Sugar Chains Decoding the Functions of Glycans

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Tadashi Suzuki • Kazuaki Ohtsubo
Naoyuki Taniguchi
Editors

Sugar Chains
Decoding the Functions of Glycans
Editors
Tadashi Suzuki Kazuaki Ohtsubo
Glycometabolome Team, Systems Department of Analytical Biochemistry
Glycobiology Research Group Faculty of Life Sciences
RIKEN-Max-Planck Joint Research Center Kumamoto University
for Systems Chemical Biology Kumamoto, Japan
RIKEN Global Research Cluster
Wako, Saitama, Japan
Naoyuki Taniguchi
Disease Glycomics Team, Systems
Glycobiology Research Group
RIKEN-Max-Planck Joint Research Center
for Systems Chemical Biology
RIKEN Global Research Cluster
Wako, Saitama, Japan

ISBN 978-4-431-55380-9 ISBN 978-4-431-55381-6 (eBook)

DOI 10.1007/978-4-431-55381-6

Library of Congress Control Number: 2015931251

Springer Tokyo Heidelberg New York Dordrecht London

© Springer Japan 2015
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Preface

When the title of this book, Sugar Chains, was first proposed by the publisher, for a
moment I thought that it may sound a bit too non-scientific, but after pondering it,
I found that this may be a perfect title for the book, as it somewhat reflects how
glycoscience or glycobiology has been perceived by those who are non-experts. It is
just an enigmatic “sugar” thing (in a strict sense, it actually represents sucrose!)
attached to lipids or proteins that gives all of us, whether experts or not, a headache
because of its incredible structural diversity as well as the lack of simple analytical
methods.
It has been more than two decades since the word “glycobiology” was coined.
While this research area, due to its unique methodology not compatible with most
biochemists, has long been regarded as a very specialized field with limited interest,
the recent explosive research progress has provided countless examples of critical
roles for glycan chains in various important biological phenomena. For instance,
cell surface glycans can be regarded as a “face” of cells, and their structures are
known to change depending on developmental stages or environment. Therefore,
cell surface glycans are utilized for identification of stem cells such as induced
pluripotent stem (iPS) cells or embryonic stem (ES) cells, or as valuable biomarkers
in diagnosis/detection of cancer.
In this book, recent breakthrough results have been introduced regarding the
roles of glycans in quality control or intracellular trafficking of proteins, immunology,
viral infection, stem cell biology, neuroscience, and various diseases such as cancer,
diabetes, chronic obstructive pulmonary disease (COPD), muscular dystrophy, or
schizophrenia. In each chapter, outstanding “glyco-related” questions are also
posed, so that researchers not familiar with glycoscience will have a clearer idea
about what the future direction for further clarification of the role of glycans in
respective research fields will be. We are proud of the fact that quite an impressive
line-up of articles is gathered here. We do hope that this book will serve as a good
“textbook” especially for those who are not familiar with, but nevertheless inter-
ested in, sugar chains in diverse research fields.

v
vi Preface

We editors would like to thank Mr. Kaoru Hashimoto and Mr. Yasutaka
Okazaki (both of Springer Japan), for giving us the opportunity of editing this book;
Ms. Momoko Asawa and Ms. Yuko Matsumoto (both of Springer Japan); and Ms.
Kotoko Ueno (Glycometabolome Team, RIKEN) for their devoted help in the
editing process.

Wako, Japan Tadashi Suzuki (for the editors)

