scholarly journals Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis

2018 ◽  
Vol 115 (38) ◽  
pp. 9557-9562 ◽  
Author(s):  
Hua Lu ◽  
Charles S. Fermaintt ◽  
Natalia A. Cherepanova ◽  
Reid Gilmore ◽  
Nan Yan ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Site Occupancy ◽  
Liver Cells ◽  
Acceptor Site ◽  
Catalytic Subunits ◽  
Immunity Factor ◽  
Free Oligosaccharides ◽  
Oligosaccharide Hydrolysis

Oligosaccharyltransferases (OSTs) N-glycosylate proteins by transferring oligosaccharides from lipid-linked oligosaccharides (LLOs) to asparaginyl residues of Asn-Xaa-Ser/Thr acceptor sequons. Mammals have OST isoforms with STT3A or STT3B catalytic subunits for cotranslational or posttranslational N-glycosylation, respectively. OSTs also hydrolyze LLOs, forming free oligosaccharides (fOSs). It has been unclear whether hydrolysis is due to one or both OSTs, segregated from N-glycosylation, and/or regulated. Transfer and hydrolysis were assayed in permeabilized HEK293 kidney and Huh7.5.1 liver cells lacking STT3A or STT3B. Transfer by both STT3A-OST and STT3B-OST with synthetic acceptors was robust. LLO hydrolysis by STT3B-OST was readily detected and surprisingly modulated: Without acceptors, STT3B-OST hydrolyzed Glc3Man9GlcNAc2-LLO but not Man9GlcNAc2-LLO, yet it hydrolyzed both LLOs with acceptors present. In contrast, LLO hydrolysis by STT3A-OST was negligible. STT3A-OST however may be regulatory, because it suppressed STT3B-OST–dependent fOSs. TREX1, a negative innate immunity factor that diminishes immunogenic fOSs derived from LLOs, acted through STT3B-OST as well. In summary, only STT3B-OST hydrolyzes LLOs, depending upon LLO quality and acceptor site occupancy. TREX1 and STT3A suppress STT3B-OST–dependent fOSs. Without strict kinetic limitations during posttranslational N-glycosylation, STT3B-OST can thus moonlight for LLO hydrolysis. In contrast, the STT3A-OST/translocon complex preserves LLOs for temporally fastidious cotranslational N-glycosylation.

scholarly journals Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Harriet Lane-Serff ◽  
Paula MacGregor ◽  
Edward D Lowe ◽  
Mark Carrington ◽  
Matthew K Higgins
Keyword(s):  
Innate Immunity ◽  
Trypanosoma Brucei ◽  
Molecular Basis ◽  
Structural Basis ◽  
Β Subunit ◽  
Trypanosome Infection ◽  
Lateral Mobility ◽  
African Trypanosomes ◽  
Distal Half ◽  
Immunity Factor

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

scholarly journals Evolution-Guided Identification of Antiviral Specificity Determinants in the Broadly Acting Interferon-Induced Innate Immunity Factor MxA

2012 ◽  
Vol 12 (4) ◽  
pp. 598-604 ◽  
Author(s):  
Patrick S. Mitchell ◽  
Corinna Patzina ◽  
Michael Emerman ◽  
Otto Haller ◽  
Harmit S. Malik ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Immunity Factor ◽  
Specificity Determinants

scholarly journals Cation channel conductance and pH gating of the innate immunity factor APOL1 are governed by pore-lining residues within the C-terminal domain

2020 ◽  
Vol 295 (38) ◽  
pp. 13138-13149 ◽  
Author(s):  
Charles Schaub ◽  
Joseph Verdi ◽  
Penny Lee ◽  
Nada Terra ◽  
Gina Limon ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Cation Channel ◽  
Planar Lipid Bilayers ◽  
Channel Formation ◽  
Cation Selectivity ◽  
Terminal Domain ◽  
Immunity Factor ◽  
Pore Lining

