Autoproteolytic Cleavage Site Research Articles

Engineering recombinant replication-competent bluetongue viruses expressing reporter genes for in vitro and non-invasive in vivo studies.

Bluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Therefore, fluorescent signal or luciferase activity is only detected after virus replication and expression of non-structural proteins. Fluorescence or luminescence signals were detected in cells infected with rBTV/Venus or rBTV/NLuc, respectively. Moreover, the marking of NS2 protein confirmed that reporter genes were only expressed in BTV-infected cells. Growth kinetics of rBTV/NLuc and rBTV/Venus in Vero cells showed replication rates similar to those of wild-type and rBTV. Infectivity studies of these recombinant viruses in IFNAR(-/-) mice showed a higher lethal dose for rBTV/NLuc and rBTV/Venus than for rBTV indicating that viruses expressing the reporter genes are attenuated in vivo. Interestingly, luciferase activity was detected in the plasma of viraemic mice infected with rBTV/NLuc. Furthermore, luciferase activity quantitatively correlated with RNAemia levels of infected mice throughout the infection. In addition, we have investigated the in vivo replication and dissemination of BTV in IFNAR (-/-) mice using BTV/NLuc and non-invasive in vivo imaging systems.IMPORTANCEThe use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein has significantly contributed to the in vitro and in vivo study of viral infections and the development of novel therapeutic approaches. In this work, we have generated rBTV that express fluorescent or luminescence proteins to track BTV infection both in vitro and in vivo. Despite the availability of vaccines, BTV and other related orbivirus are still associated with a significant impact on animal health and have important economic consequences worldwide. Our studies may contribute to the advance in orbivirus research and pave the way for the rapid development of new treatments, including vaccines.

Generation and Characterization of an Influenza D Reporter Virus.

Influenza D virus (IDV) can infect various livestock animals, such as cattle, swine, and small ruminants, and was shown to have zoonotic potential. Therefore, it is important to identify viral factors involved in the broad host tropism and identify potential antiviral compounds that can inhibit IDV infection. Recombinant reporter viruses provide powerful tools for studying viral infections and antiviral drug discovery. Here we present the generation of a fluorescent reporter IDV using our previously established reverse genetic system for IDV. The mNeonGreen (mNG) fluorescent reporter gene was incorporated into the IDV non-structural gene segment as a fusion protein with the viral NS1 or NS2 proteins, or as a separate protein flanked by two autoproteolytic cleavage sites. We demonstrate that only recombinant reporter viruses expressing mNG as an additional separate protein or as an N-terminal fusion protein with NS1 could be rescued, albeit attenuated, compared to the parental reverse genetic clone. Serial passaging experiments demonstrated that the mNG gene is stably integrated for up to three passages, after which internal deletions accumulate. We conducted a proof-of-principle antiviral screening with the established fluorescent reporter viruses and identified two compounds influencing IDV infection. These results demonstrate that the newly established recombinant IDV reporter virus can be applied for antiviral drug discovery and monitoring viral replication, adding a new molecular tool for investigating IDV.

Crystal Structure of the Catalytic Domain of a Botulinum Neurotoxin Homologue from Enterococcus faecium: Potential Insights into Substrate Recognition.

Clostridium botulinum neurotoxins (BoNTs) are the most potent toxins known, causing the deadly disease botulism. They function through Zn2+-dependent endopeptidase cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, preventing vesicular fusion and subsequent neurotransmitter release from motor neurons. Several serotypes of BoNTs produced by Clostridium botulinum (BoNT/A-/G and/X) have been well-characterised over the years. However, a BoNT-like gene (homologue of BoNT) was recently identified in the non-clostridial species, Enterococcus faecium, which is the leading cause of hospital-acquired multi-drug resistant infections. Here, we report the crystal structure of the catalytic domain of a BoNT homologue from Enterococcus faecium (LC/En) at 2.0 Å resolution. Detailed structural analysis in comparison with the full-length BoNT/En AlphaFold2-predicted structure, LC/A (from BoNT/A), and LC/F (from BoNT/F) revealed putative subsites and exosites (including loops 1-5) involved in recognition of LC/En substrates. LC/En also appears to possess a conserved autoproteolytic cleavage site whose function is yet to be established.

Context-dependent requirement of G protein coupling for Latrophilin-2 in target selection of hippocampal axons.

