Quantitative glycoproteomics reveals cellular substrate selectivity of the endoplasmic reticulum protein quality control sensors UGGT1 and UGGT2

The protein quality control sensors UDP-glucose: glycoprotein glucosyltransferase (UGGT) 1 and 2 are proposed to act as gatekeepers of the early secretory pathway. They initiate rebinding to the carbohydrate-dependent chaperones calnexin and calreticulin that associate with proteins possessing monoglucosylated glycans. The UGGTs control glycoprotein exit from the endoplasmic reticulum (ER) for trafficking to the Golgi or ER retention to provide additional folding opportunities. A quantitative glycoproteomics strategy was used to identify cellular glycoproteins modified by the UGGTs at endogenous levels and delineate the specificities of UGGT1 and UGGT2. UGGT substrates were comprised of seventy-one mainly large multidomain and heavily glycosylated proteins when compared to the general N-glycome. UGGT1 was the dominant glucosyltransferase with a preference towards large plasma membrane proteins whereas UGGT2 favored the modification of smaller, soluble lysosomal proteins. This study provides insight into the cellular secretory load that utilizes multiple rounds of carbohydrate-dependent chaperone intervention for proper maturation. Overall design: 5 different cell lines are analyzed (ALG6-/-, ALG6/UGGT1-/-, ALG6/UGGT2-/-, ALG6/UGGT1/2-/-, WT. Each sample has 3 biological replicates. Each biological replicate has 4 technical replicates. WT is used as a reference set.