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J Biol Chem, Vol. 273, Issue 52, 35016-35022, December 25, 1998
From the The interactions of lectins with multivalent
carbohydrates often leads to the formation of highly ordered
cross-linked lattices that are amenable to structural studies. A
particularly well ordered, two-dimensional lattice is formed from
fucose-specific isolectin A from Lotus tetragonolobus
cross-linked with difucosyllacto-N-neohexaose, an
oligosaccharide possessing the Lewisx determinant, which is
an oncofetal antigen. A combination of electron microscopy, x-ray
diffraction, simulation of electron micrographs, and molecular model
building was used to determine the relative positions of the tetrameric
lectin and bivalent carbohydrate within the lattice. X-ray diffraction
from unoriented pellets was used to determine the lattice dimensions
and analysis of electron micrographs was used to determine the lattice
symmetry. Molecular models of the lattice were constructed based on the
known structure of the jack bean lectin concanavalin A and the high
degree of sequence homology between the two lectins. Using the symmetry and dimensions of the lattice and its appearance in filtered electron micrographs, molecular models were used to determine the orientation of
the lectin in the lattice, and to define the range of
lectin-oligosaccharide interactions consistent with the structural
data. The present study provides the first description of a highly
ordered, two-dimensional, cross-linked lattice between a tetravalent
lectin and a bivalent carbohydrate.
The carbohydrate moieties of glycoproteins and glycolipids have
been shown to be involved in a variety of biological recognition processes including cell-cell and cell-substratum interactions, immunity, apoptosis, and metastasis of tumor cells (1-5). The composition and structures of the oligosaccharides correlate with cell
differentiation and transformation (cf. Ref. 6). For
example, the expression of oligosaccharides possessing specific Lewis
blood group antigenic determinants is developmentally regulated and altered as a result of differentiation and oncogenic transformation (7). Some antigens such as the Lewisx
(Lex)1 antigen
are transiently expressed during ontogeny and reappear in tumors, and
have hence been termed oncofetal antigens (cf. Ref. 5).
The molecular recognition properties of the oligosaccharide chains of
glycoproteins and glycolipids are often characterized in terms of their
interactions with lectins (8). A number of mammalian lectins are
involved in receptor-mediated endocytosis of glycoproteins
(cf. Ref. 2), while others have been implicated in cellular
recognition processes including apoptosis (4) and metastasis (9,
10).
The biological signal transduction properties of lectins appear to be
due to their ability to bind and cross-link specific glycoprotein and
glycolipid receptors on cells. For example, lectin-induced cross-linking of receptors often leads to mitogenic responses in cells
(11), in the arrest of bulk transport in ganglion cell axons (12), in
the induction of mating reactions in fungi (13), in the molecular
sorting of glycoproteins in the secretory pathways of cells (14), and
in the apoptosis of activated human T-cells (4). Furthermore,
lectin-induced cross-linking of transmembrane glycoproteins leads to
changes in their interactions with cytoskeletal proteins and
concomitant alterations in the mobility and aggregation of other
surface receptors (15, 16).
Studies have shown that lectins undergo two general types of
cross-linking interactions with multivalent carbohydrates, designated type 1 and type 2 complexes (17). In a type 1 complex, binding between
a divalent lectin and a divalent carbohydrate results in one
dimensional cross-linking (e.g. helical). In a type 2 complex, binding between a multivalent lectin and multivalent
carbohydrate, where the valency of either the lectin or carbohydrate is
greater than 2, results in two-dimensional (planar or tubular) or
three-dimensional cross-linking (crystalline). Importantly, type 2 interactions can lead to the formation of homogeneous cross-linked
complexes, even in the presence of mixtures of the molecules
(cf. Ref. 17). Hence, type 2 interactions are an important
source of binding specificity between lectins and glycoconjugate receptors.
