N-glycan LLO assembly II — ER-lumenal face + glucosylation (to Glc3Man9GlcNAc2-PP-dolichol); ALG3/ALG9/ALG12/ALG6/ALG8/ALG10

After the Man5GlcNAc2-PP-dolichol intermediate is flipped from the cytoplasmic to the lumenal face of the endoplasmic-reticulum membrane (by the RFT1 flippase), the dolichol-linked oligosaccharide (LLO) is completed inside the ER lumen to the mature Glc3Man9GlcNAc2-PP-dolichol donor for protein N-linked glycosylation. Unlike the cytoplasmic-face steps, the lumenal glycosyltransferases use lipid-linked donors: dolichyl-phosphate-mannose (Dol-P-Man) for the mannose additions and dolichyl-phosphate-glucose (Dol-P-Glc) for the glucose additions. Four mannoses are added: ALG3 (alpha-1,3) adds the sixth mannose, ALG9 (alpha-1,2) adds the seventh and, later, the ninth mannose, and ALG12 (alpha-1,6) adds the eighth, giving Man9GlcNAc2-PP-dolichol. Three glucoses then cap the glycan: ALG6 (alpha-1,3) adds the first glucose, ALG8 (alpha-1,3) the second, and ALG10 (alpha-1,2) the third and terminal glucose, producing the mature Glc3Man9GlcNAc2-PP-dolichol. The terminal glucose is the recognition signal for efficient transfer of the glycan to nascent proteins by the oligosaccharyltransferase (and, after transfer, for glucosidase trimming and calnexin/calreticulin quality control). Inherited defects in each step cause a congenital disorder of glycosylation (CDG type I): ALG3-CDG (CDG-Id), ALG9-CDG (CDG-Il / Gillessen-Kaesbach-Nishimura syndrome), ALG12-CDG (CDG-Ig), ALG6-CDG (CDG-Ic, one of the commonest CDG-I subtypes) and ALG8-CDG (CDG-Ih).