The formation of neural circuits requires extensive interactions of cell-surface proteins to guide axons to their correct target neurons. Trans-cellular interactions of the adhesion G protein-coupled receptor latrophilin-2 (Lphn2) with its partner teneurin-3 instruct the precise assembly of hippocampal networks by reciprocal repulsion. Lphn2 acts as a repulsive receptor in distal CA1 neurons to direct their axons to the proximal subiculum, and as a repulsive ligand in the proximal subiculum to direct proximal CA1 axons to the distal subiculum. It remains unclear if Lphn2-mediated intracellular signaling is required for its role in either context. Here, we show that Lphn2 couples to Gα12/13 in heterologous cells; this coupling is increased by constitutive exposure of the tethered agonist. Specific mutations of Lphn2's tethered agonist region disrupt its G protein coupling and autoproteolytic cleavage, whereas mutating the autoproteolytic cleavage site alone prevents cleavage but preserves a functional tethered agonist. Using an in vivo misexpression assay, we demonstrate that wild-type Lphn2 misdirects proximal CA1 axons to the proximal subiculum and that Lphn2 tethered agonist activity is required for its role as a repulsive receptor in axons. By contrast, neither tethered agonist activity nor autoproteolysis were necessary for Lphn2's role as a repulsive ligand in the subiculum target neurons. Thus, tethered agonist activity is required for Lphn2-mediated neural circuit assembly in a context-dependent manner.

Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3

Adhesion G protein-coupled receptor latrophilin 3 (ADGRL3), a cell adhesion molecule highly expressed in the central nervous system, acts in synapse formation through trans interactions with its ligands. It is largely unknown if these interactions serve a purely adhesive function or can modulate G protein signaling. To assess how different structural elements of ADGRL3 (e.g., the adhesive domains, autoproteolytic cleavage site, or tethered agonist (TA)) impact receptor function, we require constructs that disrupt specific receptor features without impacting others. While we showed previously that mutating conserved Phe and Met residues in the TA of ADGRL3-C-terminal fragment (CTF), a CTF truncated to the G protein-coupled receptor proteolysis site, abolishes receptor-mediated G protein activation, we now find that autoproteolytic cleavage is disrupted in the full-length version of this construct. To identify a construct that disrupts TA-dependent activity without impacting proteolysis, we explored other mutations in the TA. We found that mutating the sixth and seventh residues of the TA, Leu and Met, to Ala impaired activity in a serum response element activity assay for both full-length and CTF constructs. We confirmed this activity loss results from impaired G protein coupling using an assay that acutely exposes the TA through controlled proteolysis. The ADGRL3 mutant expresses normally at the cell surface, and immunoblotting shows that it undergoes normal autoproteolysis. Thus, we found a construct that disrupts tethered agonism while retaining autoproteolytic cleavage, providing a tool to disentangle these functions invivo. Our approach and specific findings are likely to be broadly applicable to other adhesion receptors.

Abstract A102: Rescue of lost skin dendritic cells in melanoma is key for the resuscitation of antitumor T-cell responses

Abstract One major characteristic of skin-related cancers is the loss of skin-resident dendritic cell populations. In the tg(Grm1)EPv mouse model, ectopic expression of the metabotropic glutamate receptor-1 in melanocytes leads to a highly proliferative and antiapoptotic phenotype, resulting in melanoma formation within the dermis. The slow progression of these tumors allows for the in depth analysis of the immune infiltrate in growing tumors. We here show that total DCs (CD11c+ cells) are gradually lost as the tumor progresses. Mainly the dermal CD11b+ DCs are affected, whereas epidermal Langerhans cells remain unchanged. We hypothesized that extrinsic cell death of the DCs may be induced within the growing tumor. Indeed, the tumor tissue upregulated the expression of Fas ligand (FasL) that can induce extrinsic apoptosis in immature DCs that express Fas (CD95). Fas-mediated apoptosis depends on the activation of caspase-8 and mice in which the autoproteolytic cleavage site D387 is mutated to alanine (casp8D387A/casp8D387A) show strong resistance to Fas-mediated death. We compared the apoptosis induction in the different DC subsets in casp8WT/casp8WT and in casp8D387A/casp8D387A upon intradermal administration of an agonistic anti-CD95 antibody and we found that, indeed, CD11b+ DCs in WT mice are susceptible to apoptosis via CD95. In order to retrieve the intratumoral DCs, we treated tg(Grm1)EPv mice carrying tumor lesions with Flt3L and the percentages of the CD11b+ subset could be restored back to the levels of tumor-free mice. At the same time, T-cells from both the tumor and the tumor-draining lymph node were able to produce more cytotoxic cytokines. Our data thus indicate that the loss of DCs in melanoma depends on induction of apoptosis within the tumor; rescue of skin DCs may be a promising strategy in order to enhance the efficacy of existing immunotherapeutic strategies against skin-related cancers. Citation Format: Anastasia Prokopi, Christoph H. Tripp, Bart Tummers, Kerstin Komenda, Katharina Hutter, Giuseppe Cappellano, Lydia Bellmann, Mirjana Efremova, Zlatko Trajanoski, Suzie Chen, Bjorn E. Clausen, Douglas R. Green, Patrizia Stoitzner. Rescue of lost skin dendritic cells in melanoma is key for the resuscitation of antitumor T-cell responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A102.