In order to understand the molecular basis of these cross-linking
interactions, x-ray crystallography has been used to investigate several lectin-carbohydrate cross-linked complexes. These include two
type 1 complexes involving dimeric animal lectins cross-linked with
divalent oligosaccharides (18, 19). Of type 2 complexes that have been
determined, the three-dimensional crystal structures of the tetrameric
soybean agglutinin (SBA) cross-linked with four biantennary
carbohydrates have been reported (20, 21). The x-ray crystallographic
images of those structures, refined to 2.4-2.8-Å resolution, indicate
that the stability of the lattices are due to protein-carbohydrate
interactions, with essentially no direct lectin-lectin interactions.
The results also demonstrate that each carbohydrate cross-linked
complex with SBA is a unique lattice, in agreement with previous work
on type 2 complexes (cf. Ref. 17). The structure of the
snowdrop lectin cross-linked with a Man5 oligomannose glycopeptide (22)
is another example of a type 2 complex. Interestingly, the glycopeptide
exhibits two modes of binding, one involved in cross-linking and one
involved in extended site interactions with individual subunits. More
recently, the crystal structure of the tetrameric Maclura
pomifera agglutinin cross-linked with the T-antigen disaccharide
at 2.2-Å resolution has been reported (23). The T-antigen disaccharide
functions as a divalent ligand in the complex, even though its binding
epitopes are asymmetric.
An exception to the above type 1 and type 2 complexes is the x-ray
crystal structure reported for the wheat germ agglutinin cross-linked
complex with a glycopeptide isolated from glycophorin (24). The
cross-linked complex consists of divalent-divalent lectin-carbohydrate
interactions, in addition to carbohydrate-carbohydrate stabilizing
interactions in the complex. It thus resembles a type 1 interaction
with secondary homophilic interactions of the carbohydrate ligand.
In the present paper, a structural analysis of the type 2 complex
formed by the Fuc-specific tetrameric isolectin A from the seeds of
Lotus tetragonolobus (LTL-A) with
difucosyllacto-N-neohexaose (Fuc-octa) is described using
x-ray diffraction, electron microscopy, image analysis and model
building. Our previous work has demonstrated that Fuc-octa binds as a
bivalent ligand to LTL-A, and is able to cross-link and precipitate the
lectin along with two other analogs of the oligosaccharide (25). The
molecular recognition properties of Fuc-octa are important since it is
a naturally occurring biantennary fucosyl oligosaccharide possessing
the dimeric Lex blood group antigen (Fig.
1). The Lex determinant is an
oncofetal antigen since it is transiently expressed during ontogeny and
reappears later in a variety of neoplastic diseases (cf.
Ref. 5). For example, elevated levels of the Lex antigen
are present in patients with lung cancer, and colon and liver
adenocarcinomas (5).
LTL-A is a member of the legume family of lectins, which have been
widely used to explore the properties of membranes from both normal and
transformed cells (26, 27). The x-ray crystal structures of many
members of this family have been determined, including concanavalin A
(ConA) (28-30), favin (31), pea lectin (32), lentil lectin (33),
Lathyrus ochrus isolectins I and II (34, 35),
Griffonia simplicifolia lectin IV (36), Erythrina corallodendron lectin (37), peanut agglutinin (38), SBA (20), and
phytohemagglutinin-L (39). The results demonstrate that this family of
proteins have very similar monomeric structures (40), but that they
often differ in their dimeric and quaternary structures, particularly
among tetrameric lectins such as ConA, the peanut agglutinin, and SBA.
Like most legume lectins, LTL-A is highly homologous to other members
of this family of proteins, including ConA, the prototypical Man/Glc-specific lectin from jack bean (41). LTL-A is a tetramer possessing one carbohydrate binding site per subunit of
Mr 28,000; hence, it is tetravalent in its
carbohydrate binding activity (42). Precipitation of LTL-A by three
different fucosyl biantennary oligosaccharides, including Fuc-octa,
produces three distinct, highly organized, cross-linked lattices as
observed by negative stain electron microscopy (25). The molecular
basis for the stability and uniqueness of these lattices has not been
established. The LTL-A/Fuc-octa cross-linked lattice was chosen for
analysis because of its highly regular structure as observed by
electron microscopy (25). Furthermore, unlike previously described type 2 complexes, which possess three-dimensional lattice structures (20-23), the LTL-A/Fuc-octa complex is the first example of a
two-dimensional lattice that is of the type likely to occur on the
surface of a cell. The present study thus describes the structure of
the lattice in some detail.
