M-type lectins
The thiodisaccharide substrate analogue is shown in green and the Ca2+ ion in dark blue. Protein Data Bank structure ID: 1X9D.
N-linked glycan structures in ERAD
Introduction
The M-type lectins are members of the glycoside hydrolase family 47 protein structural group. They are closely related to the the alpha-mannosidases of the ER and cis-Golgi and function alongside these proteins in the handling of N-linked glycoproteins. M-type lectins are type II transmembrane proteins with very short cytoplasmic tails (although in some cell types the expressed protein may lack the transmembrane domain). In common with the ligand/substrate binding domains of other glycoside hydrolase family 47 proteins, such as human ER mannosidase, left, the M-type CRD is a barrel-like structure with both alpha-helices and beta-sheets. Some of the M-type lectins have C-terminal extensions after the M-type CRD that are not related to known protein folds.
Evolution and function of alpha-mannosidases
Humans have four alpha-mannosidases: ER mannosidase and Golgi mannosidases IA, IB and IC (gene names Man1B1, Man1A1, Man1A2 and Man1C1 respectively). ER mannosidase mediates the first mannose trimming reaction in the processing of N-linked glycans, reducing a Man9GlcNAc2 oligosaccharide to Man8GlcNAc2 by removal of a specific terminal mannose residue (see diagram, left). ER mannosidase is conserved in eukaryotes; the yeast protein is known as Mns1. The trio of cis-Golgi mannosidases performs further specific trimming reactions on the Man8GlcNAc2 oligosaccharide, reducing the glycan to a Man5GlcNAc2 core that may be elaborated upon by a number of glycosyltransferases in the medial and trans-Golgi. The three Golgi mannosidases are conserved in vertebrates only and often have just a single homologue in invertebrate organisms.
Evolution of M-type lectins
The M-type lectins are related to the alpha-mannosidases but lack key catalytic residues, as well as a key disulphide bond thought to be essential for enzymatic activity. As a result they bind to high mannose glycans attached to glycoproteins in the ER lumen, but have no catalytic function. Like other intracellular lectin families, the M-type lectin family is modest in size. Mammals have three M-type lectins, EDEM1, EDEM2 and EDEM3 (ER-associated degradation-enhancing alpha-mannosidase-like proteins), and these are generally conserved in metazoa, although EDEM1 is missing in Drosophila for example. Yeast has a single M-type lectin, Mnl1, that is related to EDEM1. Different M-type lectins are found in plants.
M-type lectins in ER-associated glycoprotein degradation (ERAD)
The ER-associated degradation (ERAD) system enables glycoproteins that have remained unfolded or incorrectly folded in the ER lumen for an extended period of time to be exported to the cytoplasm for proteosomal degradation. The ERAD system is upregulated under stress conditions, when a greater proportion of proteins remain unfolded or misfolded, and serves to clear the secretory pathway of 'waste' proteins and thus prevent clogging. EDEM1 was the first M-type lectin established as having a role in recognizing unfolded or misfolded proteins in ERAD. More recently, EDEMs 2 and 3 have also been shown to accelerate ERAD, but EDEM3 is unique in retaining mannosidase activity, suggesting it enhances ERAD by a different mechanism to EDEMs 1 and 2.
The folding of glycoproteins in the ER lumen is assisted by chaperones including the calnexin family lectins. In the calnexin cycle of assisted protein folding, a glucosyltransferase enzyme distinguishes between folded and unfolded glycoproteins bearing Man9GlcNAc2 N-linked glycans. Proteins which have yet to fold correctly are re-glucosylated to signal that re-binding to calnexin is necessary. Proteins which have achieved the correct folded state remain deglucosylated as a signal that they are ready to proceed to the Golgi. Deglucosylated glycoproteins are substrates for ER mannosidase, a slower-acting enzyme which trims a specific terminal mannose residue from Man9GlcNAc2 glycans to generate a Man8GlcNAc2 structure (see diagram, left). Mannose trimming is an essential part of normal glycoprotein maturation in the ER, and is the final glycan processing step before glycoproteins are transported to the Golgi. However, during the calnexin cycle, unfolded proteins also spend some time deglucosylated, when they can be acted on by ER mannosidase. Trimming of mannose from a Man9GlcNAc2 glycan on an unfolded glycoprotein is the signal that the glycoprotein should be exported from the ER to the cytoplasm for degradation, and concordantly inhibits further participation of the glycoprotein in the calnexin cycle.
The slower rate of action of ER mannosidase, relative to the components of the calnexin cycle, means that only proteins which have failed to fold correctly after multiple attempts are targeted for degradation. Some proteins which exhibit delayed folding, either as an intrinsic property or as the result of a mutation (in cystic fibrosis, for example), are therefore targeted by ERAD, resulting in reduced levels of protein expression. All glycoproteins - folded or unfolded - bear a Man8GlcNAc2 glycan following ER mannosidase processing, so ERAD receptors are required to distinguish between folded and unfolded glycoproteins bearing this structure, in order to trigger degradation of the unfolded ones only. The EDEM proteins are believed to fulfil this role, probably alongside other proteins such as the P-type lectin OS-9. In a modification to the scheme depicted (left), deglucosylation may occur while a glycoprotein is bound to calnexin, allowing direct handover of unfolded glycoproteins from calnexin to EDEM; calnexin and EDEM1 are known to associate via transmembrane domain interactions. EDEMs in turn hand over bound glycoproteins to the translocon machinery for retrotranslocation into the cytoplasm, where the glycoproteins are recognized by proteins including F-box lectins and targeted for proteasomal degradation. That the protein generating the Man8GlcNAc2 degradation signal (ER mannosidase) and the proteins recognizing the signal (EDEMs) are structurally related points to concurrent evolution of both aspects of the ERAD system in a very early eukaryotic ancestor.
Ligand binding
The structure of ER mannosidase with a bound substrate analogue suggests that the ligand binding site in M-type CRDs is a deep cleft at one end of the barrel. This unusually deep binding site for a lectin allows selective interaction with high mannose glycans rather than just terminal residues.