BspK, a Serine Protease from the Predatory Bacterium Bdellovibrio bacteriovorus with Utility for Analysis of Therapeutic Antibodies.

ABSTRACTThe development of therapeutic and diagnostic antibodies is a rapidly growing field of research, being the fastest expanding group of products on the pharmaceutical market, and appropriate quality controls are crucial for their application. We have identified and characterized the serine protease termed BspK (Bdellovibrio serine protease K) from Bdellovibrio bacteriovorus and here show its activity on antibodies. Mutation of the serine residue at position 230 rendered the protease inactive. Further investigations of BspK enzymatic characteristics revealed autoproteolytic activity, resulting in numerous cleavage products. Two of the autoproteolytic cleavage sites in the BspK fusion protein were investigated in more detail and corresponded to cleavage after K28 and K210 in the N- and C-terminal parts of BspK, respectively. Further, BspK displayed stable enzymatic activity on IgG within the pH range of 6.0 to 9.5 and was inhibited in the presence of ZnCl2. BspK demonstrated preferential hydrolysis of human IgG1 compared to other immunoglobulins and isotypes, with hydrolysis of the heavy chain at position K226 generating two separate Fab fragments and an intact IgG Fc domain. Finally, we show that BspK preferentially cleaves its substrates C-terminally to lysines similar to the protease LysC. However, BspK displays a unique cleavage profile compared to several currently used proteases on the market.IMPORTANCE The rapid development of novel therapeutic antibodies is partly hindered by difficulties in assessing their quality and safety. The lack of tools and methods facilitating such quality controls obstructs and delays the process of product approval, eventually affecting the patients in need of treatment. These difficulties in product evaluations indicate a need for new and comprehensive tools for such analysis. Additionally, recent concerns raised regarding the limitations of established products on the market (e.g., trypsin) further highlight a general need for a larger array of proteases with novel cleavage profiles to meet current and future needs, within both the life science industry and the academic research community.

Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines.

Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease which is most effectively prevented through vaccination. Segments 7 (M) and 8 (NS) of the influenza virus genome encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing M or NS viral segments individually or modified M or NS viral segments combined in which the overlapping open reading frames of matrix 1 (M1)/M2 for the modified M segment and the open reading frames of nonstructural protein 1 (NS1)/nuclear export protein (NEP) for the modified NS segment were split by using the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at nonpermissive high temperatures, whereas high viral titers could be obtained at permissive low temperatures (33°C). Furthermore, viruses containing the M split segment were highly attenuated in vivo, while they retained their immunogenicity and provided protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and that such attenuated viruses represent an excellent option as safe, immunogenic, and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of the M segment to encode M1 and M2 from two separate, nonoverlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product. Vaccination represents our best therapeutic option against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal, and novel approaches are necessary for the prevention of disease caused by this important human respiratory pathogen. In this work, we describe a novel approach to generate safer and more efficient live-attenuated influenza virus vaccines (LAIVs) based on recombinant viruses whose genomes encode nonoverlapping and independent M1/M2 (split M segment [Ms]) or both M1/M2 and NS1/NEP (Ms and split NS segment [NSs]) open reading frames. Viruses containing a modified M segment were highly attenuated in mice but were able to confer, upon a single intranasal immunization, complete protection against a lethal homologous challenge with wild-type virus. Notably, the protection efficacy conferred by our viruses with split M segments was better than that conferred by the current temperature-sensitive LAIV. Altogether, these results open a new avenue for the development of safer and more protective LAIVs on the basis of the reorganization of spliced viral RNA segments in the genome.