Specimen Preparation--
Seeds of L. tetragonolobus
(syn. Tetragonolobus purpureas) were purchased from
Schumacher & Co. The native lectin mixture was purified from the crude
extract (prepared according to Ref. 42) by affinity chromatography as
described previously (43). The major isolectin, LTL-A, was separated by
DEAE-cellulose chromatography (44). Fuc-octa was obtained from BioCarb
Chemicals, Lund, Sweden. The concentrations of sugars were measured by
the phenol-sulfuric acid method (45, 46). The monosaccharides were
obtained from Sigma. LTL-A/Fuc-octa precipitations were performed at
4 °C in a final volume of 0.1 ml by using 0.1 M Tris-HCl
buffer, pH 7.2, containing 0.9 M KCl, 1 mM
MnCl2, and 1 mM CaCl2 as described previously (54). Concentrations of LTL-A (monomer) and Fuc-octa were
100 µM and 90 µM, respectively.
X-ray Diffraction--
Pellets of the LTL-A/Fuc-octa precipitate
were aspirated into clean, thin-walled glass capillaries (0.5 or 0.7 mm
diameter), and sealed with wax. X-ray diffraction patterns from the
precipitates were obtained using radiation from a rotating anode x-ray
source (Rigaku, RU200). The double-mirror camera system (47) was used (CuKa; Electron Microscopy--
Precipitates were negatively stained on
300-mesh, carbon-coated Parlodion grids which had been freshly glow
discharged, touched to filter paper, floated on a drop of 1%
phosphotungstic acid, pH 7.0, and blotted immediately. Samples were
observed at 80 kV in a JEOL 1200EX electron microscope (25).
Electron micrographs were digitized using an Eikonix EC850 digital
imaging camera system. Optical densities of the electron micrographs in
the range of 0-2 O.D. were coded into 8 bits (integers 0-255). At the
setting used, each pixel corresponded to 2.07 Å in the structure. The
areas selected from the micrographs for digital filtering contained
~260 unit cells.
To extract the periodic information and determine the symmetry of the
lattice in projection, the electron micrographs were filtered to
improve the signal to noise ratio (48). This procedure was implemented
by computing the Fourier transform of the image, multiplying the
transform by a set of Gaussian functions (one around each of the
reciprocal lattice points), and then computing a back Fourier transform
to obtain the filtered image.
Electron Micrograph Simulation--
To investigate the
orientation of the LTL-A tetramer in the lattice, a computer simulation
of the filtered electron micrographs was employed. Negative staining
was simulated by computing the projected volume of the molecules, in
essence to computationally surround the protein with "stain" of
uniform density in a manner similar to that used by Steven and Navia
(49). No attempt was made to augment the density near charged groups
(which would correspond to positive staining). The structure of the
protein tetramer was modeled by using the three-dimensional atomic
coordinates of the ConA tetramer (28), giving each atom a "weight"
proportional to its van der Waals volume. ConA tetramers were rotated
computationally into orientations consistent with the lattice symmetry
and then projected onto the viewing plane. Calculations were made to
determine the possible effect of multiple molecular layers on the
images obtained. Further, the relative positions of the two dimers
making up the tetramer of LTL-A were varied as suggested by differences that have been observed in the dimer-dimer interactions in the homologous leguminous lectins. Mean phase differences were used as a
criterion to evaluate the quality of simulations as detailed by
Cabral-Lilly et al. (50).
