(Received for publication, December 19, 1995; and in revised form, February 26, 1996)
From the
Calreticulin was identified by immunochemical and sequence
analyses to be the higher molecular mass (60 kDa) component of the
polypeptide doublet previously observed in a rat liver Golgi
endomannosidase preparation obtained by chromatography on a
Glc1
3Man-containing matrix. The affinity for this saccharide
ligand, which paralleled that of endomannosidase and was also observed
with purified rat liver calreticulin, suggested that this chaperone has
lectin-like binding properties. Studies carried out with immobilized
calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with
this protein were limited to monoglucosylated polymannose components.
Although optimal binding occurred with
Glc
Man
GlcNAc, substantial interaction with
calreticulin was retained after sequential trimming of the polymannose
portion down to the Glc
Man
GlcNAc stage. The
1
6-mannose branch point of the oligosaccharide core,
however, appeared to be essential for recognition as
Glc
Man
GlcNAc did not interact with the
calreticulin. The carbohydrate-peptide linkage region had no
discernible influence on binding as monoglucosylated oligosaccharides
in N-glycosidic linkage interacted with the chaperone to the
same extent as in their unconjugated state. The immobilized
calreticulin proved to be a highly effective tool for sorting out
monoglucosylated polymannose oligosaccharides or glycopeptides from
complex mixtures of processing intermediates. The copurification of
calreticulin and endomannosidase from a Golgi fraction in comparable
amounts and the strikingly similar saccharide specificities of the
chaperone and the processing enzyme have suggested a tentative model
for the dissociation through glucose removal of
calreticulin-glycoprotein complexes in a post-endoplasmic reticulum
locale; in this scheme, deglucosylation would be brought about by the
action of endomannosidase rather than glucosidase II.
It has become apparent in recent years that N-linked
oligosaccharides at an early stage of processing can play an important
role in the quality control of the secretory pathway by influencing the
folding and assembly of the proteins to which they are
attached(1, 2, 3) . More specifically,
attention has been focused on the glucose residues that are initially
present on the N-linked carbohydrate unit
(GlcMan
GlcNAc
), since it has been
noted that inhibition of glucose trimming through a blockade of the
ER(
)-situated glucosidases may lead to accelerated
degradation (4, 5) or delayed secretion (6, 7, 8) of various glycoproteins. The
finding that retention of the triglucosyl sequence can result in such a
profound effect on newly synthesized glycoproteins can be rationalized
by reports that proteins with N-linked oligosaccharides bind
to certain molecular chaperones only subsequent to the processing of
the carbohydrate units to the monoglucosylated state(1) . Most
of the evidence for this lectin-like activity has been obtained through
studies on calnexin(9, 10) , a membrane-associated
chaperone of the ER, although very recent observations, made while our
investigations were in progress, have suggested on the basis of
electrophoretic examinations that the lumenal chaperone calreticulin
can also bind glycoproteins after partial deglucosylation(11) .
Our attention was drawn to the latter chaperone by the quite unexpected finding, from sequence analyses that the larger (mass of 60 kDa) of the two previously observed polypeptide components present in the ligand affinity chromatography-purified rat liver Golgi endomannosidase preparation (12) was indistinguishable from calreticulin. The copurification of endomannosidase and calreticulin on a Glc-Man-Affi-Gel suggested that the chaperone has a saccharide affinity and provided the impetus for undertaking a detailed definition of its lectin-like properties toward N-linked oligosaccharides at various processing stages. The results obtained from studies with immobilized calreticulin provided some striking parallels between the saccharide specificity of this chaperone to those previously observed for endomannosidase (13, 14) and suggested the possibility that the two proteins may work in tandem in a post-ER location.
For the preparation of
GlcMan
GlcNAc isomers, the purified
[
C]Glc
Man
GlcNAc
(
100,000 dpm) was incubated with rat liver ER membranes (750
µg of protein) for 20 h at 37 °C in 100 µl of 0.1 M NaMES buffer, pH 6.5, containing 0.2% (v/v) Triton X-100 in the
presence of 2 mM castanospermine (a gift from Dr. M. Kang,
Merrell Dow Research Institute, Cincinnati, OH) and either 2 mM 1,4-dideoxy-1,4-imino-D-mannitol (Oxford Glycosystems) or
5 µM kifunensine (Toronto Research Chemical). In presence
of the 1,4-dideoxy-1,4-imino-D-mannitol, which inhibits ER
mannosidase II(25) , Glc
Man
GlcNAc,
isomer B, in which the terminal mannose of the middle chain is missing,
is generated while kifunensine by blocking the action of ER mannosidase
I (26) results in the formation of
Glc
Man
GlcNAc, isomer C, in which the residue
terminating the
1,6-linked chain has been excised. The respective
Glc
Man
GlcNAc isomers were isolated from the
digests by preparative thin layer chromatography after deproteinization
with 80% ethanol and desalting by passage through Dowex 50
(H
) and Dowex 1 (acetate) as described previously (26) .