Contents

1 N-Glycans and Quality Control of Proteins ......................................... 1

Nobuko Hosokawa and Tadashi Suzuki
2 Glycan-Mediated Protein Transport
from the Endoplasmic Reticulum .......................................................... 21
Morihisa Fujita, Xiao-Dong Gao, and Taroh Kinoshita
3 Gangliosides and T-Cell Immunity ........................................................ 35
Masakazu Nagafuku and Jin-ichi Inokuchi
4 Gangliosides Regulate Tumor Properties: With Focus
on the Suppression of Metastasis-Associated
ppGalNAc-T13 with GM1 ...................................................................... 55
Koichi Furukawa, Yasuyuki Matsumoto, Qing Zhang,
and Keiko Furukawa
5 Role of Glycans in Viral Infection ......................................................... 71
Tadanobu Takahashi and Takashi Suzuki
6 Discovery and Applications of a Novel Human
Pluripotent Stem Cell-Specific Lectin Probe rBC2LCN ..................... 95
Hiroaki Tateno and Jun Hirabayashi
7 Glycan Structure and Neural Plasticity ................................................ 107
Tadahisa Mikami and Hiroshi Kitagawa
8 The Involvement of Midkine, a Heparin-Binding
Growth Factor, in Cancer Development ............................................... 127
Satoshi Kishida and Kenji Kadomatsu
9 Tumor-Associated Glycans and Their
Functional Roles in the Multistep Process
of Human Cancer Progression ............................................................... 139
Reiji Kannagi, Keiichiro Sakuma,
Bi-He Cai, and Shin-Yi Yu

vii
viii Contents

10 Mammalian Sialidase and Tumor Development .................................. 159

Taeko Miyagi, Kohta Takahashi, Kazuhiro Shiozaki,
and Kazunori Yamaguchi
11 Roles of Glycans in Immune Evasion
from NK Immunity ................................................................................. 177
Shigeru Tsuboi
12 Glycomic Analysis of Cancer ................................................................. 189
Yasuhide Miyamoto
13 Glyco-Predisposing Factor of Diabetes ................................................. 209
Kazuaki Ohtsubo
14 Macrophages Govern Ganglioside GM3 Expression
in Adipocytes to Regulate Adipogenesis and Insulin
Signaling in Homeostatic and Pathogenic Conditions ......................... 219
Jin-ichi Inokuchi
15 O-Mannosyl Glycan and Muscular Dystrophy .................................... 235
Hiroshi Manya and Tamao Endo
16 Glycans and Chronic Obstructive Pulmonary
Disease (COPD) ....................................................................................... 259
Congxiao Gao and Naoyuki Taniguchi
17 α1,6-Fucosyltransferase Knockout Mice
and Schizophrenia-Like Phenotype....................................................... 267
Wei Gu, Tomohiko Fukuda, and Jianguo Gu

Index ................................................................................................................. 281

Chapter 1
N-Glycans and Quality Control of Proteins

Nobuko Hosokawa and Tadashi Suzuki

Abstract Glycosylation is one of the most ubiquitous posttranslational modifica-

tions for eukaryotic proteins. There are numerous examples of the attachment of
glycans to carrier proteins resulting in changes in their physicochemical properties,
such as solubility or heat stability, as well as physiological properties, such as bio-
activity or intra- or intercellular trafficking. In addition, recent studies have revealed
that N-glycans can act as a readout for the folding status of glycoproteins in the
endoplasmic reticulum (ER), so that only proper amounts of functional proteins are
made and delivered to their respective destinations. This process is often called the
glycoprotein quality control system, as a part of the ER protein homeostasis machin-
ery. After misfolded glycoproteins are targeted for destruction in the ER, they are
eventually retrotranslocated into the cytosol for proteasomal degradation. In the
cytosol, glycans are again used for recognition by ubiquitin ligases but are eventu-
ally removed from glycoproteins in order to efficiently degrade misfolded glycopro-
teins. In the present review, a particular focus will be on the mannose-trimming
processes, whereby N-glycan-dependent ER-associated degradation signals are cre-
ated and recognized in the ER, as well as on the function of Fbs proteins and PNGase
that recognize N-glycans in the cytosol.

Keywords Endoplasmic reticulum (ER) • ER-associated degradation (ERAD) •

EDEM • Fbs protein • Glycoprotein • Mannose trimming • N-glycans • PNGase •
Protein quality control • Ubiquitin ligase

N. Hosokawa (*)
Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences,
Kyoto University, 53 Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
e-mail: [email protected]
T. Suzuki
Glycometabolome Team, Systems Glycobiology Research Group, RIKEN-Max-Planck Joint
Research Center for Systems Chemical Biology, RIKEN Global Research Cluster,
RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
e-mail: [email protected]

