(Received for publication, January 17, 1996; and in revised form, February 6, 1996)
From the
Several lines of evidence indicate that calreticulin has
lectin-like properties. As a molecular chaperone, calreticulin binds
preferentially to nascent glycoproteins via their immature
carbohydrates; this property closely resembles that seen for calnexin,
a chaperone with extensive molecular identity to calreticulin. A cell
surface form of calreticulin also exhibits lectin-like properties,
binding specific oligomannosides including those covalently linked to
laminin. In the present study we examined the interaction between
calreticulin and laminin by means of surface plasmon resonance. The
results show that calreticulin specifically binds to glycosylated
laminin but fails to specifically bind tunicamycin-derived
unglycosylated laminin or bovine serum albumin. Calreticulin binding to
glycosylated laminin requires calcium and is abolished in the presence
of EDTA. Scatchard analysis of binding yields an apparent association
constant, K, of 2.1 ± 0.9
10
M
while kinetic analysis
yields an estimate of the association on rate, (K
), as 2
10
M
s
. The composite
results support calreticulin's lectin-like properties as well as
its proposed role in laminin recognition, both in the cell interior and
on the cell surface.
Calreticulin is found in many different locations in various
eukaryotic cells, including the lumen of the endoplasmic reticulum
(ER), ()the cell surface, perinuclear areas, and cytosolic
granules(1) . Some of these locations appear cell-specific,
that is not all cells exhibit calreticulin at each location. The ER
lumen is the most common location of calreticulin, a site where it is
found in abundance(2) . Given its strong avidity for calcium,
calreticulin has been proposed to serve as a major calcium-sequestering
protein within cells. Recently, calreticulin has been implicated as a
molecular chaperone for nascent glycoproteins(3, 4) ;
this activity resembles that of calnexin, a glycoprotein-selective
chaperone whose domain structure substantially overlaps with that of
calreticulin(5, 6) . Both chaperones appear to
selectively bind immature N-glycosyl groups of a nascent
glycoprotein in addition to binding hydrophobic regions of the protein
itself. Cell surface calreticulin also binds specific carbohydrates,
recognizing oligomannoside structures that are identical to those of
nascent glycoproteins(7, 8, 9) .
These
emerging lines of evidence describe lectin-like properties of
intralumenal and cell surface calreticulin. Ligand binding studies
indicate that intralumenal calnexin preferentially recognizes the
immature glycosyl structure,
GlcMan
GlcNac
(10) ; indirect
data suggest that intralumenal calreticulin recognizes similar
structures (3, 4) . Binding studies of intact cells
show that cell surface calreticulin recognizes mannan, Man
,
Man
, and laminin oligomannosides but not mannose or
Man
(7, 8) . In the present study we
examine the interaction of calreticulin with glycosylated
Engelbreth-Holm-Swarm tumor laminin, which contains a repertoire of
immature to mature N-linked carbohydrates ranging from
oligomannosides to complex triantennary saccharides (11, 12, 13) , or with tunicamycin-derived
laminin, lacking such carbohydrates. Surface plasmon resonance was used
to detect and measure binding affinity; this method has the advantages
of high sensitivity and generation of real-time data(14) .
Calreticulin was kindly provided by R. Freedman; it had been purified from bovine liver ER(2) . Glycosylated laminin was purified from mouse Engelbreth-Holm-Swarm tumor while unglycosylated laminin was purified from a mouse cell line incubated in tunicamycin(15) . Tunicamycin-derived laminin lacks detectable N-linked carbohydrates, and its protein molecular structure appears virtually identical to glycosylated laminin(15) .
Binding analysis of the interaction between calreticulin and laminin
was performed on either a BIAcore or BIAcore 2000 biosensor (Pharmacia
Biosensor, Uppsala, Sweden) using contemporary technology(14) .
Experiments were performed at 25 °C in 10 mM HEPES-buffered saline, 150 mM NaCl, and 0.005% surfactant
P20 (Pharmacia) either with calcium (2 mM CaCl) or
without (10 mM EDTA). Proteins were coupled to the sensor chip
through free amino groups. The carboxymethylated dextran surface
(sensor chip CM5, Pharmacia) was first activated by addition of 0.2 MN-ethyl-N`-(3-diethylaminopropyl)-carbodiimide
and 0.05 MN-hydroxysuccinimide (Pharmacia amine
coupling kit), followed by addition of protein, either laminin,
unglycosylated laminin, or bovine serum albumin (BSA), at a
concentration of 20 µg/ml in 10 mM sodium acetate, pH 4.5.
Remaining N-hydroxysuccinimide esters were blocked by the
addition of 1.0 M ethanolamine hydrochloride, pH 8.5. Several
different protein concentrations were immobilized in order to optimize
conditions. In the experiments shown immobilization conditions were
controlled such that all three proteins gave approximately 3000
resonance units of immobilized material.
