The N-glycoproteins folding pathway

Step 1Assembly of the precursor complex,Glc3Man9GlcNAc2

The assembly of the precursor complex

Flippase

OST

The N-glycoproteins folding pathway

Step 2Transfer of Precursor onto the Asn-X-Thr motif on the emerging protein

The N-glycoproteins folding pathway

Step 3Trimming of the first two glucose residues by ER glucosidase I and II

The N-glycoproteins folding pathwayStep 4The protein enters the Calnexin/Calreticulum cycle

The Calnexin/Calreticulum cycle

The N-glycoproteins folding pathway

Step 5ASecretion of Mature proteins to Golgi

Step 5BDegradation of terminally misfolded proteins through ER-Associated Degradation (ERAD)

ER-Associated Degradation (ERAD)

Fate of Free Oligosaccharides (FOS)

Effect of NB-DNJ on the N-glycoprotein folding pathway

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

R&D – Lipid Linked OligosaccharidesSpecies Detected

Control

1mM NB-DNJ

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

R&D – Intracellular Free OligosaccharidesSpecies Detected

Control

1mM NB-DNJ

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

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?

Control Cells

Medium only

24 hours

36 hours

48 hours

72 hours

96 hours

1mMNB-DNJ

Medium only

24 hours

36 hours

48 hours

72 hours

96 hours

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??

R&D – Protein-linked Oligosaccarides (Intracellular)Species Detected

Control

1mM NB-DNJ

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….

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

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??

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

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…

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%

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%