Proteolytic release of membrane-bound endo-(1,4)-β-glucanase activity associated with cell wall softening inAchlya ambisexualis

2002 ◽  
Vol 48 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Terry W Hill ◽  
Darlene M Loprete ◽  
Kim N Vu ◽  
Susan P. Bayat Mokhtari ◽  
L Vanessa Hardin
Keyword(s):  
Cell Wall ◽  
Membrane Vesicles ◽  
Cysteine Proteases ◽  
Proteolytic Processing ◽  
Cysteine Protease Inhibitor ◽  
Glucanase Activity ◽  
Molecular Masses ◽  
Detergent Treatment ◽  
Membrane Bound

Branching and other cell wall softening events in fungi and oomycetes are thought to involve the activity of secreted enzymes, which are packaged in membrane vesicles and delivered to sites of cell expansion, there to work in a carefully regulated manner upon the structure of the wall. Here we demonstrate a latent endo-(1,4)-β-glucanase activity in a mixed membrane fraction of the oomycete Achlya ambisexualis, which can be released by cysteine proteases with an increase of apparent activity. In addition, a similar endogenous process is strongly inhibited by the cysteine protease inhibitor iodoacetamide, while inhibitors of other types of proteases have a much smaller effect. Detergent treatment of membranes releases two glucanases detectable by electrophoretic activity staining, with apparent molecular masses of about 164 and 35 kDa. Proteolysis produces several activity bands, with major species having apparent molecular masses of about 149, 133, 48, 35, and 25 kDa. The ca. 35- and 25-kDa bands migrate in parallel with glucanases secreted during wall softening in vivo. We propose that the initiation of wall softening in Achlya involves the proteolytic processing and solubilization of at least some secreted endoglucanases. We also propose that the solubilization component of this process functions not just to provide the enzymes with access to wall matrix substrates but also may provide a mechanism for the eventual termination of their biological function.Key words: apical growth, hyphal branching, proteases, cell walls, protein secretion.

scholarly journals Complex Proteolytic Processing Acts on Delta, a Transmembrane Ligand for Notch, during Drosophila Development

1998 ◽  
Vol 9 (7) ◽  
pp. 1709-1723 ◽  
Author(s):  
Kristin M. Klueg ◽  
Todd R. Parody ◽  
Marc A.T. Muskavitch
Keyword(s):  
Cell Fate ◽  
Cultured Cells ◽  
Cellular Responses ◽  
Proteolytic Processing ◽  
Cell Fate Specification ◽  
A Cell ◽  
Membrane Bound ◽  
Delta Functions ◽  
Intracellular Domains

Delta functions as a cell nonautonomous membrane-bound ligand that binds to Notch, a cell-autonomous receptor, during cell fate specification. Interaction between Delta and Notch leads to signal transduction and elicitation of cellular responses. During our investigations to further understand the biochemical mechanism by which Delta signaling is regulated, we have identified four Delta isoforms inDrosophila embryonic and larval extracts. We have demonstrated that at least one of the smaller isoforms, Delta S, results from proteolysis. Using antibodies to the Delta extracellular and intracellular domains in colocalization experiments, we have found that at least three Delta isoforms exist in vivo, providing the first evidence that multiple forms of Delta exist during development. Finally, we demonstrate that Delta is a transmembrane ligand that can be taken up by Notch-expressing Drosophila cultured cells. Cell culture experiments imply that full-length Delta is taken up by Notch-expressing cells. We present evidence that suggests this uptake occurs by a nonphagocytic mechanism.

scholarly journals Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.

1975 ◽  
Vol 67 (3) ◽  
pp. 835-851 ◽  
Author(s):  
G Blobel ◽  
B Dobberstein
Keyword(s):  
Light Chain ◽  
Light Chains ◽  
Discussion Section ◽  
Heterologous System ◽  
In Vitro Processing ◽  
Nascent Chain ◽  
Detergent Treatment ◽  
Membrane Bound

Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.

scholarly journals Role of Proteolysis in Determining Potency ofBacillus thuringiensis Cry1Ac δ-Endotoxin

2000 ◽  
Vol 66 (12) ◽  
pp. 5174-5181 ◽  
Author(s):  
Daniel J. Lightwood ◽  
David J. Ellar ◽  
Paul Jarrett
Keyword(s):  
Membrane Vesicles ◽  
Apparent Molecular Weight ◽  
Pieris Brassicae ◽  
Proteolytic Processing ◽  
Mamestra Brassicae ◽  
Ofbacillus Thuringiensis ◽  
Domain I

ABSTRACT Bacillus thuringiensis protein δ-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac δ-endotoxin towards Pieris brassicae (family: Pieridae) andMamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of ∼56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of ∼58, ∼40, and ∼20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, butM. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.

