We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.
The following protocol is intended for analysis of sugar chains of total glycoproteins or of a specific glycoprotein of interest. The procedure is basically the same for both cases with a few alterations as stated throughout the protocol.
Chase (h) | 0 | 4 |
Released with endo H (cpm) a | 90000 | 16800 |
Released with N-glycosidase F (cpm) b | 20400 | 3900 |
Glycoproteins in retentate (cpm) c | 371700 | 127695 |
Dolichol-oligosaccharides in retentate (cpm) d | 4350 | 540 |
Dolichol-oligosaccharides in retentate (%) e | 1.4 ± 0.7 | 0.2 ± 0.2 |
Table 1. Analysis of release of N-linked sugar chains with deglycosidases and of the presence of dolichol-oligosaccharides in glycoprotein samples.
We applied the pulse-chase procedure to a lysate from NIH 3T3 cells. After Microcon filtration, labeled N-linked oligosaccharides were released mainly with endo H after pulse labeling (high mannose type) and with further treatment with N-glycosidase F after a chase period, which would correspond to complex type (Golgi-processed glycans), endo H resistant oligosaccharides. This quantitative analysis determined that dolichol-oligosaccharides, accounted for an insignificant fraction of the label in the initial retentate.
Notes:
Representative Results
Figure 1. Pulse-chase analysis of total NIH 3T3 glycoproteins in untreated cells and upon proteasomal inhibition. After 1h pulse-labeling with [2-3H] we obtained an expected profile with some glucosylated precursors remaining (Glc2Man9GlcNAc2 (G2M9) and G1M9), but most of the label present in M9 and M8 species, the latter being the result of trimming of all the glucose and one mannose residue (Figure 1 A). No free mannose or other small precursors were detected, attesting to the thoroughness of the purification. Following a rather long 8h chase there was more extensive trimming to M7-6 and a small amount of M5, but the major species continued to be M9 and M8 (Figure 1 B). If the chase was done in the presence of a proteasomal inhibitor (30 μM MG-132), there was only a minor accumulation of these same trimmed species (Figure 1 C).
Figure 2. Quantitative analysis of sugar chains of an ERAD substrate compared to total glycoproteins. (A) In an experiment similar to the one in Figure 1, relative molar amounts of each oligosaccharide species were calculated based on mannose content. We converted the cpm values obtained for each glycan species, the percent of each species relative to the total sum of the relative molar amounts of all species present was then plotted as a function of chase time for an average of two experiments. (B) Similar to (A) except that glycans released from the ERAD substrate ASGPR H2a were analyzed after the pulse labeling and chase for up to 4h, in the presence or absence of the proteasomal inhibitor MG-132. (C) Values for 4h chase in the presence of MG-132 from (A) and (B) are compared for ASGPR H2a and the glycoprotein pool. (D) Scheme of N-linked sugar chain trimming processes in the ER. Sugar trimming processes that lead to M6-5 linked to proteins (R) that are targeted to ERAD compared to M9-8 on those that exit to the Golgi and beyond. The results indicate that the ERAD process is associated with the mannose trimming of N-glycans to yield species with 5-6 mannose residues remaining.
The pulse-chase analysis of glycans in live cells with HPLC separation provides a method for studying the dynamics of oligosaccharide structural alterations throughout the life of a glycoprotein. There is growing evidence that such alterations are involved in producing the signals for ER folding, quality control and trafficking systems 2-5. The method can be applied not only for a specific glycoprotein of interest but also for analyzing the dynamics of the structure of total glycoproteins, as illustrated in this article, where we compared the contrasting fate of a specific ERAD substrate and that of the cellular glycoprotein pool. The purification technique is simple but nonetheless specific. By using strict deglycosidases and molecular filtration it is ensured that only N-linked glycans released from glycoproteins are obtained, leaving behind other macromolecules labeled with [2-3H]Man, such as O-linked sugar chains and GPI-anchored proteins. Oligosaccharides released from the dolichol precursor are a negligible contaminant (Table 1).
