Dataset Information

Glycosylation Limits Forward Trafficking of the Tetraspan Membrane Protein PMP22

ABSTRACT: Peripheral myelin protein 22 (PMP22) is a tetraspan integral membrane protein for which mistrafficking-causing mutations are linked to the inherited peripheral neuropathy, Charcot-Marie-Tooth disease (CMTD). Wild type (WT) PMP22 is an inefficient folder with ~20% of the protein trafficking to the plasma membrane. We discovered that N-linked glycosylation significantly limits forward trafficking of WT and disease variants of PMP22. N-glycosylation of WT PMP22 was found to occur primarily post-translationally. Glycosylation inhibition dramatically increased PMP22 trafficking efficiency. Quantitative proteomics identified novel PMP22 interacting proteins that may impact trafficking. Our results suggest that critical quality control decisions for unstable L16P PMP22 occur at earlier stages in the trafficking pathway than for the WT protein. Knock-out cell lines of likely PMP22 interactors led to the discovery that calnexin limits trafficking of stable PMP22 variants, UGGT1 promotes trafficking and RER1 limits trafficking of all PMP22 variants. This work establishes N-glycosylation as a key determinant of PMP22 retention in the ER, ultimately limiting forward surface-trafficking.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Kidney

DISEASE(S): Charcot-marie-tooth Disease

SUBMITTER: Madison Wright

LAB HEAD: Charles R. Sanders

PROVIDER: PXD023091 | Pride | 2021-04-27

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20181214_Marinko_SandersLab_PMP22_IP_TMT6plex_.msf	Msf
	20181214_Marinko_SandersLab_PMP22_IP_TMT6plex_0C.raw	Raw
	20181214_Marinko_SandersLab_PMP22_IP_TMT6plex_100C.raw	Raw
	20181214_Marinko_SandersLab_PMP22_IP_TMT6plex_10C.raw	Raw
	20181214_Marinko_SandersLab_PMP22_IP_TMT6plex_30C.raw	Raw

Items per page:

1 - 5 of 28

Similar Datasets

Project description:The amino acid sequences of immunodominant domains of hemagglutinin (HA) on the surface of influenza A virus (IAV) evolve rapidly, producing viral variants. HA mediates receptor recognition, binding and cell entry, and serves as the target for IAV vaccines. Glycosylation, a post-translational modification that places large, branched polysaccharide molecules on proteins, can modulate the function of HA, and can shield antigenic regions allowing for viral evasion from immune responses. Our previous work showed that subtle changes in the HA protein sequence can have a measurable change in glycosylation. Thus, being able to measure quantitatively how glycosylation changes in viral variants is critical for understanding how HA function may change throughout viral evolution. Moreover, understanding quantitatively how the choice of viral expression systems affects glycosylation can help in the process of vaccine design and manufacture. Although IAV vaccines are most commonly expressed in chicken eggs, cell-based vaccines have many advantages and the adoption of more cell-based vaccines would be an important step in mitigating seasonal influenza and protecting against future pandemics. Here, we have investigated the use of data-independent acquisition (DIA) mass spectrometry for quantitative glycoproteomics. We found that DIA improved the sensitivity of glycopeptide detection for four variants of A/Switzerland/9715293/2013 IAV: WT and mutant, each expressed in chicken eggs and MDCK cells. We used the Tanimoto similarity metric to quantify changes in glycosylation between WT and mutant, and between egg-expressed and cell-expressed virus. Our DIA site-specific glycosylation similarity comparison of WT and mutant expressed in eggs confirmed our previous analysis while achieving greater depth of coverage. We found that sequence changes resulting from different viral expression systems affected distinct glycosylation sites of HA. Our methods can be applied to track glycosylation changes in circulating IAV variants to bolster the genomic surveillance already being done, for a more complete understanding of IAV evolution.

Dataset Information

Glycosylation Limits Forward Trafficking of the Tetraspan Membrane Protein PMP22

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets