N-glycoproteomics reveals distinct glycosylation alterations in NGLY1-deficient patient-derived dermal fibroblasts

Rohit Budhraja, Mayank Saraswat, Diederik De Graef, Wasantha Ranatunga, Madan G. Ramarajan, Jehan Mousa, Tamas Kozicz, Akhilesh Pandey, Eva Morava

Research output: Contribution to journalArticlepeer-review


Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated. We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell-derived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 N-glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG.

Original languageEnglish (US)
Pages (from-to)76-91
Number of pages16
JournalJournal of inherited metabolic disease
Issue number1
StatePublished - Jan 2023


  • NGLY1
  • congenital disorder of deglycosylation
  • congenital disorder of glycosylation
  • glyco
  • mass spectrometry
  • proteomics
  • proteomics

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)


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