifically expressed in B cells of pancreatic islets.

The distribution of Kexin family proteases in adult rat pancreatic islets was investigated by immunohistochemical means using a series of specific antibodies specific for PC1, PC2, PC6, Furin, PACE4A and a recently identified member of the Kexin family, PACE4C. PACE4C expression was limited to B cells of the pancreatic islets. PC2 was found in A and in some D cells more than in B cells and PC1 was evident only in B cells. Furin and PC6 were weakly and evenly expressed in the entire islet. PACE4A was hardly found in the islets. These findings indicated that individual Kexin family proteases are uniquely distributed in the islets and suggested that these proteases share roles in these cells as follows: PC2 is involved in the peptide hormone precursor processing in A cells and in D cells, and PACE4C, PC1 and PC2 (mainly PACE4C and PC1) are responsible for the processing event(s) specific to B cells.

Classification of serine proteases derived from steric comparisons of their active sites.

The steric arrangements of the amino acyl residues in the catalytic triads and tetrads of the active site are compared with each other by means of systematic analysis of the conformation of the serine proteases stored in the Brookhaven Protein Data Bank. On this basis a differentiation between the representatives of the (chymo)trypsin family on the one hand and those of the subtilisin family on the other hand is found. The enzyme tonin distinguishes from representatives of the (chymo)trypsin family and should be classified to a new subclass of this family. Thermitase represents a new subclass of the subtilisins. The spatial orientation of the amino acyl residues of the active site of tonin suggests a new mechanism of enzyme catalysis that possibly also occurs in dipeptidyl peptidase IV.

GAP-releasing enzyme is a member of the pro-hormone convertase family of precursor protein processing enzymes.

The recent discovery of mammalian endoproteinases which show extensive sequence homology with the yeast Kex 2 gene product (kexin) has lead to the hypothesis that processing enzymes of pro-hormone precursor proteins belong to a family of calcium dependent, subtilisin-like serine proteinases. We previously showed that hypothalamic GAP-releasing enzyme shares these characteristics and possesses the requisite specificity to be considered as a processing enzyme of progonadotropin releasing hormone (pro-GnRH) precursor protein. Thus, GAP-releasing enzyme (and other non-related proteins) were tested for their immunological reactivity with antisera raised against pituitary pro-hormone convertase 1/3 (PC1/3) and insulinoma PC2. On the basis of indirect enzyme-linked immunosorbent (ELISA) and Western blot assays, GAP-releasing enzyme is now shown to be immunologically related to PC1/3. We can conclude that GAP-releasing enzyme is also likely to be a member of the pro-hormone convertase family and should be considered the physiologically relevant processing enzyme of pro-GnRH. It is possible that GAP-releasing enzyme represents bovine hypothalamic PC1/3.

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases.

Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.