Intracellular functions of N-glycans

Immediately after transfer to Asn residues on glycoproteins, membrane anchored glucosidase-I trims oligosaccharides to GlcNAc₂Man₉Glc₂. During glycoprotein folding, the second and third Glc residues are cleaved by the soluble glucosidase-II enzyme whereas soluble UDP-Glc:glycoprotein Glc-transferase (UGGT) transfers back a Glc residue to GlcNAc₂Man₉ on misfolded glycoproteins. This cycle of glucosylation-deglucosylation is part of the quality control of glycoprotein folding and enables interaction of misfolded glycoproteins with the Glc-specific lectins calnexin and calreticulin. These lectins are associated with the thiol-disulfide oxidoreductase ERp57, which assist in refolding glycoproteins.

Figure 33. Quality control of N-glycoprotein folding in the ER. Mono-glucosylated N-glycoproteins interact with the lectins calreticulin (CRT) and calnexin (CNX, not shown), which also bind to the oxidoreductase chaperone ERp57. Glucosidase-II (PPKCSH) cleaves the terminal Glc residue on folded and unfolded proteins. The latter are re-glucosylated by UDP-Glc:glycoprotein Glc-transferase (UGGT), which enables CRT/CNX binding and additional rounds of ERp57-mediated folding. After cleavage of the inner terminal Man residue, folded N-glycoproteins are bound by trafficking proteins such as ERGIC53 and transported out of the ER.

The transition from GlcNAc₂Man₉ to GlcNAc₂Man₇ represents another signal for the intracellular trafficking of glycoproteins. The ER mannosidase-I enzyme cleaves the terminal Man residue of the inner branch of N-glycans, yielding GlcNAc₂Man₈. Properly folded glycoproteins will leave the ER carrying this GlcNAc₂Man₈ glycan. If the glycoprotein remains unfolded, a second mannosidase called HTM1 in yeasts (EDEM1 to EDEM3 in mammals) will cleave terminal Man from the outer α1-6 linked branch yielding GlcNAc₂Man₈, which is recognized by the Yos9p lectin (OS-9 in mammals). Yos9p/OS-9 is associated with the HRD ubiquitin ligase complex involved in the retro-translocation of misfolded proteins out of the ER and of their degradation by the proteasome complex in the cytosol.

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After leaving the ER, glycoproteins are transferred to the ER-Golgi intermediary compartment (ERGIC). This process is in part mediated by the N-glycan lectin ERGIC53, which is also abbreviated LMAN1 for lectin, mannose-binding, 1. Deficiency of ERGIC53/LMAN1 causes a bleeding disorder (OMIM 227300) characterized by the defective secretion of coagulation factors V and VIII, which are N-linked glycoproteins. N-glycans also carry the Man-6-P signal mediating the targeting of proteins to lysosomes. This lysosomal targeting signal is produced in two steps, consisting first of the addition of phospho-GlcNAc to Man residues on N-glycans by the Golgi-localized GlcNAc-1-phosphotransferase complex. This enzyme is a hexamer comprising two α-, two β-, and 2 γ-subunits, where the α- and β-subunits are catalytically active and ensure the recognition of the lysosomal protein substrates. The contribution of the g-subunits are unclear, but required for the full activity of the complex. Mutations in the GNPTAB gene encoding the polypeptide yielding the α- and β-subunits cause I-cell disease (OMIM 252500) and pseudo-Hurler polydystrophy (OMIM 252600), which are also classified as mucolipidosis II and mucolipidosis III. Mutations in GNPTG encoding the γ-subunit cause a variant form of mucolipidosis III (OMIM 252605). After transfer of phospho-GlcNAc to Man, an uncovering enzyme localized in the trans Golgi network removes GlcNAc, thereby leaving the phosphate group at the 6’-position of Man. The Man-6-P signal is recognized by cognate Man-6-P receptors, which capture the glycoproteins bearing the signal and transports them to the lysosomes. The presence of Man-6-P receptors at the plasma membrane enables the treatment of defective lysosomal enzymes by intravenous administration of Man-6-P bearing recombinant enzymes.