Molecular Model Building--
Using atomic coordinates for
ConA (28) obtained from the Brookhaven Protein Data Bank, a fragment of
the cross-linked lattice was constructed using the molecular software
package FRODO on a Silicon Graphics IRIS workstation. Lattice
dimensions were taken from the results of x-ray powder diffraction and
the positions of the LTL-A dimers in the lattice from comparison of
simulated and actual electron micrographs. Models for the lattice were
examined to determine the presence of forbidden contacts and the
relative positions of the carbohydrate binding sites.
X-ray Diffraction--
X-ray diffraction patterns (Fig.
2) obtained from pelleted samples of the
LTL-A/Fuc-octa precipitates consist of a set of concentric reflections
falling at the reciprocal spacings listed in Table
I. All the observed reflections can be
indexed on an orthorhombic lattice with lattice dimensions 85.1 × 76.1 × 121.8 Å. As shown below, the 76.1 × 121.8 Å dimensions correspond closely to those observed in electron micrographs
of the lattice in projection. The 85.1 Å dimension corresponds to the
thickness of the two-dimensional lattice. Only two "meridonal"
reflections (second and third order of the 85.1 Å repeat) are
observed. This suggests a partial stacking of the lattices in the
pellet. The remainder of the observed reflections correspond to
"equatorial" reflections from the two-dimensional lattice. This is
typical of diffraction from two-dimensional arrays in which the
meridional and equatorial reflections usually dominate the diffraction
pattern, with off-equatorial reflections other than the meridionals
being very weak. The presence of partial stacking of the lattices was
confirmed using electron microscopy of frozen-fratured pellets (data
not shown). These micrographs indicate that in most cases the lattices
stack in register.
Electron Micrographs--
In electron micrographs of negatively
stained specimens (e.g. Fig.
3A), the stain-excluding
regions appear to be concentrated along two sets of intersecting lines
rotated by 62-65° relative to one another. Dimensions determined
from the micrographs varied slightly, but fell within expected errors
of the dimensions obtained in the x-ray powder diffraction experiments.
Stain is concentrated in diamond- or triangular-shaped regions between
these two linear arrays. Some lattices exhibited substantially greater
contrast than others, suggesting the presence of multiple layers.
However, the overall distribution of stain-excluding material in the
lattices appeared independent of the contrast, suggesting that if
lattice stacking did occur, the lattices stack in register. This is
consistent with the indexing of the x-ray powder diffraction patterns
indicating an orthorhombic lattice and electron micrographs of
frozen-fractured pellets.
The power spectrum (square of the Fourier transform) of the electron
micrograph in Fig. 3B (enlargement of Fig. 3A) is
shown in Fig. 3C. Peaks fall on a lattice with dimensions of
approximately 1/76 × 1/122 Å Electron Micrograph Simulations--
LTL-A is 41.7% identical in
sequence to ConA, 39.6% identical to favin, and 40.8% identical to
pea lectin (41). The three-dimensional structure of the dimers of ConA,
favin, and pea lectin are very similar; the root-mean-square difference
in
To generate all possible orientations of the LTL-A tetramer consistent
with the observed symmetry of the lattice, models were constructed
using the known molecular structure of ConA. Each of the three
molecular two-fold axes of the tetramer were aligned with the
crystallographic axis in turn. The tetramer was then rotated about that
axis in 3° steps, and a simulated micrograph was calculated. The
resolution of the simulation was limited to 19 Å to match that of the
electron micrographs. Fourier transforms of the simulated micrographs
were calculated and a mean phase difference between Fourier transforms
of simulated and observed electron micrographs was calculated. Plots of
mean phase difference indicated models most consistent with the
electron micrographs, but the correspondence between simulated and
actual micrographs remained unsatisfactory for even the best models.
The failure of the original modeling to adequately simulate the
electron micrographs indicated the need to consider additional modeling
parameters. Two that were considered were the possibility of multiple
layers and variation of the dimer-dimer contact angle.
To test for the effect of multiple layers, two- and three-layer models
were simulated and searches identical to that described above were
carried out for a broad range of relative positions of the layers.
Correspondence between calculated and observed micrographs became
progessively worse for shifts of multiple layers relative to one
another. All multi-layer models resulted in poorer correspondence
between calculated and observed than for the one-layer models.