Exploring the structural constraints at cleavage site of mucin 1 isoform through molecular dynamics simulation.

In silico alanine scanning mutagenesis on the cleavable isoform of mucin 1 revealed isoleucine 67 as one of the key factors contributing to the strain at the autoproteolytic cleavage site. In this study, we demonstrate the structural basis of isoleucine-induced rigidity towards the strain-driven autoproteolysis at G(-1)S(+1) cleavage site of mucin 1. We further evaluated the gain in flexibility upon isoleucine 67 mutation through molecular dynamics and essential dynamics studies. The results show that the mutant exhibits stability in its secondary structural elements while the native displays a less-bonded network, however the cleavage site of native remains constrained. Essential dynamics revealed that large motions of the mutant were confined to the loop although the internal domain of the structure remains unaffected. Also, the mutation exerted a larger effect on the intraprotein interactions and consequently resulted in a stabilized motif at the cleavage. Analyses on MD trajectory conformations illustrate a completely disrupted motif in native as an effect of the peptide strain. The study also revealed that in mutant, the cleavage competent catalytic groups C=O and OG were in geometrical aspects unfavorable for a nucleophilic attack. The results support the earlier speculation that the presence of bulky isoleucine proximal G(-1)S(+1) cleavage site limits the conformational sampling of residues and therefore maintains the residues in a torsionally restrained conformation.

Determination of the second autoproteolytic cleavage site of cephalosporin C acylase and the effect of deleting its flanking residues in the α-C-terminal region

Self-activation through two-step intra-molecular cleavages is of great importance for the synthesis of mature and functional cephalosporin acylase in the N-terminal nucleophile (Ntn) hydrolase superfamily. A synthetic gene for cephalosporin C acylase (sCPCAcy) from Pseudomonas sp. SE83 was overexpressed, and the self-activated sCPCAcy was produced in Escherichia coli JM109(DE3)/pET28-sCPCAcy. The first autoproteolytic cleavage site of Pre-sCPCAcy was determined to be G239–S240 according to the common features of Ntn hydrolases. The second cleavage site was identified as A232–S233 by C-terminus tandem MS/MS analysis of the purified α-subunit, which released a 7-aa spacer peptide with the generation of the α and β subunits of the mature sCPCAcy. The effect of the cleavage-site-flanking residues in the α-C-terminal region of sCPCAcy on its activation and characteristics was further evaluated. Residue G229 was found to be crucial for the first cleavage of Pre-sCPCAcy. Deletions in the α-C-terminal region were performed, and 14 mutant proteins were constructed. The majority of the fragment-deleted mutant proteins completely lost their activity due to failure of the first autocleavage, but this loss was not observed in mutant proteins D2 (227-AM-228 deletion) and D4 (212-ADLA-215 deletion), which formally activated into mature sCPCAcy with high activity. The Kcat/Km values of mutant proteins D2 and D4 were 46% and 102% higher than that of the original control, respectively.

Degenerate evolution of the hedgehog gene in a hemichordate lineage

The discovery of a set of highly conserved genes implicated in patterning during animal development represents one of the most striking findings from the field of evolutionary developmental biology. Existence of these "developmental toolkit" genes in diverse taxa, however, does not necessarily imply that they always perform the same functions. Here, we demonstrate functional evolution in a major toolkit gene. hedgehog (hh) encodes a protein that undergoes autocatalytic cleavage, releasing a signaling molecule involved in major developmental processes, notably neural patterning. We find that the hh gene of a colonial pterobranch hemichordate, Rhabdopleura compacta, is expressed in a dramatically different pattern to its ortholog in a harrimaniid enteropneust hemichordate, Saccoglossus kowalevskii. These represent two of the three major hemichordate lineages, the third being the indirect developing ptychoderid enteropneusts. We also show that the normally well-conserved amino acid sequence of the autoproteolytic cleavage site has a derived change in S. kowalevskii. Using ectopic expression in Drosophila, we find that this amino acid substitution reduces the efficiency of Hh autocatalytic cleavage and its signaling function. We conclude that the Hh sequence and expression in S. kowalevskii represent the derived state for deuterostomes, and we argue that functional evolution accompanied secondary reduction of the central nervous system in harrimaniids.