The human innate immunity factor apolipoprotein L-I (APOL1) protects against infection by several protozoan parasites, including Trypanosoma brucei brucei. Endocytosis and acidification of high-density lipoprotein–associated APOL1 in trypanosome endosomes leads to eventual lysis of the parasite due to increased plasma membrane cation permeability, followed by colloid-osmotic swelling. It was previously shown that recombinant APOL1 inserts into planar lipid bilayers at acidic pH to form pH-gated nonselective cation channels that are opened upon pH neutralization. This corresponds to the pH changes encountered during endocytic recycling, suggesting APOL1 forms a cytotoxic cation channel in the parasite plasma membrane. Currently, the mechanism and domains required for channel formation have yet to be elucidated, although a predicted helix-loop-helix (H-L-H) was suggested to form pores by virtue of its similarity to bacterial pore-forming colicins. Here, we compare recombinant human and baboon APOL1 orthologs, along with interspecies chimeras and individual amino acid substitutions, to identify regions required for channel formation and pH gating in planar lipid bilayers. We found that whereas neutralization of glutamates within the H-L-H may be important for pH-dependent channel formation, there was no evidence of H-L-H involvement in either pH gating or ion selectivity. In contrast, we found two residues in the C-terminal domain, tyrosine 351 and glutamate 355, that influence pH gating properties, as well as a single residue, aspartate 348, that determines both cation selectivity and pH gating. These data point to the predicted transmembrane region closest to the APOL1 C terminus as the pore-lining segment of this novel channel-forming protein.

scholarly journals Crystal structure of the mouse innate immunity factor bacterial permeability-increasing family member A1

2018 ◽  
Vol 74 (5) ◽  
pp. 268-276 ◽  
Author(s):  
Michael S. Little ◽  
Matthew R. Redinbo
Keyword(s):  
Crystal Structure ◽  
Innate Immunity ◽  
Ion Channel ◽  
Family Member ◽  
Mus Musculus ◽  
Biological Function ◽  
Secreted Proteins ◽  
Upper Airways ◽  
Immunity Factor ◽  
Lipopolysaccharide Binding

Bacterial permeability-increasing family member A1 (BPIFA1) is an innate immunity factor and one of the most abundantly secreted proteins in the upper airways. BPIFA1 is multifunctional, with antimicrobial, surfactant and lipopolysaccharide-binding activities, as well as established roles in lung hydration. Here, the 2.5 Å resolution crystal structure of BPIFA1 from Mus musculus (mBPIFA1) is presented and compared with those of human BPIFA1 (hBPIFA1) and structural homologs. Structural distinctions between mBPIFA1 and hBPIFA1 suggest potential differences in biological function, including the regulation of a key pulmonary ion channel.

scholarly journals A Cytosolic Reductase Pathway is Required for Efficient N-Glycosylation of an STT3B-Dependent Acceptor Site

2021 ◽  
Author(s):  
Marcel van Lith ◽  
Marie Anne Pringle ◽  
Bethany Fleming ◽  
Giorgia Gaeta ◽  
Jisu Im ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Site Occupancy ◽  
Secretory Protein ◽  
Glycosylation Site ◽  
Redox Conditions ◽  
In Vitro Translation ◽  
Translation System ◽  
Acceptor Site ◽  
Reductive Pathway

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A and STT3B-dependent glycosylation. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

scholarly journals Author response: Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor

2014 ◽  
Author(s):  
Harriet Lane-Serff ◽  
Paula MacGregor ◽  
Edward D Lowe ◽  
Mark Carrington ◽  
Matthew K Higgins
Keyword(s):  
Innate Immunity ◽  
Author Response ◽  
Structural Basis ◽  
Immunity Factor

Novel cross-talk within the IKK family controls innate immunity

2011 ◽  
Vol 434 (1) ◽  
pp. 93-104 ◽  
Author(s):  
Kristopher Clark ◽  
Mark Peggie ◽  
Lorna Plater ◽  
Ronald J. Sorcek ◽  
Erick R. R. Young ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Protein Kinases ◽  
Gene Transcription ◽  
Molecular Mechanisms ◽  
Interleukin 1 ◽  
Nuclear Factor Κb ◽  
Regulatory Subunit ◽  
Tumour Necrosis Factor Receptor ◽  
Catalytic Subunits ◽  
Physiological Substrates

Members of the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} family play a central role in innate immunity by inducing NF-κB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKϵ/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKβ, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKKϵ and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-κB essential modulator), which is associated with reduced IKKα/β activity and NF-κB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105, RelA (IKKα and IKKβ) and IRF3 (IKKϵ and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity.

scholarly journals A Cytosolic Reductase Pathway is Required for Complete N-Glycosylation of an STT3B-Dependent Acceptor Site

2021 ◽  
Author(s):  
Marcel van Lith ◽  
Marie Anne Pringle ◽  
Bethany Fleming ◽  
Giorgia Gaeta ◽  
Jisu Im ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protein Translocation ◽  
Site Occupancy ◽  
Secretory Protein ◽  
Glycosylation Site ◽  
Redox Conditions ◽  
Translation System ◽  
Acceptor Site ◽  
Post Translational Modification

AbstractN-linked glycosylation of proteins entering the secretory pathway is an essential post-translational modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide bond, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is dependent on the STT3A-containing oligosaccharide transferase. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

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