For the preparation of radiolabeled
GlcMan
GlcNAc, Glc
-
Man
GlcNAc, and Glc
Man
GlcNAc,
digestion of
[
C]Glc
Man
GlcNAc (85,000
dpm) was carried out with jack bean
-mannosidase (2 units, Sigma)
in 300 µl of 0.15 M sodium citrate buffer, pH 5.2, for 72
h at 37 °C in the presence of toluene. The oligosaccharides were
isolated from the deproteinized and desalted digests by preparative
thin layer chromatography, and in each case their monoglucosylated
state was verified by the release of Glc
Man
through endomannosidase digestion (14) .
The
heptasaccharide fraction obtained by preparative thin layer
chromatography from the cytosolic oligosaccharides of
[C]glucose-labeled HepG2 cells provided a
mixture of
[
C]Glc
Man
GlcNAc and
[
C]Man
GlcNAc(27) . The
preparation of Glc
Man
GlcNAc was effected by
submitting Glc
Man
GlcNAc (115,000 dpm) to
acetolysis as described previously(13) ; the oligosaccharide
was resolved from other fragmentation products by thin layer
chromatography.
Mild acid treatment of the oligosaccharide-lipid
fraction from [C]glucose-labeled thyroid slices (28) yielded
[
C]Glc
Man
GlcNAc
and
[
C]Glc
Man
GlcNAc
,
which were purified by preparative thin layer chromatography and
converted to their Glc
Man
GlcNAc
and
Glc
Man
GlcNAc
derivatives by endo H
digestion(28) .
Glycopeptides containing a mixture of
incompletely processed N-linked oligosaccharides were obtained
by Pronase digestion of the delipidated protein from thyroid slices
labeled with [C]glucose during a 3-h incubation
as described previously(22) ; by subsequent passage through a
Dowex 1-X2 200-400-mesh (acetate) column, the glycopeptides were
enriched in glucosylated and deglucosylated polymannose carbohydrate
units.
Figure 1:
Immunochemical identification of
calreticulin in the ligand affinity chromatographically purified rat
liver endomannosidase preparation and the Golgi membranes from which it
was obtained. Subsequent to polyacrylamide gel electrophoresis in SDS,
the endomannosidase obtained by Glc-Man-Affi-Gel chromatography (0.3
µg of protein, AG) as well as unfractionated rat liver
Golgi membranes (50 µg of protein, GOL) and calreticulin
standard (0.7 µg of protein, CRT) were immunoblotted with
antiserum against rat calreticulin as described under
``Experimental Procedures.'' The components were detected by
autoradiography after reaction of the bound antibody with I-labeled protein. For comparison the components of an
aliquot of the endomannosidase preparation (1 µg of protein) were
visualized by silver staining (AG, Silver) after the
electrophoresis. The designated molecular size markers expressed as kDa
were Escherichia coli
-galactosidase (116,000), bovine
serum albumin (66,000), hen ovalbumin (45,000), and bovine erythrocyte
carbonic anhydrase (29,000).
In order to determine if calreticulin by itself binds to the Glc-Man-Affi-Gel matrix, we chromatographed a sample of the purified protein on this column under the same conditions as employed for the Triton-solubilized Golgi membranes and observed on the basis of immunoblotting that this protein was indeed retained and eluted under the same conditions as the endomannosidase activity (Fig. 2). Moreover, when a Golgi extract was placed on the Affi-Gel column, the immunologically detected calreticulin and enzymatically monitored endomannosidase activity emerged in the same fractions (data not shown).
Figure 2: Immunochemical detection of calreticulin in the eluted fractions from a Glc-Man-Affi-Gel column. Polyacrylamide gel electrophoresis in SDS, followed by immunoblotting with anti-calreticulin serum, was carried out on concentrated aliquots (300 µl) of neutralized fractions eluted with the glycine HCl, pH 3.0, buffer from a GlcMan-Affi-Gel column(12) , which had been loaded with 10 µg of purified rat calreticulin. The numbers on top of the lanes refer to the fraction (4 ml) emerging from the column upon application of the glycine buffer as described previously(12) . When Triton-solubilized Golgi membranes were chromatographed on this column, a similar immunoblot of the emerging fractions was obtained; the calreticulin and endomannosidase activity peaks (fraction 2) coincided. The detection of the components by autoradiography and the molecular size markers were the same as in Fig. 1. The lane designated as CRT contained a calreticulin standard.
Figure 3:
Separation of monoglucosylated and
unglucosylated ManGlcNAc on a calreticulin-Sepharose
column. A mixture of
C-labeled
Glc
Man
GlcNAc and Man
GlcNAc (14,000
dpm), which was incompletely resolved by thin layer chromatography, was
applied to an immobilized calreticulin column under the conditions
described under ``Experimental Procedures.'' Fractions of 1
ml were collected and monitored for radioactivity by scintillation
counting (left panel). After desalting equal amounts of the
unbound (UN) and bound (BD) peaks as well as a
portion of the initial (IN) sample were chromatographed on a
silica-coated plate in Solvent System B for 26 h (right
panel). The components were detected by fluorography and their
migration compared to standard oligosaccharides. The abbreviations
employed were as follows: G
M
,
Glc
Man
GlcNAc; M
,
Man
GlcNAc; M
,
Man
GlcNAc; M
,
Man
GlcNAc; M
,
Man
GlcNAc.
Figure 4:
Assay of the binding capacity of several
monoglucosylated polymannose oligosaccharides to a column of
immobilized calreticulin. Purified C-labeled
oligosaccharides (2,000-20,000 dpm), together with a
H-labeled Glc
Man
GlcNAc internal
standard, were chromatographed on a calreticulin-Sepharose column as
described under ``Experimental Procedures.'' The
radioactivity in each 1-ml fraction was determined by double channel
scintillation counting and is plotted for the
C-labeled
(
) and
H-labeled (
) oligosaccharide. The scale
refers only to the
C radioactivity; the
H-labeled Glc
Man
GlcNAc standard was
present at approximately 3-fold greater radioactivity than the
C-labeled components. The abbreviations employed were as
follows: G
M
,
Glc
Man
GlcNAc; G
M
,
Glc
Man
GlcNAc; G
M
,
Glc
Man
GlcNAc (isomer B); G
M
,
Glc
Man
GlcNAc; G
M
(Red), Glc
Man
GlcNAc reduced
with NaBH
.
Figure 5:
Assessment of the oligosaccharide binding
specificity of calreticulin. Purified C-labeled
oligosaccharides and glycopeptides were chromatographed on
calreticulin-Sepharose as in Fig. 4, and their extent of binding
to this immobilized protein relative to
Glc
Man
GlcNAc was determined on the basis of
their emergence from the column. The abbreviations used are as follows: G
M
,
Glc
Man
GlcNAc; G
M
,
Glc
Man
GlcNAc; G
M
,
Glc
Man
GlcNAc; G
M
,
Glc
Man
GlcNAc, isomer B; G
M
,
Glc
Man
GlcNAc, isomer C; G
M
,
Glc
Man
GlcNAc; G
M
,
Glc
Man
GlcNAc; G
M
,
Glc
Man
GlcNAc; G
M
,
Glc
Man
GlcNAc; G
M
(Red), NaBH
-reduced
Glc
Man
GlcNAc; M
, Man
GlcNAc; G
M
-pept and M
-pept,
Glc
Man
GlcNAc
and
Man
GlcNAc
, respectively, linked N-glycosidically to peptide.
The immobilized
calreticulin column also proved to be highly effective in removing
GlcMan
GlcNAc from complicated mixtures of
polymannose intermediates even with the additional presence of the tri-
and diglucosylated Man
GlcNAc components (Fig. 6).
Thin layer chromatography indicated that the unbound material was
specifically freed from the Glc
Man
GlcNAc
oligosaccharide, which was recovered in the bound fraction (Fig. 6). The minor saccharide component migrating ahead of the
Glc
Man
GlcNAc in the bound fractions was
identified as Glc
Man
GlcNAc, which has been
reported to occur as an N-linked processing
intermediate(33) .