Co-occurrence of both l-asparaginase subtypes in Arabidopsis: At3g16150 encodes a K+-dependent l-asparaginase

L-asparaginases (EC 3.5.1.1) are hypothesized to play an important role in nitrogen supply to sink tissues, especially in legume-developing seeds. Two plant L-asparaginase subtypes were previously identified according to their K(+)-dependence for catalytic activity. An L-asparaginase homologous to Lupinus K(+)-independent enzymes with activity towards beta-aspartyl dipeptides, At5g08100, has been previously characterized as a member of the N-terminal nucleophile amidohydrolase superfamily in Arabidopsis. In this study, a K(+)-dependent L-asparaginase from Arabidopsis, At3g16150, is characterized. The recombinants At3g16150 and At5g08100 share a similar subunit structure and conserved autoproteolytic pentapeptide cleavage site, commencing with the catalytic Thr nucleophile, as determined by ESI-MS. The catalytic activity of At3g16150 was enhanced approximately tenfold in the presence of K(+). At3g16150 was strictly specific for L-Asn, and had no activity towards beta-aspartyl dipeptides. At3g16150 also had an approximately 80-fold higher catalytic efficiency with L-Asn relative to At5g08100. Among the beta-aspartyl dipeptides tested, At5g08100 had a preference for beta-aspartyl-His, with catalytic efficiency comparable to that with L-Asn. The phylogenetic analysis revealed that At3g16150 and At5g08100 belong to two distinct subfamilies. The transcript levels of At3g16150 and At5g08100 were highest in sink tissues, especially in flowers and siliques, early in development, as determined by quantitative RT-PCR. The overlapping spatial patterns of expression argue for a partially redundant function of the enzymes. However, the high catalytic efficiency suggests that the K(+)-dependent enzyme may metabolize L-Asn more efficiently under conditions of high metabolic demand for N.

Thermostability of an alkaline protease, AprP, is enhanced by replacements of Ser307 and Ser331 at the cleavage sites

The thermostability of an alkaline protease, AprP from Pseudomonas sp. KFCC 10818, was improved by replacing Ser307 and Ser331 at the autoproteolytic cleavage sites with various amino acids. Six mutant enzymes were purified and characterized. Two of these had half-lives four and three times longer than the wild-type protease at 55 °C in the presence of 1 mM CaCl2. Three mutant enzymes had half-lives twice as long as the wild-type under the same condition.

Enhanced thermal stability of an alkaline protease, AprP, isolated from a Pseudomonas sp. by mutation at an autoproteolysis site, Ser-331.

The thermal stability of the alkaline protease extracellular subtilisin-type serine protease (AprP) from Pseudomonas sp. KFCC 10818 was improved by altering an amino acid residue at an autoproteolytic cleavage site. N-terminal sequence analysis of the autoproteolytic products of the protein revealed the presence of two cleavage sites, Ser-307 and Ser-331. To increase the thermal stability of the enzyme, serine residues of these sites were replaced with aspartate. The S331D mutant enzyme was successfully purified and characterized whereas the S307D mutant was not. The half-lives of the S331D mutant at 55 degrees C and 60 degrees C were 1.5 and 2.4 times longer than that of the wild-type enzyme, respectively. In addition, the catalytic efficiency was also enhanced.

Factors affecting autolysis of a subtilisin-like serine proteinase secreted by Ophiostoma piceae and identification of the cleavage site

The extracellular serine proteinase secreted by Ophiostoma piceae was degraded by autoproteolysis under certain conditions. At elevated temperatures the mature protein of 33 kDa was rapidly degraded without any accumulation of protein breakdown products. Glycerol, calcium ions and ammonium sulfate raised the heat stability of the enzyme, increasing its half-life at 45°C from 1.9 min to 9.4 min, 40.4 min and 2 h, respectively. Thermal unfolding of the proteinase also occurred at higher temperatures in the presence of calcium ions and ammonium sulfate. Under conditions of heating, altered pH or partial depletion of protein-bound ions by EDTA, the structure of the proteinase was more susceptible to proteolysis. The major hydrolytic fragments of 19 kDa and 14 kDa, had N-terminal sequences of Ala 1-Tyr 2-Thr 3-Thr 4-Gln 5-Thr 6-Gly 7-Ala 8-Pro 9-and Ser 170-Glu 171-Pro 172-Se 173-Val 174-X-Thr 176-Val 177-Gly 178-Ala 179-, respectively. Since the former sequence was identical to the N-terminus of the native protein, the major autoproteolytic cleavage site for a class II subtilase appeared to be the N-side of Ser 170 using numbering based on the sequence of proteinase K. The secondary structure of the proteinase from O. piceae was similar to that of proteinase K based on circular dichroic spectra in the far ultraviolet region. This cleavage site was in a similar region to that identified for class I subtilases, which was located in an outer exposed loop of the tertiary structure of subtilisin-like serine proteinases.

