Substrate Specificity of the Oxidoreductase ERp57 Is Determined Primarily
by Its Interaction with Calnexin and
Calreticulin |
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Authors: | Catherine E Jessop Timothy J Tavender Rachel H Watkins Joseph E Chambers and Neil J Bulleid |
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Institution: | Faculty of Life Sciences, The Michael Smith Building, University of Manchester, Manchester, M13 9PT, United Kingdom |
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Abstract: | The formation of disulfides within proteins entering the secretory pathway
is catalyzed by the protein disulfide isomerase family of endoplasmic
reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to
catalyze the isomerization of non-native disulfide bonds formed in
glycoproteins with unstructured disulfide-rich domains. Here we investigated
the mechanism underlying ERp57 specificity toward glycoprotein substrates and
the interdependence of ERp57 and the calnexin cycle for their correct folding.
Our results clearly show that ERp57 must be physically associated with the
calnexin cycle to catalyze isomerization reactions with most of its
substrates. In addition, some glycoproteins only require ERp57 for correct
disulfide formation if they enter the calnexin cycle. Hence, the specificity
of ER oxidoreductases is not only determined by the physical association of
enzyme and substrate but also by accessory factors, such as calnexin and
calreticulin in the case of ERp57. These conclusions suggest that the calnexin
cycle has evolved with a specialized oxidoreductase to facilitate native
disulfide formation in complex glycoproteins.The ability to form disulfide bonds within proteins entering the secretory
pathway is essential for cell survival and occurs within the endoplasmic
reticulum (ER).3 For
proteins with few disulfides, the process can be catalyzed by oxidation of
cysteine residues to form the correct, native disulfide; however, for proteins
with several disulfides, an isomerization reaction is also required to correct
non-native disulfides formed following oxidation
(1). Both these reactions are
catalyzed by a group of ER-resident proteins that belong to the protein
disulfide isomerase (PDI) family, which comprises over 17 members
(2). It is well established
that PDI and several other family members are able to catalyze the formation
and isomerization of disulfides in vitro, although the exact function
of each of the family members in vivo is unknown. It is still an open
question as to whether they all catalyze similar reactions and have distinct
substrate specificities or whether they have distinct enzymatic functions
related to the breaking and formation of disulfides.For one member of the PDI family, the function and substrate specificity is
a little clearer. ERp57 has been shown previously to interact specifically
with glycoproteins during their folding
(3). The enzyme is physically
associated with either calnexin or calreticulin
(4) and is therefore ideally
placed to catalyze correct disulfide formation within proteins entering the
calnexin/calreticulin cycle (referred to subsequently just as the calnexin
cycle). In addition, the ability of ERp57 to catalyze the refolding of
substrates in vitro is greatly enhanced if the substrate is bound to
calnexin (5). Recently,
substrates for the reduction or isomerization reaction catalyzed by ERp57 have
been identified by trapping mixed disulfides between enzyme and substrate
(6). Strikingly, there was an
overrepresentation of substrate proteins with cysteine-rich domains containing
little secondary structure, suggesting that the main function of ERp57 is in
the isomerization of non-native disulfides. ERp57 has also been shown to
function independently from the calnexin cycle. It is a component of the MHC
class I loading complex where it forms a disulfide-linked complex with tapasin
and is thought to either stabilize the complex or facilitate correct assembly
of class I molecules (7,
8). Recently, ERp57 has been
demonstrated to isomerize interchain disulfides in the major capsid protein,
VP1, of simian virus 40 (9).
The ability to dissociate VP1 pentamers by ERp57 does not require the
substrate to interact with the calnexin cycle. Hence, it is still unclear how
ERp57 recognizes its substrates, and in particular, whether this recognition
is solely determined by an interaction with the calnexin cycle.The recognition of substrates by PDI is somewhat clearer in that one
particular domain within the protein (the b′ domain) has been shown to
be primarily responsible for substrate recognition and peptide binding
(10). The corresponding domain
within ERp57 has been shown to be responsible for interaction with the
calnexin cycle (11),
suggesting that for ERp57, substrate recognition must occur outside this
domain or is determined solely by substrate interaction with calnexin via its
oligosaccharide side chain. Hence, the aim of our study was to evaluate the
necessity of the calnexin cycle both for ERp57 to recognize its substrates and
for correct folding of glycoproteins. ERp57 was found to be required for the
efficient folding of one substrate, influenza virus hemagglutinin (HA), but
only when it entered the calnexin cycle. HA did not require ERp57 to fold if
it was blocked from entering the calnexin cycle. In contrast, β1-integrin
does not fold efficiently either if ERp57 was depleted or if ERp57 is blocked
from entering the calnexin cycle
(6). Although ERp57 may be
dispensable for the folding of some glycoproteins, the interaction with
calnexin commits them to an ERp57-dependent fate. We also found that the
majority of ERp57 substrates need to enter the calnexin cycle to be acted upon
by the enzyme, demonstrating that substrate specificity is primarily dependent
upon substrate entry into the calnexin cycle. |
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