scholarly journals Proteolytic processing of the α-subunit of rat endopeptidase-24.18 by furin

1995 ◽  
Vol 309 (2) ◽  
pp. 683-688 ◽  
Author(s):  
P E Milhiet ◽  
S Chevallier ◽  
D Corbeil ◽  
N G Seidah ◽  
P Crine ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Consensus Sequence ◽  
Proteolytic Processing ◽  
Alpha Subunit ◽  
Structural Domain ◽  
Α Subunit ◽  
Alpha Subunits ◽  
Golgi Compartment ◽  
Membrane Bound

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.

scholarly journals Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins

Insects ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 223
Author(s):  
Junxiang Wang ◽  
Xiaozhen Yang ◽  
Huan He ◽  
Jingru Chen ◽  
Yuanyuan Liu ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Reverse Genetics ◽  
Membrane Vesicles ◽  
Molecular Evidence ◽  
Bacillus Thuringiensis Subsp ◽  
Membrane Bound ◽  
Homozygous Strain ◽  
High Binding Affinity ◽  
Single Knockout

The insecticidal Cry4Ba and Cry11Aa crystal proteins from Bacillus thuringiensis subsp. israelensis (Bti) are highly toxic to Ae. aegypti larvae. The glycosylphosphatidylinositol (GPI)-anchored APN was identified as an important membrane-bound receptor for multiple Cry toxins in numerous Lepidoptera, Coleoptera, and Diptera insects. However, there is no direct molecular evidence to link APN of Ae. aegypti to Bti toxicity in vivo. In this study, two Cry4Ba/Cry11Aa-binding Ae. aegypti GPI-APN isoforms (AeAPN1 and AeAPN2) were individually knocked-out using CRISPR/Cas9 mutagenesis, and the AeAPN1/AeAPN2 double-mutant homozygous strain was generated using the reverse genetics approach. ELISA assays showed that the high binding affinity of Cry4Ba and Cry11Aa protoxins to the midgut brush border membrane vesicles (BBMVs) from these APN knockouts was similar to the background from the wild-type (WT) strain. Likewise, the bioassay results showed that neither the single knockout of AeAPN1 or AeAPN2, nor the simultaneous disruption of AeAPN1 and AeAPN2 resulted in significant changes in susceptibility of Ae. aegypti larvae to Cry4Ba and Cry11Aa toxins. Accordingly, our results suggest that AeAPN1 and AeAPN2 may not mediate Bti Cry4Ba and Cry11Aa toxicity in Ae. aegypti larvae as their binding proteins.

scholarly journals CdtC-Induced Processing of Membrane-Bound CdtA Is a Crucial Step inAggregatibacter actinomycetemcomitansCytolethal Distending Toxin Holotoxin Formation

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Keiko Tsuruda ◽  
Oranart Matangkasombut ◽  
Masaru Ohara ◽  
Motoyuki Sugai
Keyword(s):  
Mammalian Cells ◽  
Proteolytic Processing ◽  
Content Type ◽  
Protein Toxin ◽  
Sequential Event ◽  
Membrane Bound ◽  
Processed Form ◽  
Rapid Processing

ABSTRACTAggregatibacter actinomycetemcomitansis an oral pathogen causing periodontal disease and bacterial endocarditis. It produces cytolethal distending toxin (CDT) that could damage mammalian cells and tissues. CDT is a tripartite protein toxin composed of CdtA, CdtB, and CdtC. We have previously indicated that CdtA is a lipoprotein and that the proteolytic processing of CdtA is important for biogenesis and secretion of CDT holotoxin. Here, we established anin vitroprocessing assay of CdtA and investigated the interactions of CdtA with other Cdt subunits. This assay demonstrated that incubation of membrane-bound CdtA (MCdtA), CdtB, and CdtC immediately generated a processed form of CdtA (CdtA′), which is recovered from the soluble fraction. In contrast, incubation of soluble membrane-unbound CdtA with CdtB and CdtC did not yield any CdtA′. Furthermore, incubation of CdtC with MCdtA was enough to induce rapid processing of MCdtA, whereas CdtB alone was unable to induce the processing. Coimmunoprecipitation demonstrated that CdtA′ and CdtC formed a complex. Furthermore, subsequent addition of CdtB to this reaction mixture resulted in complete CDT holotoxin complex. The cytolethal distending activity assay demonstrated that CDT complex containing CdtA′ showed far stronger cytotoxicity than that containing CdtA. Collectively, our data suggest that CDT holotoxin formationin vivois a sequential event: interaction of MCdtA and CdtC induces proteolytic processing of MCdtA, and the released CdtA′ forms a complex with CdtC. Subsequent binding of CdtB to the CdtA′/CdtC complex results in CDT holotoxin formation.