Endoproteolytic Cleavage of Its Propeptide Is a Prerequisite for Efficient Transport of Furin Out of the Endoplasmic Reticulum

The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1 + P4 + P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.

Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants.

The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its N- and C-terminal regions, respectively. We reported previously that a 30-kDa protease is derived by autoproteolytic processing of a polyprotein which is the translation product of the entire UL80 open reading frame (E. Z. Baum, G. A. Bebernitz, J. D. Hulmes, V. P. Muzithras, T. R. Jones, and Y. Gluzman, J. Virol. 67:497-506, 1993). Three autoproteolytic cleavage sites within the UL80 polyprotein were characterized; site 143 is within the protease domain and inactivates the protease. In this article, we report (i) expression analyses of UL80 in infected cells, including the processing kinetics of the UL80 polyprotein; (ii) the existence of an additional cleavage site (site 209) within the protease domain of the UL80 polyprotein; and (iii) the effect of mutagenesis at each of the cleavage sites upon proteolytic activity and steady-state levels of the UL80 processing products. During the course of infection, UL80 polyprotein processing begins at cleavage site 643 and follows at sites 256 and 143. Cleavage at site 643 and/or 256 within the polyprotein is not a prerequisite for efficient protease activity, since all three proteases (85-, 80-, and 30-kDa proteins) were equally active in cleaving the assembly protein precursor to its mature form. Inhibition of cleavage at site 143 resulted in a three- to sixfold increase in the steady-state level of the 30-kDa protease, supporting the hypothesis that cleavage at this site may represent a mechanism by which cytomegalovirus regulates the level of active protease.

Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.

Primary structures of platelet aggregation inhibitors (disintegrins) autoproteolytically released from snake venom hemorrhagic metalloproteinases and new fluorogenic peptide substrates for these enzymes.

A hemorrhagic protein (60 kDa), HR1B, present in the venom of Trimeresurus flavoviridis is a mosaic protein consisting of an NH2-terminal metalloproteinase-domain, a disintegrin (platelet aggregation inhibitor)-like domain, and a unique COOH-terminal Cys-rich domain. Since the gross structures of HR1B and protein precursors of disintegrins, trigramin, and rhodostomin, all of which contain the metalloproteinase domain, are similar, many disintegrins so far detected in snake venoms are assumed to be autoproteolytic fragments released from precursors. In ongoing related experiments, the newly purified hemorrhagic metalloproteinases, HR1A from T. flavoviridis venom and HT-1 from Crotalus ruber ruber venom, in addition to HR1B, were autoproteolyzed, in the absence of Ca2+, at 37 degrees C for 3-12 h. Under these conditions, HR1A, HR1B, and HT-1 each released a single major fragment of 32, 34, and 31 kDa, respectively. The entire amino acid sequences of the isolated fragments indicated the presence of disintegrin-like and Cys-rich domains in the COOH-terminal regions of HR1A, HR1B, and HT-1, respectively. It seems likely that so-called disintegrins probably originate from various metalloproteinases present in venom. On the bases of peptide sequences close to the autoproteolytic cleavage sites of these metalloproteinases and the sites of fibrinogen cleaved by these enzymes, we synthesized new intramolecularly quenched fluorogenic peptide substrates. Among the 10 peptides tested, 2-aminobenzoyl (Abz)-Ser-Pro-Met-Leu-2,4-dinitroanilinoethylamide (Dna) proved to be the best substrate for venom metalloproteinase, as deduced from kinetic analyses.

Identification of autoproteolytic cleavage site in the Asp-49 mutant subtilisin J by site-directed mutagenesis

Ser-49 located at the exposed surface loop of subtilisin J was replaced with Asp and Arg. Proteinase activity of the Asp-49 mutant was similar to that of wild-type, but the Arg mutant was inactivated. At 37°C, mature subtilisin J protein of the Asp-49 mutant rapidly degraded, and specific breakdown products were accumulated. These proteins were analyzed by SDS-PAGE, and the N-terminal sequences were determined for the mature and deleted protein. We identified the autoproteolytic cleavage site in the mature Asp-49 mutant protein from sequencing data.