Figure 6:
Selective retention of
GlcMan
GlcNAc on a calreticulin-Sepharose column
from mixtures of oligosaccharide processing intermediates. A mixture of
C-labeled oligosaccharides consisting of similar amounts
(
8,000 dpm) of Glc
Man
GlcNAc and
Man
GlcNAc components was chromatographed on a
calreticulin-Sepharose column as described under ``Experimental
Procedures,'' and the radioactivity in 1-ml fractions was
determined by scintillation counting (left panel). After
desalting equal aliquots of the bound (BD) and unbound (UN) peaks as well as a portion of the initial mixture (IN) were chromatographed on a silica-coated plate in Solvent
System B for 20 h, and the components were detected by fluorography (G
M
to M
, right panel). A mixture of
Glc
Man
GlcNAc and Man
GlcNAc
was similarly chromatographed on an immobilized calreticulin column,
and the initial, bound, and unbound oligosaccharides were examined by
thin layer chromatography (G
M
to M
, right panel). The
abbreviations are the same as in Fig. 3, except for the
following: G
,
Glc
Man
GlcNAc; G
,
Glc
Man
GlcNAc; G
,
Glc
Man
GlcNAc; M
,
Man
GlcNAc.
The effectiveness of the
calreticulin-Sepharose in resolving metabolic intermediates was further
made apparent when the cytosolic heptasaccharide fraction from HepG2
cells, which has been reported to consist of
GlcMan
GlcNAc and
Man
GlcNAc(27) , was loaded onto the column (Fig. 7). Thin layer chromatography after endomannosidase
digestion did indeed reveal that the unabsorbed material consisted
primarily of Man
GlcNAc, which was resistant to the action
of this enzyme, while the bound oligosaccharide represented
Glc
Man
GlcNAc, which was converted to
Man
GlcNAc through the release of Glc
Man
(Fig. 7).
Figure 7:
Separation of cytosolic heptasaccharide
components from metabolically radiolabeled HepG2 cells by
calreticulin-Sepharose chromatography. The heptasaccharide fraction
(8,500 dpm) from the cytosol of
[C]glucose-labeled HepG2 cells, which is known
to consist of Glc
Man
GlcNAc and
Man
GlcNAc(27) , was chromatographed on an
immobilized calreticulin column as described under ``Experimental
Procedures'' and the emerging radioactivity was monitored by
scintillation counting (left panel). The desalted unbound (UN) and bound (BD) oligosaccharide fractions as well
as several monoglucosylated standards were then treated with
endomannosidase and subsequently chromatographed on cellulose-coated
plates for 20-24 h (middle and right panels).
The components were detected by fluorography and their migration
compared to standards. The abbreviations employed are as follows: G
M
,
Glc
Man
; M
,
Man
GlcNAc; M
,
Man
GlcNAc; M
,
Man
GlcNAc.
It is apparent from the present investigation that calreticulin cofractionates with endomannosidase during affinity chromatography of Triton-solubilized Golgi proteins on a Glc-Man-Affi-Gel column. This was surprising, particularly in view of the fact that only two polypeptide components in approximately equal amounts were retained by this matrix from a complex mixture of components, and alerted us to the possibility that calreticulin has a lectin-like binding capacity.
Studies carried out with immobilized
calreticulin and an array of oligosaccharides derived from N-linked carbohydrate units revealed that protein-saccharide
interactions were limited to monoglucosylated polymannose components.
While the presence of a terminal mannose-linked glucose residue was
clearly critical for the binding to take place, substantial interaction
was retained after extensive trimming of the two unglucosylated chains
of the polymannose unit. Indeed, even after truncation of
GlcMan
GlcNAc to
Glc
Man
GlcNAc, about 65% of the initial binding
capacity was still observed.
Preservation of the 1
6-inked
mannose residue of the latter oligosaccharide was, however, essential
to lectin recognition, as when this inner branch point was excised to
yield Glc
Man
GlcNAc
(Glc
1
3Man
1
2Man
1
2Man
1
3Man
1
4GlcNAc) interaction with calreticulin could no longer
be detected. On the other hand, the carbohydrate-peptide linkage region
appeared to have no discernible influence on binding, as
monoglucosylated oligosaccharides in N-glycosidic linkage or
in their unconjugated state terminating in either N-acetylglucosamine or di-N-acetylchitobiose
interacted with the calreticulin to the same extent.