Better results were obtained when the relative orientation of the two
LTL-A dimers in a tetramer were varied relative to one another. This
involved rotating the dimers about the two-fold axis that relates one
monomer to the other in each dimer. This variation was suggested by the
fact that the structural homology among lectin dimers is very high, but
the relative orientations of the dimers relative to one another is
somewhat variable. Models were generated by rotating dimers up to
25.0° relative to one another. The best correspondence between
calculated and observed micrographs and the lowest mean phase
difference occurred for a relative rotation of the dimers of about
20° in a direction that brought them more nearly parallel to one
another. Correspondence of the simulated micrograph with a filtered
electron micrograph is demonstrated in Fig.
4.
Molecular Model Building--
Construction of a molecular model of
the cross-linked lattice made it possible to evaluate potential models
for the lattice based on other physical criteria. A viable model for
the lattice must have monomers with sugar binding sites separated by an
appropriate distance for cross-linking by Fuc-octa. Therefore, the size
of the cross-linking carbohydrate provides a constraint on the
molecular structure of the lattice. The distance between the two
binding epitopes in the oligosaccharide must match the distance between the two carbohydrate-binding sites on adjacent proteins in the lattice.
Three rotamer conformations about the
The second criteria for modeling the lattice is to exclude those
exhibiting molecular interpenetration (impossibly close contacts) between the protein molecules. Using a molecular model of LTL-A based
on a pair of ConA dimers rotated relative to one another, potential
models were evaluated on these two criteria. Many models were excluded
based on the separation of adjacent binding sites. Forbidden contacts
provided less constraint on the possible models as the protein-protein
contacts in the lattice are remote from the center of the tetramer. The
model with the lowest mean phase difference proved to have acceptable
relative positions of the carbohydrate binding sites, and essentially
no forbidden contacts.
Molecular Model of the LTL-A Lattice--
The candidates for the
best model for the cross-linked lattice were selected using the
amplitude-weighted mean phase difference and the carbohydrate-binding
site distance as criteria. These candidates were then displayed using
an optimized gray scale to allow visual comparison of the simulated
images with the filtered electron micrographs. The model having the
best overall agreement between calculated and observed features is
obtained using the dimers of ConA rotated relative to one another by
20.0° about the x-axis (the two-fold axis relating
monomers in a single dimer). The molecular y-axis in ConA
corresponds to the crystallographic two-fold axis (mirror plane) in the
two-dimensional lattice. The best simulation corresponds to a 12°
rotation of the tetramer about this y-axis. The thickness of
the two-dimensional array modeled in this way was calculated to be 85 Å. This thickness corresponds with the thickness calculated from the
positions of meridional x-ray reflections observed in the powder
pattern (Table I). The resulting simulation of an electron micrograph
is compared with a filtered image in Fig. 4, and the corresponding
molecular model is drawn in Fig. 6.
Fig. 6 shows the relative positions of four lectin tetramers in the
lattice. Only The model of the LTL-A/Fuc-octa lattice presented in Fig. 6
provides substantial information about the way in which LTL-A is
cross-linked by Fuc-octa. The full 222-point group symmetry of the
lectin is not reflected in the lattice symmetry, as only one molecular
two-fold axis is crystallographic. The structure of Fuc-octa is
apparently not consistent with a lattice in which the point group
symmetry of the LTL-A is optimally crystallographic. Rotation of the
LTL-A tetramer by 12° about the y-axis would result in a lattice that
would correspond to a single layer of a lattice with C222 symmetry. In
this lattice, the cross-linking carbohydrate would span a two-fold
axis. Fuc-octa with its The LTL-A tetramer is made up of a pair of dimers rotated approximately
20° relative to one another as compared with dimers in the highly
homologous ConA tetramers. This conclusion was reached by comparison of
the calculated and observed electron micrographs of tetramers
constructed from dimers at different relative orientations. The
accuracy of the model presented here depends on the structures of ConA
and LTL-A being very similar. The difference in monomer structure
between ConA and LTL-A should be small (40, 41), making the ConA
monomer an adequate model for the simulation of electron micrographs at
20 Å resolution. The present findings that suggest a different
quaternary structure for LTL-A relative to ConA are supported by known
differences in the quaternary structures of ConA (cf. Ref.