The method provides a high sensitivity of detection using a very small amount of starting material. When expecting a very low yield, the sensitivity can be further increased by reducing the volume of the HPLC fractions using SpeedVac. Thus, the solubility limitation of the scintillation fluid can be overcome, and the entire content of the fractions can be monitored in the scintillation counter.
We thank Zehavit Frenkel and Sandra Tolchinsky for technical assistance. Research related to this work is supported by grants from the Israel Science Foundation (1229/07) and German-Israeli Project Cooperation (DIP-DFG).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
600E Multisolvent delivery System controller | Waters | WAT062710 | ||
Acetonitrile LiChrosolv (gradient grade for liquid chromatography) | Merck Bioscience | 1.0003 | ||
Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30 | Millipore | UFC503024 or 4208 | ||
Concentrator 5301, incl. 48 x 1.5 / 2.0 ml fixed-angle rotor | Eppendorf | 5301 000.016 | ||
Dialyzed Foetal Calf Serum | Biological Industries | 04-011-1A | ||
Dulbecco’s Modified Eagle’s Medium | Gibco Invitrogen Cell Culture | 41965039 | ||
Dulbecco’s Modified Eagle’s Medium – glucose free | Sigma-Aldrich | D5030 | ||
Dulbecco’s Phosphate Buffered Saline (PBS) | Sigma-Aldrich | D1408 | ||
EDTA disodium salt (Titriplex Iii) | Merck | 1084180250 | ||
Endo Hf Kit (1,000,000 units/ml) | New England Biolabs | p0703S | ||
Foetal Calf Serum (FCS) | Biological Industries | 04-001-1A | ||
Frac-100 fraction collector | Amersham Biosciences | 18-1000-77 | ||
LS 6500 Liquid Scintillation Counting systems | Beckman Coulter | 510720 | ||
Mannose, D-[2-3H(N)]- (Specific Activity: 15-30Ci/mmol) | PerkinElmer Life Sciences | NET570A | ||
N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132) | Calbiochem | 474790 | ||
N-glycosidase F (1000 units/ml) | Roche | 11365177 | ||
N-octylglucoside | Sigma-Aldrich | O3757 | ||
NIH 3T3 cells | American Type Culture Collection (ATCC) | CRL-1658 | ||
Opti-Flour | PerkinElmer Life Sciences | 6013199 | ||
Phosphoric acid solution ( 49-51%, for HPLC) | Fluka | 79607 | ||
Protease Inhibitor Cocktail | Sigma-Aldrich | P2714 | ||
Protein A-Sepharose beads | Repligen | IPA300 | ||
Rabbit polyclonal anti-H2 carboxy-terminal 6 | ||||
Sodium deoxycholate | Sigma-Aldrich | 30970 | ||
Sodium dodecyl sulfate | Bio-RAD | 161-0301 | ||
Sodium phosphate | Sigma-Aldrich | 342483 | ||
Sodium pyruvate solution (100mM) | Sigma-Aldrich | S8636 | ||
Spherisorb NH2 Column, 5 μm, 4.6 x 250 mm | Waters | PSS831115 | ||
Triton X-100 | BDH | 306324N | ||
Vibra-Cell ultrasonic processors VCX 750 | Sonics and Materials, Inc. | 690-003 | ||
Buffer A: 1% Triton X-100, 0.5% w/v sodium deoxycholate, Protease inhibitor cocktail 2% v/v in PBS | ||||
Buffer B: 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS | ||||
Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside | ||||
NIH 3T3 stably expressing H2a 6 | ||||
Buffer D: 0.5% Triton X-100, 0.25% w/v sodium deoxycholate, 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS | ||||
Buffer E: 0.5% w/v SDS, 1% v/v 2-Mercaptoethanol, Protease inhibitor cocktail 2% v/v in PBS | ||||
2-mercaptoethanol | Sigma-Aldrich | M3148 |