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

Abstract

UDP-glucose: glycoprotein glucosyltransferase (UGGT) 1 and 2 are central hubs in the chaperone network of the endoplasmic reticulum (ER), acting as gatekeepers to the early secretory pathway yet little is known about their cellular clients. These two quality control sensors control lectin chaperone binding and glycoprotein egress from ER. A quantitative glycoproteomics strategy was deployed to identify cellular substrates of the UGGTs at endogenous levels in CRISPR-edited HEK293 cells. The seventy-one UGGT substrates identified were 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 sheds light on differential specificities and roles of UGGT1 and UGGT2 and provides insight into the cellular reliance on carbohydrate-dependent chaperone intervention

Effects of the investigational drug sodium phenylbutyrate-TUDCA (AMX0035) on the transcriptional and metabolic landscape of sporadic ALS fibroblasts

“…Another major class of pathways enriched in genes from the vehicle Black module was intracellular localization/transport (Fig 5F). Furthermore, individual Black module genes that had the strongest correlations with disease traits included SELENOO , a mitochondrial redox-sensitive selenoprotein (65), LONP1 , a mitochondrial protease that degrades misfolded or oxidatively damaged proteins (66), UGGT1 , which recognizes unfolded glycoproteins in the ER (67), ORAI1 , an essential component of ER store-operated calcium entry (68), and TRAM2 and TMEM147 , members of the ER translocon complex (69) (Table S3). Finally, the most commonly identified transcription factors with binding sites enriched in genes from the Black module were Elk1, E2F1, Elf1, Sp1, and Erg1 (Table S11), the same transcription factors identified in the differential expression analysis.…”

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

“…The ALG6 -/-HEK293-6E cells were treated with increasing concentrations of 5M-8OH-Qand -following incubation with the molecule -glucosylation of known UGGT substrate glycoproteins was analyzed by isolating monoglucosylated glycoproteins from the cell lysate. After GST-CRT precipitation, the eluate was probed for two known substrates of UGGT: the proprotein of human IFGR1 (ProIFGR1, a UGGT1 substrate (35)) and the proprotein of HexB (ProHexB, a UGGT2 substrate (35)) and their glucosylation levels quantified. The amount quantified in each GST-CRT pulldown was divided by the total amount found within the sample's whole cell lysate (WCL), resulting in the percent glucosylation at that dose of 5M-8OH-Q (35).…”

“…After GST-CRT precipitation, the eluate was probed for two known substrates of UGGT: the proprotein of human IFGR1 (ProIFGR1, a UGGT1 substrate (35)) and the proprotein of HexB (ProHexB, a UGGT2 substrate (35)) and their glucosylation levels quantified. The amount quantified in each GST-CRT pulldown was divided by the total amount found within the sample's whole cell lysate (WCL), resulting in the percent glucosylation at that dose of 5M-8OH-Q (35).…”

“…Next, we asked whether 5M-8OH-Q inhibits both human paralogues of UGGT (UGGT1 and UGGT2 (35,40,41)). Double knock-out (KO) cells (ALG6/UGGT1 -/and ALG6/UGGT2 -/-(35))…”

Glycan quality control in and out of the endoplasmic reticulum of mammalian cells

“…The innermost glucose residue is removed by the ER glucosidase II to generate non-glucosylated glycans (Man 9 GlcNAc 2 , Figure 2, step VI) at much slower rates than the rate of removal of the middle glucose residue [43], driving the dissociation of glycoproteins from CNX and CRT. If glycoproteins are still unfolded, the non-glucosylated glycans are re-glucosylated by UDP-glucose:glycoprotein glucosyltransferases (UGGTs) [44][45][46][47][48][49][50] and recognized again by CNX and CRT (Figure 2, step VII) [51]. This CNX/CRT cycle is a system that functions for the productive folding of glycoproteins but the cycle can be terminated when the client glycoproteins are recognized as being terminally misfolded.…”