28), the peanut agglutinin (38), and SBA (20).
The structure of the LTL-A/Fuc-octa lattice can be compared with the
structures of the cross-linked lattices formed between SBA and four
biantennary pentasaccharides (20, 21). In those structures, each of the
four three-dimensional lattices formed by SBA and the oligosacharides
are completely stabilized by carbohydrate cross-links that span the
inter-protein space. There are no protein-protein interactions between
lectin tetramers in the crystals. The presence of any protein-protein
interactions at the tetramer-tetramer interface of the LTL-A/Fuc-octa
lattice is difficult to assess based on the model constructed here.
Interactions between adjacent tetramers are focussed at the ends of
LTL-A also forms cross-linked lattices with at least two other
biantennary fucosyl oligosaccharides (25). One is similar to the
lattice described here, while the other forms a helical aggregate in
which the topology of interactions is substantially different from that
described here. The affinity of LTL-A for all three fucosyl
oligosaccharides is similar, which suggests that their interactions
with the protein are similar. This suggest that differences in the
overall structures of the carbohydrates account for differences in the
structures of the respective cross-linked lattices. Similar findings
have been observed in the structures of cross-linked lattices formed
between SBA and four analogs of the blood group I carbohydrate antigen
(21).
The present study provides insight into the structure of the
LTL-A/Fuc-octa cross-linked complex. The results indicates that LTL-A
and Fuc-octa form a highly organized two-dimensional cross-linked lattice. The findings demonstrate that the structure and symmetry of
the LTL-A/Fuc-octa lattice is dependent on the geometry and symmetry of
the binding sites of the lectin and the binding epitopes of the
carbohydrate. The present study thus provides evidence for the
formation of a highly ordered two-dimensional type 2 cross-linked complex between a tetravalent lectin and a bivalent carbohydrate of the
type likely to occur on the surface of a cell. The ability of lectins
to form two- and three-dimensional type 2 organized clusters with
specific carbohydrate receptors may relate to the biological activities
of these proteins. In addition, since Fuc-octa possesses the dimeric
Lex determinant, which is an oncofetal antigen, the
cross-linking properties of the oligosaccharide may be important in its
biological activities.
We thank Lyn Dean and Jane Fant, Analytical
Ultrastructure Center, Albert Einstein College of Medicine, for
assistance in preparing the electron micrographs. We thank Terence
Russell, Physics Department, Boston University, for help in x-ray
diffraction and preliminary model building.
*
This work was supported by National Institutes of Health
(NIH) Grant CA-16054 (to C. F. B.), NIH Grant GM-29829 and
National Science Foundation grants (to L. M.), and NIH Core Grant
P30 CA-13330 (to C. F. B).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Current address: Dept. of Biophysics, Boston University School of
Medicine, Boston, MA 02118-2526.
**
To whom correspondence may be addressed: Depts. of Molecular
Pharmacology, Microbiology and Immunology, Albert Einstein College of
Medicine, Bronx, NY 10461.
The abbreviations used are:
Lex, Lewisx blood group; LTL-A, isolectin A from the seeds of
L. tetragonolobus; ConA, lectin from jack bean
(Canavalia ensiformis); SBA, lectin from soybean
(Glycine max); Fuc-octa, difucosyllacto-N-neohexaose. All sugars are in the
D-configuration except L-fucose.
Electron Microscopy and X-ray Diffraction Studies of
Lotus tetragonolobus A Isolectin Cross-linked with a
Divalent Lewisx Oligosaccharide, an Oncofetal Antigen*
,§,
,
Department of Physics, Boston University,
Boston, Massachusetts 02215, the ¶ Departments of Molecular
Pharmacology, Microbiology and Immunology, Albert Einstein College of
Medicine, Bronx, New York 10461, and the
Institute of Molecular Biophysics, Florida
State University, Tallahassee, Florida 32306-3015
ABSTRACT
Top
Abstract
Introduction
Results
Discussion
Conclusions
References
INTRODUCTION
Top
Abstract
Introduction
Results
Discussion
Conclusions
References
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Fig. 1.