© Springer Japan 2015 1

T. Suzuki et al. (eds.), Sugar Chains, DOI 10.1007/978-4-431-55381-6_1
2 N. Hosokawa and T. Suzuki

1.1 N-Glycans as Signals for Glycoprotein Quality Control

in the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is an organelle where the majority of secretory

and membrane proteins are synthesized, with approximately one-third of genes
encoding proteins that enter the secretory pathway. Most of these proteins are mod-
ified with glycosylation, resulting in changes in their solubility and stability, or
biological activities. An N-glycan comprised of 14 sugars (Glc3Man9GlcNAc2) is
covalently attached en bloc to the Asn residue in the consensus sequence (Asn-
Xaa-Ser/Thr) of newly synthesized proteins in mammalian or yeast ER (Fig. 1.1a)
(Kornfeld and Kornfeld 1985). However, immediately after the addition, process-
ing of glucose from the N-glycan begins, followed by mannose processing
(Helenius and Aebi 2004; Parodi 2000). Normally, it is believed that glycoproteins
leave the ER after the removal of a (and potentially more) mannose residue(s) from
the N-glycans. The specific sugar moieties are recognized by lectins in the ER
(Fig. 1.1b). Nascent polypeptides are eventually folded with the assistance of chap-
erone proteins, oxidoreductases and other enzymes in the ER. The ER quality con-
trol mechanism strictly monitors the folding states of proteins, ensuring that only
correctly folded or assembled proteins are sorted further to the secretory pathway

a
B b ch
ch

h
nc
ran
ran

α1,2
bra
Ab

α1,3
C

α1,3
α1,2 α1,2 α1,2 Glucose
α1,2 Mannose
α1,3 α1,6
N-Acetylglucosamine
α1,3 α1,6

b
Glucosidase II ER ManI
EDEMs
Glucosidase I,II ER ManI Golgi ManI

α1,6
α1,6

G3M9 G1M9 M9 M8B M7A M6 M5

UGGT
XTP3-B ? OS-9/XTP3-B
CNX/CRT

Fig. 1.1 Structure of N-glycan (a) and processing/recognition of N-glycans during quality control
of glycoproteins (b). α1,2-Linked mannoses are shown in light green, and α1,6-linked mannoses
are shown in olive green in b
1 N-Glycans and Quality Control of Proteins 3

(Ellgaard and Helenius 2003; Sitia and Braakman 2003). Polypeptides that fail to
obtain their native conformations or have unfolded structures under stress condi-
tions are prevented from being secreted. They reenter the folding cycle, whereupon
terminally misfolded polypeptides are eventually degraded. ER-associated degra-
dation (ERAD) is a mechanism whereby misfolded ER proteins are retrotranslo-
cated to the cytosol and degraded by proteasomes (McCracken and Brodsky 2003;
Plemper and Wolf 1999). During these processes, N-glycans act as signals for the
quality control of glycoproteins.
In the ER, monoglucosylated N-glycan (Glc1Man9GlcNAc2) is recognized by the
lectin calnexin (CNX) and its soluble homologue calreticulin (CRT), thereby assist-
ing protein folding (Helenius and Aebi 2001; Maattanen et al. 2010). Once the ter-
minal glucose is removed by glucosidase II, glycoproteins are released from CNX/
CRT. UDP-glucose:glycoprotein-glucosyltransferase (UGGT) is an enzyme that
adds monoglucose back to N-glycans on the polypeptides that still have not acquired
their native conformations, thus, directing the nonnative conformer back to CNX/
CRT (CNX/CRT cycle or monoglucose cycle) (Caramelo and Parodi 2008;
D’Alessio et al. 2010). Since S. cerevisiae possesses calnexin (Cne1p) but lacks
UGGT, glycoproteins do not enter this extensive folding cycle in this organism.
Glycoproteins that fail to obtain their native conformations are removed from the
ER, thereby preventing deleterious effects such as aggregation formation or inter-
ference with the folding of newly synthesized polypeptides. Mannoses are pro-
cessed from N-glycans on the polypeptides while remaining in the ER, and hence
unfolded glycoproteins with mannose-processed N-glycans are targeted for degra-
dation (mannose timer model) (Helenius 1994; Jakob et al. 1998). Removal of the
mannose from the A branch terminates reentry to the CNX/CRT cycle. Recently,
N-glycans lacking the terminal mannose of the C branch, or the exposure of the
α1,6-linked mannose, have been identified as the signal for ERAD (Aebi et al. 2010;
Hosokawa et al. 2010a; Lederkremer 2009; Smith et al. 2011). Yos9p in S. cerevi-
siae and OS-9 and XTP3-B/Erlectin in mammals are resident ER proteins that con-
tain sugar recognition domains for mannose 6-phosphate homology (MRH)
(Castonguay et al. 2011; Munro 2001) and recognize N-glycan signals for ERAD.
Membrane-embedded ubiquitin ligases (E3) play central roles in ERAD. The
Hrd1p-Hrd3p ubiquitin ligase complex in S. cerevisiae and its mammalian homo-
logue HRD1-SEL1L form large membrane complexes by the association of various
ERAD components including ubiquitination enzymes (E2) and Der1p (Derlins in
mammals) (Claessen et al. 2012; Hampton and Sommer 2012; Smith et al. 2011). In
the luminal side of the complex, a large domain of Hrd3p/SEL1L acts as a scaffold
for the recognition of misfolded cargo by the association of lectins and chaperone
proteins. Hrd3p/SEL1L also directly binds to the misfolded proteins; thus, in coor-
dination with lectins and chaperones, the client proteins are transported to the cyto-
sol through the retrotranslocation channel.
In the following sections, we will review the current knowledge of the role of
various mannosidases/mannose-binding lectins for the creation/recognition of
“degradation signals” on misfolded glycoproteins.
4 N. Hosokawa and T. Suzuki