The selectivity of the immobilized calreticulin for monoglucosylated polymannose oligosaccharides or glycopeptides made it a highly effective tool for sorting out these components from complex mixtures of processing intermediates. Indeed, the high specificity of the calreticulin stands out in contrast to the mannose/glucose-binding lectins, such as concanavalin A(34) , which cannot discriminate between glucosylated and unglucosylated polymannose oligosaccharides(35) .
While calreticulin and
endomannosidase are believed to function quite differently, namely as
molecular chaperone (1, 36, 37) and
processing enzyme(13, 14) , respectively, our study
demonstrates some intriguing similarities that merit comment. The
selective retention of these two proteins on a matrix containing
Glc1
3Man substituents was an expression of a common specific
interaction with monoglucosylated polymannose oligosaccharides. The
inability of calreticulin to bind tri- and diglucosylated
oligosaccharides was mirrored by the previously reported low in
vitro reactivity of endomannosidase with such saccharide species.
Also relevant was the finding that monoglucosylated oligosaccharides
with extensively truncated mannose chains could still effectively
interact with both the chaperone and the enzyme(14) ,
particularly since this property stands in pronounced contrast to the
specificity of glucosidase II, which is known to require the untrimmed
mannose branches for interaction with its substrate(38) .
Although calreticulin is generally believed to be primarily situated in the ER(32) , our finding of this protein in the Golgi is consistent with reports indicating its presence at the cell surface (39, 40) and other subcellular compartments(41, 42, 43) . Indeed, it is apparent that calreticulin takes part in intracellular trafficking which accounts for this wide distribution (43, 44) and distinguishes it from calnexin, the other lectin-like chaperone, which is a membrane-bound ER-resident protein(45) .
The presence
of molecular chaperones with affinity for proteins with
monoglucosylated N-linked oligosaccharides has provided the
basis for a model (1) that accounts for their preferential
association with glycoproteins at an early stage of processing and
explains the observed accelerated protein degradation due to impaired
folding or oligomerization during a glucosidase
blockade(4, 5) . However, as this scheme also
postulated that dissociation of glycoproteins from the chaperone is
brought about by the action of glucosidase II, its relevance would be
limited to the ER locale where this enzyme is situated(46) . If
deglucosylation is required to dissociate the calreticulin-glycoprotein
complexes in a more distal location, which would be either the Golgi
itself or an ER-Golgi intermediate compartment, another mechanism is
required. This has prompted us to propose a tentative modified model (Fig. 8) for such a more distal site in which removal of the
glucose is achieved by endomannosidase through the excision of a
Glc1
3Man disaccharide. This scheme would take into account
the occurrence of calreticulin and endomannosidase in comparable
amounts in this location and, more importantly, the fact that
endomannosidase in marked contrast to glucosidase II has the capacity
to interact with N-linked oligosaccharides in which the
mannose chains have been trimmed. The latter characteristic is
relevant, as glycoproteins that exit from the ER will have already
undergone a substantial degree of processing though the action of
ER-resident mannosidases(25, 26) . Although the ER may
be the primary site for protein folding and oligomerization to take
place, a number of instances have already been described in which such
quality controlling events take place in more distal
compartments(47, 48) .
Figure 8:
Schematic proposal for two distinct
mechanisms of dissociating glycoproteins with monoglucosylated N-linked oligosaccharides from their interaction with
calreticulin subsequent to folding of their polypeptide chain. This
hypothetical model suggests that when the binding with the lumenal
chaperone takes place in the ER compartment, glucosidase II removes the
glucose (G) residue, while if this association occurs in a
Golgi or possibly ER-Golgi intermediate (ERGIC) location
endomannosidase is involved in the deglucosylation by excising a
Glc1
3Man (G-M)
disaccharide.
The highly specific lectin-like interaction of molecular chaperones like calreticulin and calnexin with the N-linked oligosaccharides of glycoproteins represents an intriguing example of the biological role of saccharide chains and in particular extends the function of the polymannose-linked glucose residues beyond that of their well known involvement in the process of cotranslational N-glycosylation(49, 50) . Since it is quite likely, however, that the carbohydrate-protein interaction is only one manner in which the binding of chaperones to polypeptide intermediates is mediated(10, 45, 51) , definition of the mechanisms utilized by various cell types for their diverse secretory proteins will require extensive further investigation.