Figure 1:
Binding of calreticulin in the presence
of calcium. In separate experiments, calreticulin solution ranging from
0.5 10
to 2.0
10
M was applied to three different protein surfaces. Upper panel, glycosylated laminin surface; middle
panel, unglycosylated laminin surface; lower panel,
bovine serum albumin surface. RU, resonance
units.
Figure 2: Calreticulin binding to laminin. Calreticulin binding to glycosylated and unglycosylated laminin was measured in the presence of calcium or EDTA. RU, resonance units.
Figure 3: Scatchard analysis of calreticulin binding to glycosylated laminin in the presence of calcium. RU, resonance units.
Figure 4: Binding of calreticulin in the presence of EDTA. Titration was the same as described for Fig. 1. RU, resonance units.
The objective of this study was to further explore interactions between calreticulin and the N-linked carbohydrates of laminin. Conceivably, intralumenal glycosylated laminin chains and/or assembled laminin molecules may transiently bind to molecular chaperones, including those which recognize glycoproteins. The present results provide a firm basis for potential ER intralumenal binding of glycosylated laminin molecules to calreticulin. Presumably, individual laminin subunits bind to calreticulin, followed by laminin molecular assembly, perhaps while the subunits are still complexed to the chaperone. Notably, laminin synthesized in the presence of tunicamycin fails to be secreted(15) , perhaps reflecting the inability of certain chaperones such as calreticulin to properly interact with the unglycosylated protein.
Lectins require a suitable cation, often calcium, for sustaining their carbohydrate binding properties. Oligomannosides have been specifically implicated in cell surface calreticulin binding to laminin(7) . The present results bolster the interpretation that calreticulin has lectin-like activity by demonstrating that it binds to glycosylated laminin in the presence of calcium while EDTA abolishes such binding. Calreticulin fails to bind to unglycosylated laminin, further substantiating its lectin-like properties. Intralumenal calreticulin binds nascent transferrin(4) , a glycoprotein, and appears to interact with nascent myeloperoxidase via that glycoprotein's N-linked oligomannosides(3) , thereby resembling the binding of calnexin to nascent glycoproteins(20) . Presumably, both intralumenal calreticulin and calnexin rely upon calcium to support their lectin-like activities.
Binding of carbohydrate ligands to both
plant (24) and animal lectins (25) has been evaluated
by surface plasmon resonance. Association on rates ranging from 1.63
10
to 5.7
10
M
s
were found,
and association constants ranging from 6.2
10
to
4.3
10
M were reported. Our results for
calreticulin binding to glycosylated laminin yield an on rate and
association constant, which differ from those values, perhaps
reflecting biological variation between various lectins and their
ligands. Given the disparate molecular sizes of calreticulin (43 kDa)
and laminin (about 900 kDa) and the magnitude of the sensorgram
signals, which reflect their binding, more than one calreticulin
molecule may bind each laminin molecule. Additional studies will be
needed to quantitatively substantiate this interpretation.
In mouse
melanoma cells the calreticulin-laminin complex itself may reach the
cell surface, accounting for calreticulin appearance on the surface (9) and consistent with the observation that these cells
produce and release laminin(21) . A precedent for postulating
such a pathway is that intralumenal calnexin, complexed to antigen
receptor proteins, reaches the surface of immature
thymocytes(22) . This complex transits to the thymocyte surface
from the ER, due to impairment of internal recycling of calnexin. The
authors speculate that surface calnexin may mediate cell-cell
lectin-dependent interactions and may also generate intracellular
signals. It is already clear that surface calreticulin recognizes
laminin (9) and fibrinogen (23) ; such recognition
leads to specific cellular responses in each instance. Mouse melanoma
cells, adherent to laminin, will spread once their surface calreticulin
binds to a suitable oligomannoside(7) . Human fetal fibroblasts
bind the fibrinogen B chain via surface calreticulin, thereby
stimulating cell proliferation(23) . Thus, cell signaling may
be mediated by a new class of cell surface receptors, those which have
lectin-like properties.
These studies measured the binding affinity
of bovine ER-derived calreticulin and murine laminin. Given that
calreticulin structure is highly conserved (18) and that the
oligomannoside N-linked structures are virtually
species-independent, it is not surprising that cross-species binding
occurs. At present, it appears that intracellular and cell surface
calreticulins may recognize similar carbohydrate structures, but direct
comparison of these two proteins will be required to precisely define
their ligand affinities. Interestingly, calreticulin, anchored in the
ER membrane by a genetically engineered calnexin transmembrane domain,
behaved more like calnexin than did the native non-anchored form of
calreticulin(4) . The anchored calreticulin efficiently bound
the same spectrum of nascent glycoproteins as calnexin, while the
non-anchored calreticulin did so with far less efficiency. Binding of
both forms of calreticulin required the presence of appropriate
oligosaccharide structures on the nascent glycoproteins. By analogy, we
speculate that cell surface calreticulin of mouse melanoma cells, which
we find cannot be removed by exhaustive washing ()and is
therefore retained at the surface membrane, will differ in lectin
binding from intralumenal calreticulin. Studies are in progress to
investigate this possibility.