1.2 Enzymes That Process Mannose Residues

from the N-Glycans for ERAD: ER Mannosidase,
Golgi Mannosidases, and EDEM Proteins

In mammals, three subfamilies of Class I α-mannosidases (glycosyl hydrolase fam-

ily 47 (Herscovics 1999a; Mast and Moremen 2006)) are known: ER processing
α1,2-mannosidase (ER ManI), Golgi α1,2-mannosidases (Golgi ManIA, IB, and
IC), and ER degradation enhancing α-mannosidase-like proteins (EDEM1, 2, and 3)
(Fig. 1.2a). This class of α-mannosidases trims α1,2-linked mannoses, although the
mannose-processing activity of EDEM proteins remains controversial. S. cerevisiae
possesses ER mannosidase (Mns1p) and EDEM homologues (Htm1p/Mnl1p and
Mnl2p) but lacks Golgi α1,2-mannosidase.
The mannose-processing specificity of ER ManI and Golgi ManI has been well
characterized using purified recombinant proteins in vitro. ER ManI (Mns1p in
yeast) preferentially removes mannose from the B branch, creating Man8GlcNAc2
isomer B (Man8B) (Gonzalez et al. 1999; Herscovics 1999b; Tremblay and
Herscovics 1999). However, ER ManI is also able to remove mannoses from other
branches when high concentrations of this enzyme are incubated with N-glycans for
a prolonged time period (Aikawa et al. 2012; Herscovics et al. 2002). This suggests
that N-glycans that are recognized as degradation signals can be created by the sole
function of ER ManI. In agreement with this, overexpression of ER ManI in cultured
cells enhances glycoprotein ERAD (Hosokawa et al. 2003; Wu et al. 2003) and
increases mannose trimming from ERAD substrates, whereas knockdown of ER
ManI greatly inhibits mannose processing (Avezov et al. 2008). Under normal con-
ditions, ER ManI is concentrated in a specific ER subcompartment termed the ER
quality control compartment (Avezov et al. 2008), in which misfolded proteins des-
tined for degradation are enriched. ER ManI is short-lived and is degraded in the
lysosome (Wu et al. 2007). This mechanism is thought to prevent clearance of
nascent polypeptides during the folding process by the presence of excess ER ManI,
which would otherwise impair normal cellular function.
Golgi α1,2-mannosidases preferentially process mannoses from the A and C
branches once the B-branch terminal mannose is removed (Lal et al. 1998). Thus, it is
also possible that Golgi α1,2-mannosidases create the N-glycan tags recognized as
ERAD signals during the recycling of misfolded substrates between the ER and Golgi
(Hammond and Helenius 1994; Hosokawa et al. 2007). It was recently reported that
ER ManI resides in the Golgi apparatus (Pan et al. 2011), and a model was proposed
whereby misfolded glycoproteins that escaped the ER quality control checkpoint are
sorted to the Golgi apparatus, where ER ManI captures, demannosylates, and assists
the substrates sent back to the ER for degradation (Pan et al. 2013b). In this scenario,
the question is which enzyme processes mannoses from the folded proteins in the ER?
Is there another α1,2-mannosidase in the ER that normally creates N-glycan degrada-
tion signals? Several issues remain unresolved regarding mannose processing in the
early secretory pathway.
 1