scholarly journals Nippocystatin, a Cysteine Protease Inhibitor fromNippostrongylus brasiliensis, Inhibits Antigen Processing and Modulates Antigen-Specific Immune Response

2001 ◽  
Vol 69 (12) ◽  
pp. 7380-7386 ◽  
Author(s):  
Teruki Dainichi ◽  
Yoichi Maekawa ◽  
Kazunari Ishii ◽  
Tianqian Zhang ◽  
Baher Fawzy Nashed ◽  
...  
Keyword(s):  
Immune System ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Antigen Processing ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Host Immune System ◽  
Nippostrongylus Brasiliensis

ABSTRACT During infection, parasites evade the host immune system by modulating or exploiting the immune system; e.g., they suppress expression of major histocompatibility complex class II molecules or secrete cytokine-like molecules. However, it is not clear whether helminths disturb the immune responses of their hosts by controlling the antigen-processing pathways of the hosts. In this study, we identified a new cysteine protease inhibitor, nippocystatin, derived from excretory-secretory (ES) products of an intestinal nematode,Nippostrongylus brasiliensis. Nippocystatin, which belongs to cystatin family 2, consists of 144 amino acids and is secreted as a 14-kDa mature form. In vivo treatment of ovalbumin (OVA)-immunized mice with recombinant nippocystatin (rNbCys) profoundly suppressed OVA-specific proliferation of splenocytes but not non-antigen-specific proliferation of splenocytes. OVA-specific cytokine production was also greatly suppressed in rNbCys-treated mice. Although the serum levels of both OVA-specific immunoglobulin G1 (IgG1) and IgG2a were not affected by rNbCys treatment, OVA-specific IgE was preferentially downregulated in rNbCys-treated mice. In vitro rNbCys inhibited processing of OVA by lysosomal cysteine proteases from the spleens of mice. Mice with anti-nippocystatin antibodies became partially resistant to infection with N. brasiliensis. Based on these findings, N. brasiliensis appears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts.

scholarly journals Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Ryan Finethy ◽  
Sarah Luoma ◽  
Nichole Orench-Rivera ◽  
Eric M. Feeley ◽  
Arun K. Haldar ◽  
...  
Keyword(s):  
Cell Wall ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Binding Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Bacterial Cell Wall ◽  
Cell Wall Component ◽  
Gram Negative

ABSTRACT The Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as “free” LPS aggregates or actively secreted by live bacteria as a component of outer membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli. In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1β (IL-1β) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types. IMPORTANCE The bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of outer membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by bacterial infections. IMPORTANCE The bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of outer membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by bacterial infections.

Molecular organization of selected prokaryotic S-layer proteins

2005 ◽  
Vol 51 (9) ◽  
pp. 731-743 ◽  
Author(s):  
Harald Claus ◽  
Erol Akça ◽  
Tony Debaerdemaeker ◽  
Christine Evrard ◽  
Jean-Paul Declercq ◽  
...  
Keyword(s):  
Cell Wall ◽  
Molecular Sieves ◽  
Extracellular Enzymes ◽  
Protein Stabilization ◽  
Proteolytic Processing ◽  
Molecular Organization ◽  
Wall Structures ◽  
Attachment Sites ◽  
Archaeal Species ◽  
Molecular Masses

Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different species of the prokaryotic domains bacteria and archaea. S-layers usually consist of a single (glyco-)protein species with molecular masses ranging from about 40 to 200 kDa that form lattices of oblique, tetragonal, or hexagonal architecture. The primary sequen ces of hyperthermophilic archaeal species exhibit some characteristic signatures. Further adaptations to their specific environments occur by various post-translational modifications, such as linkage of glycans, lipids, phosphate, and sulfate groups to the protein or by proteolytic processing. Specific domains direct the anchoring of the S-layer to the underlying cell wall components and transport across the cytoplasma membrane. In addition to their presumptive original role as protective coats in archaea and bacteria, they have adapted new functions, e.g., as molecular sieves, attachment sites for extracellular enzymes, and virulence factors.Key words: prokaryotes, cell walls, S-layer (glyco-) proteins, protein stabilization.

scholarly journals In Vitro and In Vivo Functional Activity of Chlamydia MurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance

2003 ◽  
Vol 185 (4) ◽  
pp. 1218-1228 ◽  
Author(s):  
Andrea J. McCoy ◽  
Robin C. Sandlin ◽  
Anthony T. Maurelli
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Enzymatic Reaction ◽  
Developmental Cycle ◽  
E Coli ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Fosfomycin Resistance

ABSTRACT Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal ΔmurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.

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