Functional insights into the role of novel type I cohesin and dockerin domains from Clostridium thermocellum

2009 ◽  
Vol 424 (3) ◽  
pp. 375-384 ◽  
Author(s):  
Benedita A. Pinheiro ◽  
Harry J. Gilbert ◽  
Kazutaka Sakka ◽  
Kazuo Sakka ◽  
Vânia O. Fernandes ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Type I ◽  
Cell Wall Polysaccharides ◽  
Molecular Scaffold ◽  
Catalytic Subunits ◽  
Anaerobic Microorganisms ◽  
Binding Interfaces

Cellulosomes, synthesized by anaerobic microorganisms such as Clostridium thermocellum, are remarkably complex nanomachines that efficiently degrade plant cell wall polysaccharides. Cellulosome assembly results from the interaction of type I dockerin domains, present on the catalytic subunits, and the cohesin domains of a large non-catalytic integrating protein that acts as a molecular scaffold. In general, type I dockerins contain two distinct cohesin-binding interfaces that appear to display identical ligand specificities. Inspection of the C. thermocellum genome reveals 72 dockerin-containing proteins. In four of these proteins, Cthe_0258, Cthe_0435, Cthe_0624 and Cthe_0918, there are significant differences in the residues that comprise the two cohesin-binding sites of the type I dockerin domains. In addition, a protein of unknown function (Cthe_0452), containing a C-terminal cohesin highly similar to the equivalent domains present in C. thermocellum-integrating protein (CipA), was also identified. In the present study, the ligand specificities of the newly identified cohesin and dockerin domains are described. The results revealed that Cthe_0452 is located at the C. thermocellum cell surface and thus the protein was renamed as OlpC. The dockerins of Cthe_0258 and Cthe_0435 recognize, preferentially, the OlpC cohesin and thus these enzymes are believed to be predominantly located at the surface of the bacterium. By contrast, the dockerin domains of Cthe_0624 and Cthe_0918 are primarily cellulosomal since they bind preferentially to the cohesins of CipA. OlpC, which is a relatively abundant protein, may also adopt a ‘warehouse’ function by transiently retaining cellulosomal enzymes at the cell surface before they are assembled on to the multienzyme complex.

Analysis of Structural Signals Conferring Localisation of Pig OST48 to the Endoplasmic Reticulum

2001 ◽  
Vol 382 (7) ◽  
pp. 1039-1047 ◽  
Author(s):  
Birgit Hardt ◽  
Raquel Aparicio ◽  
Wilhelm Breuer ◽  
Ernst Bause
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Transmembrane Domain ◽  
Membrane Topology ◽  
Type I ◽  
Catalytic Subunits ◽  
Hybrid Proteins ◽  
Lysine Residues ◽  
Typical Type ◽  
Hydrophobic Transmembrane Domain

Abstract Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the cotranslational transfer of GlcNAc[2]Man[9]Glc[3] from Dol PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ERtargeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man[9] mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the Cterminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48?K) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ERresidency on OST48. The doublelysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48?K-3, when coexpressed in COS-1 cells together with myctagged ribophorin I, was quantitatively retained in the ER. By contrast, coexpression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMO?K-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the Cterminus of the Man[9]mannosidase luminal domain. Thus ERlocalisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the doublelysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in recapture of molecules which have escaped from the ER.

scholarly journals Analysis of the Role of N-glycosylation in Cell-surface expression, Function and Binding Properties of SARS-CoV-2 receptor ACE2

2021 ◽  
Author(s):  
Alberto Brandariz-Nuñez ◽  
Raymond R Rowland
Keyword(s):  
Cell Surface ◽  
Viral Entry ◽  
Biological Activities ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Type I ◽  
Protein Activity ◽  
Host Cell Membrane ◽  
S Protein

Human angiotensin I-converting enzyme 2 (hACE2) is a type-I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV and SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation plays no role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell to cell fusion mediated by SARS-CoV S protein but not SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which could be the result of low cell surface expression of the deglycosylated ACE2 receptor.

scholarly journals Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains

2016 ◽  
Vol 0 (0) ◽  
Author(s):  
Frederike Schmidt ◽  
Miryam Müller ◽  
Johannes Prox ◽  
Philipp Arnold ◽  
Caroline Schönherr ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Amyloid Β ◽  
Type I ◽  
Direct Binding ◽  
Small Intestinal ◽  
Split Ubiquitin ◽  
Biological Methods ◽  
Two Hybrid

AbstractMeprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer’s disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.

scholarly journals Role of theganSPQABOperon in Degradation of Galactan by Bacillus subtilis

2016 ◽  
Vol 198 (20) ◽  
pp. 2887-2896 ◽  
Author(s):  
Hildegard Watzlawick ◽  
Kambiz Morabbi Heravi ◽  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Abc Transporter ◽  
Plant Cell Wall ◽  
Type I ◽  
Cell Wall Polysaccharides ◽  
Pectin Degradation ◽  
Content Type ◽  
Sugar Binding ◽  
Thermal Shift Assay ◽  
Thermal Shift

ABSTRACTBacillus subtilispossesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganAandganB), two transporter component genes (ganQandganP), and the sugar-binding lipoprotein-encoding geneganS(previously known ascycB). These genes form an operon that is regulated by GanR. The degradation of galactan byB. subtilisbegins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the −35 box of the σA-type promoter of Pgan, which is located upstream ofganS.IMPORTANCEBacillus subtilisis a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied inB. subtilis. Nevertheless, theB. subtilisutilization system of galactan, which is found as the side chain of the rhamnogalacturonan type I complex in pectin, has remained partially studied. Here, we investigated the galactan utilization system consisting of theganSPQABoperon and its regulatorganR. This study improves our knowledge of the carbohydrate degradation systems ofB. subtilis, especially the pectin degradation systems. Moreover, the galactan-degrading enzymes may be exploited for the production of galacto-oligosaccharides, which are used as prebiotic substances in the food industry.

scholarly journals Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome of Clostridium thermocellum

2018 ◽  
Author(s):  
Anders Barth ◽  
Jelle Hendrix ◽  
Daniel Fried ◽  
Yoav Barak ◽  
Edward Bayer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Clostridium Thermocellum ◽  
Dynamic Equilibrium ◽  
Conformational Dynamics ◽  
Resonance Energy ◽  
Spatial Arrangement ◽  
Industrial Applications ◽  
Type I ◽  
Binding Modes ◽  
Binding Interfaces

AbstractEfficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using Förster resonance energy transfer to study the conformational dynamics of a bimodular tandem cohesin segment of the scaffoldin protein CipA ofClostridium thermocellum. Our data reveal the existence of compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the inter-cohesin linker does not significantly alter the structural dynamics. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed but the cohesin-cohesin interactions persist. This suggests that the dockerin-binding interfaces are not involved in cohesin-cohesin interactions. The formation of cohesin-cohesin interactions is also observed in all-atom molecular dynamics simulations of the system. From the simulations, we identify possible inter-cohesin binding modes, none of which show obstruction of the cohesin-dockerin binding interfaces. Our results go beyond the view of scaffoldin as “beads on a string”. We propose that both the flexibility and cohesin-cohesin interactions are important factors for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies. Hence, the flexibility of the linker region and cohesin-cohesin interactions should be considered when designing cellulosomes for industrial applications.

scholarly journals Purification, crystallization and preliminary X-ray characterization of the third ScaB cohesin in complex with an ScaA X-dockerin fromAcetivibrio cellulolyticus

2014 ◽  
Vol 70 (5) ◽  
pp. 656-658 ◽  
Author(s):  
Kate Cameron ◽  
Victor D. Alves ◽  
Pedro Bule ◽  
Luís M. A. Ferreira ◽  
Carlos M. G. A. Fontes ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Type I ◽  
Type Ii ◽  
Surface Attachment ◽  
Enzyme Binding ◽  
The Third ◽  
Entire Complex ◽  
Cell Wall Carbohydrates ◽  
Binding Interfaces

Interactions between cohesin and dockerin modules are critical for the formation of the cellulosome, which is responsible for the efficient degradation of plant cell-wall carbohydrates by anaerobes. Type I dockerin modules found in modular enzymatic components interact with type I cohesins in primary scaffoldins, enabling the assembly of the multi-enzyme complex. In contrast, type II dockerins located in primary scaffoldins bind to type II cohesins in adaptor scaffoldins or anchoring scaffoldins located at the bacterial envelope, contributing to the cell-surface attachment of the entire complex.Acetivibrio cellulolyticuspossesses an extremely complex cellulosome arrangement which is organized by a primary enzyme-binding scaffoldin (ScaA), two anchoring scaffoldins (ScaC and ScaD) and an unusual adaptor scaffoldin (ScaB). An ScaA X-dockerin mutated to inactivate one of the two putative cohesin-binding interfaces complexed with the third ScaB cohesin fromA. cellulolyticushas been purified and crystallized and data were collected to a resolution of 2.41 Å.