a b
5 25 45 532 21 115 190
Mns1p 1 549 Yos9p 1 542
(S. cerevisiae) (S. cerevisiae)
85 105 257 695
ERManI 1 699
OS-9 25 110 181 533 589
variant 1 1 667
42 62 202 640
GolgiManIA 1 653

variant 2 1 612
37 57 187 626
GolgiManIB 1 641

XTP3-B 33 111 180 342 418

23 43 181 617 long 1 483
GolgiManIC 1 630
293 348

20 42 502 short 1 428

Htm1p/Mnl1p 1 796
(S.cerevisiae)
N-Glycans and Quality Control of Proteins

13 32 172 573
Mnl2p 1 849
(S. cerevisiae) signal sequence
25 137 582
EDEM1 1 657 transmembrane region
α-mannosidase domain
21 42 480 (GH47 domain)
EDEM2 1 578 MRH domain
(PRKCSH domain)
41 59 498 674 779 ER retrieval signal
EDEM3 1 932
PA domain

Fig. 1.2 Schematic representation of the primary structures of Class I α-mannosidases (a) and MRH domain-containing lectins in the ER (b). Accession num-
bers for the protein sequences used are (a) S. cerevisiae Mns1, P32906; H. sapiens ER ManI (MAN1B1), Q9UKM7; Golgi ManIA (MA1A1), P33908; Golgi
ManIB (MA1A2), O60476; Golgi Man1C (MA1C1), Q9NR34; S. cerevisiae Htm1/Mnl1, P38888; H. sapiens EDEM1, Q92611; EDEM2, Q9BV94; and
EDEM3, Q9BZQ6 and (b) S. cerevisiae Yos9, Q99220; H. sapiens OS9, Q13438; XTP3-B, Q96DZ1. GH47, glycosyl hydrolase family 47; PRKCSH, protein
kinase C substrate 80 K-H; and PA, protease-associated domain. The domain structure is based on the conserved domain database of NCBI (http://www.ncbi.
nlm.nih.gov/cdd)
5
6 N. Hosokawa and T. Suzuki

ER ManI (gene name MAN1B1) was recently revealed to be one of the genes that
cause autosomal recessive cognitive disorders (Najmabadi et al. 2011; Rafiq et al.
2011). Homozygous nonsense mutations or missense mutations in both alleles were
identified. Furthermore, using exome analysis, MAN1B1 was identified as one of the
genes that cause congenital disorders of glycosylation (CDG) (Rymen et al. 2013),
producing developmental delay, facial dysmorphism, and obesity in addition to
intellectual disability. Involvement of ER ManI in liver disease (Pan et al. 2009)
or in liver carcinogenesis, independent of its enzyme activity, was also reported
(Pan et al. 2013a).
Mammalian EDEM1 and yeast Htm1p/Mnl1p were identified as homologues of
the ER α-mannosidase required for glycoprotein ERAD (Hosokawa et al. 2001;
Jakob et al. 2001; Nakatsukasa et al. 2001). There are three EDEM proteins in mam-
mals and two in yeast, although little is known about the recently identified Mnl2p
(Martinez Benitez et al. 2011). Several mechanisms are proposed for the require-
ment of EDEM proteins in ERAD. Initially, they were proposed to be lectins
since mannose-processing activity was not detected, although lectin activity has
not been shown in vitro either. The second mechanism is that EDEMs process man-
nose to generate N-glycan signals for degradation. A third mechanism is the glycan-
independent recognition of ERAD substrates by EDEMs. Recently, the
α-mannosidase activity of Htm1p/Mnl1p was shown to process mannose from the C
branch in yeast cells (Clerc et al. 2009; Quan et al. 2008) and was followed by an
in vitro experiment using recombinant protein purified in complex with an
oxidoreductase Pdi1p (PDI in mammals) (Gauss et al. 2011). As for mammalian
EDEMs, however, in vitro activity assays have not been successful to date. Transient
cellular expression of EDEM1 processes terminal mannose from the C branch
(Hosokawa et al. 2010b) or from the A branch (Olivari and Molinari 2007) of
N-glycans on model ERAD substrates. Also, transient expression of EDEM3
enhances mannose processing, creating M7-M6 N-glycans on total cellular glyco-
proteins as well as on misfolded ERAD substrates (Hirao et al. 2006). However,
EDEM2 mannose-processing activity appears lacking (Mast et al. 2005). Enzyme
activity and its specificity require future in vitro experimentation. Mammalian
EDEMs are also reported to recognize misfolded proteins in a glycan-independent
manner. EDEM1 point mutants lacking mannosidase activity (Cormier et al. 2009)
or deletion mutants that lack the mannosidase domain (Ron et al. 2011) are able to
associate and degrade ERAD substrates. An intrinsically disordered region close to
the N-terminus of EDEM1 is suggested to associate with the misfolded polypep-
tides (Marin et al. 2012).
In the case of EDEM1 binding to misfolded proteins independently of glycans, its
mannosidase domain is used to associate with SEL1L, which has five N-glycosylation
sites. Thus, a model was proposed whereby EDEM1 acts as a quality control receptor
that links ERAD substrate to dislocation machinery (Cormier et al. 2009). This is
similar to the proposed function of OS-9 and XTP3-B, in which the lectin domains
of OS-9 and XTP3-B recognize N-glycans on SEL1L (Christianson et al. 2008) (dis-
cussed further in the next section). Morphological study has revealed that EDEM1,
in addition to its ER localization, is concentrated in vesicles that lack the COPII coat
1 N-Glycans and Quality Control of Proteins 7