Structure of
difucosyllacto-N-neohexaose (Fuc-octa).
Fuc, Gal, GlcNAc, and GlcOH
represent fucose, galactose, N-acetylglucosamine, and
glucose residues, respectively, in the oligosaccharide.
EXPERIMENTAL PRODCEDURES
, = 1.54 Å) to produce an x-ray beam with
dimensions of approximately 0.3 × 0.3 mm at the film (Kodak
diagnostic x-ray film). Specimen-to-film distances were 70-130 mm for
most experiments, and the exposure times were usually 24-72 h.
RESULTS
Top
Abstract
Introduction
Results
Discussion
Conclusions
References
View larger version (166K):
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Fig. 2.
X-ray diffraction pattern of a pellet of the
precipitates. Approximately a dozen reflections are visible in
this pattern. These reflections are listed in Table I and are indexed
on an orthorhombic lattice with lattice dimensions 85.1 Å × 76.1 Å × 121.8 Å. Exact positions of the reflections were taken from a
densitometer trace of the diffraction pattern after circular
averaging.
X-ray spacing data for the LTL-A/Fuc-octa cross-linked complex
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Fig. 3.
A, Electron micrograph of negatively
stained precipitates of LTL-A with Fuc-octa (protein is white). The
protein forms two sets of continuous, stain-excluding strands, rotated
by 62-65° relative to one another. Each continuous strand is formed
by end-to-end binding of dimers to one another. At each vertex, a pair
of dimers, one on top of the other, form a tetramer. B,
enlargement of a portion of the electron micrograph in A.
C, power spectrum of the electron micrograph in
B. D, filtered image of electron micrograph from
B.
1. Peaks are
observable to approximately 19 Å resolution in transforms from the
most highly ordered specimens. Fig. 3D is a filtered image
of the electron micrograph of the lattice in Fig. 3B. In the
filtered images of the LTL-A lattices, the stain-excluding material is
concentrated at the vertices of the intersecting lines of
stain-excluding material noted in the original micrographs. The LTL-A
tetramer appears to be centered at these points. The micrograph in Fig.
3 has mirror lines through these points, corresponding to in-plane
two-fold axes in the lattice. The ConA tetramer has 222-point group
symmetry and it is likely that the LTL-A tetramer also exhibits this
symmetry. One of the molecular two-fold axes probably corresponds to
the observed lattice two-fold, greatly limiting the possible models for
the molecular structure of the lattice. Modeling of the lattice
structure requires the identification of the crystallographic two-fold
axis and the location of the two molecular two-fold axes that are
non-crystallographic.
-carbon positions being 0.87 Å for 185 equivalent -carbons in
ConA and pea lectin (32), and 1.4 Å for the 217 common -carbons of
ConA and favin (51). The largest differences are near the ends of the
permuted sequence in ConA, and in the vicinity of small insertions and deletions. The difference in dimer structure between ConA and LTL-A
should be comparable, making the ConA dimer structure a suitable model
for the LTL-A dimer at low resolution.
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Fig. 4.
Comparison of the simulated electron
micrograph calculated from the molecular model of LTL-A (A)
with the filtered micrograph (B). The simulated image
was calculated with ConA dimers rotated 20° about the
x-axis to form the model for the LTL-A tetramer and the
tetramer rotated 12° about the y-axis.