The role of ester groups in resistance of plant cell wall polysaccharides to enzymatic hydrolysis

1989 ◽  
Vol 20-21 (1) ◽  
pp. 45-61 ◽  
Author(s):  
K. Grohmann ◽  
D. J. Mitchell ◽  
M. E. Himmel ◽  
B. E. Dale ◽  
H. A. Schroeder
Keyword(s):  
Cell Wall ◽  
Enzymatic Hydrolysis ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cell Wall Polysaccharides

Trafficking and cell surface stability of ENaC

2001 ◽  
Vol 281 (3) ◽  
pp. F391-F399 ◽  
Author(s):  
Daniela Rotin ◽  
Voula Kanelis ◽  
Laurent Schild
Keyword(s):  
Cell Surface ◽  
Distal Colon ◽  
Type I ◽  
Surface Stability ◽  
Liddle’S Syndrome ◽  
Nonionic Detergent ◽  
Liddle's Syndrome ◽  
Targeting Signal ◽  
Increased Activity

The epithelial Na+ channel (ENaC) plays a key role in the regulation of Na+ and water absorption in several epithelia, including those of the distal nephron, distal colon, and lung. Accordingly, mutations in ENaC leading to reduced or increased channel activity cause human diseases such as pseudohypoaldosteronism type I or Liddle's syndrome, respectively. The gain of ENaC function in Liddle's syndrome is associated with increased activity and stability of the channel at the plasma membrane. Thus understanding the regulation of channel processing and trafficking to and stability at the cell surface is of fundamental importance. This review describes some of the recent advances in our understanding of ENaC trafficking, including the role of glycosylation, ENaC solubility in nonionic detergent, targeting signal(s) and hormones. It also describes the regulation of ENaC stability at the cell surface and the roles of the ubiquitin ligase Nedd4 (and ubiquitination) and clathrin-mediated endocytosis in that regulation.

scholarly journals The Suitability of Orthogonal Hosts to Study Plant Cell Wall Biosynthesis

Plants ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 516 ◽  
Author(s):  
Markus Pauly ◽  
Niklas Gawenda ◽  
Christine Wagner ◽  
Patrick Fischbach ◽  
Vicente Ramírez ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Cells ◽  
Cell Wall Polysaccharides ◽  
Plant Polysaccharides ◽  
Auxiliary Protein ◽  
Molecular Components ◽  
Selection Of

Plant cells are surrounded by an extracellular matrix that consists mainly of polysaccharides. Many molecular components involved in plant cell wall polymer synthesis have been identified, but it remains largely unknown how these molecular players function together to define the length and decoration pattern of a polysaccharide. Synthetic biology can be applied to answer questions beyond individual glycosyltransferases by reconstructing entire biosynthetic machineries required to produce a complete wall polysaccharide. Recently, this approach was successful in establishing the production of heteromannan from several plant species in an orthogonal host—a yeast—illuminating the role of an auxiliary protein in the biosynthetic process. In this review we evaluate to what extent a selection of organisms from three kingdoms of life (Bacteria, Fungi and Animalia) might be suitable for the synthesis of plant cell wall polysaccharides. By identifying their key attributes for glycoengineering as well as analyzing the glycosidic linkages of their native polymers, we present a valuable comparison of their key advantages and limitations for the production of different classes of plant polysaccharides.

scholarly journals Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains

2016 ◽  
Vol 397 (9) ◽  
pp. 857-869 ◽  
Author(s):  
Frederike Schmidt ◽  
Miryam Müller ◽  
Johannes Prox ◽  
Philipp Arnold ◽  
Caroline Schönherr ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Amyloid Β ◽  
Type I ◽  
Direct Binding ◽  
Small Intestinal ◽  
Split Ubiquitin ◽  
Biological Methods ◽  
Two Hybrid

Abstract Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer’s disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.

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