a b
EDEM1

OS-9

OS-9
EDEM1

OS-9 EDEM1
OS-9

BiP BiP
ER XTP3-B SEL1L
XTP3-B
SEL1L

Derlin Derlin
HRD1 HRD1 1/2/3
1/2/3
VIMP VIMP
cytosol ring
Herp
p97/ VCP
ring
Herp
p97/ VCP
Npl4 Npl4
Ufd1 Ufd1

Fig. 1.3 Two distinct models of N-glycan recognition by ER lectins and EDEM1 in the ERAD
process. Recognition of N-glycans on misfolded cargos (a) or on SEL1L protein (b)

(Zuber et al. 2007). These vesicles are termed EDEMosomes (Cali et al. 2008) or
ERAD tuning vesicles (Bernasconi et al. 2012) and have been demonstrated to con-
tain OS-9 and SEL1L in addition to EDEM1. Hence, these vesicles are proposed to
regulate the ERAD machinery by removing ERAD factors through lysosomal degra-
dation (Bernasconi et al. 2012). Based on these observations, N-glycans are sug-
gested to act as signals for docking with other ER resident proteins (Hebert and
Molinari 2012) (Fig. 1.3b), which awaits further verification.
A number of EDEM partner proteins have been elucidated. Yeast Htm1p/Mnl1p
tightly associates with Pdi1p through intermolecular disulfide bonding (Gauss et al.
2011; Sakoh-Nakatogawa et al. 2009), which is required for its mannosidase activ-
ity. In mammals, ERdj5, another PDI family member that functions as a disulfide
reductase in the ER, forms a trimeric complex including BiP and EDEMs for ERAD
(Ushioda et al. 2008). The association of ER ManI with EDEM1 is also reported,
which enhances glycoprotein ERAD by suppressing the degradation of ERManI
under ER stress conditions (Termine et al. 2009). SEL1L is another partner of
EDEMs (Bernasconi et al. 2012; Cormier et al. 2009). Thus, the function of EDEMs
could be modified by these partner proteins or may represent the coordinated roles
of proteins incorporated in different functional complexes.

1.3 Lectins That Recognize N-Glycan Tags for ERAD:

Yos9p, OS-9, and XTP3-B, the MRH Domain-Containing
Lectins in the ER

Misfolded polypeptides that have N-glycan tags for degradation are recognized by
specific lectins. S. cerevisiae Yos9p (yeast OS-9) was found to associate with Hrd3p
and Kar2p and discriminates ERAD cargo for degradation (Bhamidipati et al. 2005;