(1-6) linkage of Fuc-octa are
possible, with 
= 
60°, 60°, and 180° ( is the dihedral angle formed by the O-5, C-5, C-6, and O-6 atoms of the core Gal residue). The -60° conformation is considered less energetically favorable by approximately 1-2 kcal/mol by analogy with the solution structure of lacto-N-neohexaose, a biantennary carbohydrate
with LacNAc residues linked (1-6) and (1-3) to a core Gal (52). As demonstrated below, the tetramer-tetramer contact stabilized by
Fuc-octa is at a pseudo-two-fold axis. All three (1-6) rotamer conformations of Fuc-octa possess pseudo-two-fold axes with respect to
the outer Fuc residues which are the binding epitopes for LTL-A (cf. Ref. 53). However, as discussed below, the = 60°
rotamer of Fuc-octa (Fig. 5) is
consistent with the approximate two-fold symmetry of the lattice. In
this conformation, the distance between the centers of the two fucose
residues is approximately 18 Å. If the two carbohydrate-binding sites
are much greater than 18 Å apart, it would be impossible for the
oligosaccharide to cross-link adjacent tetramers. If the
carbohydrate-binding sites are too close to one another, the
oligosaccharide could not participate in an energetically favorable
cross-link. Therefore, the distance between the two
carbohydrate-binding sites on adjacent tetramers should be
approximately 18 Å.
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Fig. 5.
Corey-Pauling-Koltun (CPK) model of Fuc-octa
with rotamer conformation = 60° for its (1-6) arm as
indicated by the symmetry of the lattice. The angle is the
dihedral angle formed by the O-5, C-5, C-6, and O-6 atoms of the core
Gal residue. Fuc, Gal, GlcNAc, and
GlcOH represent fucose, galactose,
N-acetylglucosamine, and glucose residues, respectively, in
the oligosaccharide.
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Fig. 6.
Molecular model of the relative positions of
four LTL-A tetramers in the lattice. The molecular model of the
ConA dimer as determined by x-ray crystallography has been used as a
basis for predicting the molecular structure of LTL-A. Only -carbon
positions are indicated: A, view from the top; B,
edge on view of the lattice. Alternating LTL-A tetramers are colored
white and green. Fuc-octa is not shown.
-carbon positions are indicated. In Fig. 6A, the tetramers are seen from the same angle as in the
electron micrographs and simulated images. Fig. 6B is an
in-plane view of the lattice. In this view, the two tetramers at the
center are superimposed. The 12° tilt of the tetramers in the lattice is clear in this image.
DISCUSSION
Top
Abstract
Introduction
Results
Discussion
Conclusions
References
(1-6) arm in the = 60° rotamer
conformation has a pseudo-two-fold axis of symmetry with repect to the
outer Fuc residues (Fig. 5) but not an exact two-fold axis. The
relative positions of the outer Fuc residues are consistent with the
observed lattice symmetry. This can be compared with the conformation
of a biantennary pentasaccharide possessing LacNAc arms linked
(1-6) and (1-3) to a core Gal residue that was previously shown
to bind with the (1-6) arm in the = 60° rotamer conformation
in its cross-linked complex with SBA (21). In this latter case, the
pentasaccharide also possesses a pseudo-two-fold axis of symmetry with
respect to its LacNAc moieties that is consistent with the lattice
symmetry of the complex.
-sheets in a region rich with -turns. The homology between ConA
and LTL-A is lower in the loop regions than in the core, and the
locations of the interacting loops in LTL-A are not necessarily
homologous to those in ConA. A definitive answer as to whether there
are protein-protein interactions in the LTL-A lattice will have to
await higher resolution data. Nevertheless, the LTL-A/Fuc-octa lattice
is dissolved upon addition of competing monovalent carbohydrate
(i.e. Fuc), or prevented from forming in the presence of the
monosaccharide. This indicates that the stability of the lattice is
predominantly due to protein-carbohydrate interactions, as observed in
the SBA/pentasaccharide lattices (21).
CONCLUSIONS
Top
Abstract
Introduction
Results
Discussion
Conclusions
References
ACKNOWLEDGEMENTS
FOOTNOTES
Current address: Merck Sharpe & Dohme Research Laboratories,
West Point, PA 19486.
REFERENCES
Top
Abstract
Introduction
Results
Discussion
Conclusions
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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