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The N-glycoproteins folding pathway
Step 1Assembly of the precursor complex,Glc3Man9GlcNAc2
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The assembly of the precursor complex
Flippase
OST
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The N-glycoproteins folding pathway
Step 2Transfer of Precursor onto the Asn-X-Thr motif on the emerging protein
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The N-glycoproteins folding pathway
Step 3Trimming of the first two glucose residues by ER glucosidase I and II
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The N-glycoproteins folding pathwayStep 4The protein enters the Calnexin/Calreticulum cycle
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The Calnexin/Calreticulum cycle
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The N-glycoproteins folding pathway
Step 5ASecretion of Mature proteins to Golgi
Step 5BDegradation of terminally misfolded proteins through ER-Associated Degradation (ERAD)
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ER-Associated Degradation (ERAD)
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Fate of Free Oligosaccharides (FOS)
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Effect of NB-DNJ on the N-glycoprotein folding pathway
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Project AimTo investigate the effect of NB-DNJ on the following glycan
pool:1. Lipid-linked oligosaccharides (Dolichol)2. Intracellular and Extracellular FOS3. Intracellular and Extracellular Protein-linked oligosaccharides
(PLO)
To investigate the amount of protein misfolding related to N-linked glycosylation in one individual HL60 cell, in 24 hours
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R&D – Lipid Linked OligosaccharidesSpecies Detected
Control
1mM NB-DNJ
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R&D – Lipid Linked OligosaccharidesQuantitative
Amount of oligosaccharides(pmol/mg of proteins)
Standard Deviation
Control Cells (without NB-DNJ) 3.17 1.62
Treated Cells (with 1mM NB-DNJ) 3.20 0.39
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R&D – Intracellular Free OligosaccharidesSpecies Detected
Control
1mM NB-DNJ
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R&D – Intracellular FOSQuantitative
Amount of oligosaccharide(pmol/mg of protein)
Standard Deviation
Control Cells (without NB-DNJ) 180.91 5.71
Treated Cells (with 1mM NB-DNJ) 509.44 30.03
Cellular Fractionation•To identify the origin of these intracellular FOS•Result:• 50% from Cytosol• 10% from Lysosome• The rest (40%) from other cell compartments
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R&D – Extracellular Free Oligosaccharides
In 24 hours, no FOS were detected in the extracellular medium by our methods
Suggesting FOS were retained in the cells for the first 24 hours
Next Question…
How long does the cell retain the FOS before discharging them into the extracellular medium?
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Control Cells
Medium only
24 hours
36 hours
48 hours
72 hours
96 hours
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1mMNB-DNJ
Medium only
24 hours
36 hours
48 hours
72 hours
96 hours
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Conclusion
In both control and treated cells, the cell retains FOS species for at least 72 hours
96 hours intracellular v.s. extracellular FOS
R&D – Extracellular Free Oligosaccharides
Does this suggest that the excretion system
favours G3M4N over G3M5N??
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R&D – Protein-linked Oligosaccarides (Intracellular)Species Detected
Control
1mM NB-DNJ
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R&D – Protein-Linked Oligosaccharides (Intracellular)Quantitative
Amount of oligosaccharides(pmol/mg of proteins) Standard Deviation
Control Cells (without NB-DNJ) 42.70 11.65
Treated Cells (with 1mM NB-DNJ) 61.55 44.51
BUT….
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R&D – Protein-Linked Oligosaccharides (Intracellular)Quantitative (corrected)
Amount of oligosaccharides(pmol/mg of proteins) Standard Deviation
Control Cells (without NB-DNJ) 128.1 11.65
Treated Cells (with 1mM NB-DNJ) 184.65 44.51
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Bottled Medium were de-glycoproteinated by passage through a Concanavalin A (Con-A)-Sepharose 4B column
PLO extracted as per normalResults:
No PLO can be detected after 24 hours incubation (with and without 1mM NB-DNJ)
Samples were concentrated by passage through an QAE-sephadex column Still, no PLO can be detected
R&D – Protein-Linked Oligosaccharides (Extracellular)
Does this suggest that no glycoproteins were secreted into the extracellular medium in 24 hours??
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Effect of NB-DNJ on the N-glycoprotein folding pathwayControl Cells 1mM NB-DNJ
LLO 3 major + 1 minor species
3.17 pmol/mg
1 major species
3.20 pmol/mg
Intracellular FOS 3 major + numerous glucosylated species
180.91 pmol/mg
Major species in control cells decreased in amount
Major species of Glc1Man5GlcNAc1 + minor peaks
509.44 pmol/mg
Extracellular FOS Retain in the cell for at least 72 hours
Intracellular PLO Numerous species identified
128.1 pmol/mg
Species in control cells still present, but decrease in amount
3 major glucosylated species identified
184.65 pmol/mg
Extracellular PLO Cannot be detected with the methods used
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Quantitative Measure of N-Glycoprotein misfolding
Objective: To investigate the amount of protein misfolding related to N-linked
glycosylation in one individual HL60 cell, in 24 hours
Total Amount of proteins = sum of all detected oligosaccharides in different oligosaccharide pool
Misfolded protein = Cytosolic FOS + Lysosomal FOS (60% of total FOS)
Therefore…
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Quantitative Measure of N-Glycoprotein misfolding
Dolichol pool
Total FOS (amount of
cytosolic and lysosomal FOS)
Glycoproteins* Total Amount
Amount of oligosaccharides (pmol/mg) in Control cells
3.17 180.91(108.54) 128.1 312.18
Amount of oligosaccharides (pmol/mg) in Treated cells
3.20 509.44(305.66) 184.65 697.29
By applying these data to the equation…
Control Cells34.7%
Treated Cells43.8%
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Conclusion Effects of NB-DNJ to:
LLO – Affect glycan structure but not the amount Intracellular FOS – Affect both qualitatively and quantitatively Intracellular PLO – Affect glycan structure but not the amount Extracellular FOS & PLO – cannot be detected in 24 hours
34.7% of N-glycoproteins are degraded through ERAD by a single HL60 cells over 24 hours
In the presence of 1mM NB-DNJ, the amount of N-glycoproteins degraded through ERAD increased to 43.8%