(Received for publication, October 10, 1995; and in revised form, November 28, 1995)
From the
Asialoglycoprotein receptors on the surfaces of both hepatocytes and peritoneal macrophages bind terminal galactose residues of desialylated glycoproteins and mediate endocytosis and eventual degradation of these ligands. The hepatic receptor binds oligosaccharides with terminal N-acetylgalactosamine residues more tightly than ligands with terminal galactose residues, but the macrophage receptor shows no such differential binding affinity. Carbohydrate recognition domains from the macrophage receptor and the major subunit of the hepatic receptor have been expressed in a bacterial system and have been shown to retain the distinct binding selectivities of the receptors from which they derive. Binding of a series of N-acyl derivatives of galactosamine suggests that the 2-substituent of these sugars interacts with the surface of the hepatic receptor with highest affinity binding observed for the N-propionyl derivative. Chimeric sugar-binding domains have been used to identify three regions of the hepatic receptor that are essential for establishing selectivity for N-acetylgalactosamine over galactose. Based on these results and the orientation of N-acetylgalactosamine when bound to an homologous galactose-binding mutant of rat serum mannose-binding protein, a fourth region likely to interact with N-acetylgalactosamine has been identified and probed by site-directed mutagenesis. The results of these studies define a binding pocket for the 2-substituent of N-acetylgalactosamine in the hepatic asialoglycoprotein receptor.
Asialoglycoprotein receptors have been identified in both
mammalian liver and peritoneal macrophages(1, 2) . The
rat hepatic receptor, also known as the rat hepatic lectin RHL, ()is made up of three subunits: RHL-1 (41.5 kDa), RHL-2 (49
kDa), and RHL-3 (54 kDa)(3) . Each subunit is a type II
transmembrane protein, with a short NH
-terminal cytoplasmic
domain, an internal signal membrane anchor, and a COOH-terminal
Ca
-dependent carbohydrate recognition domain (CRD)
linked to the membrane through an intervening neck region. The major
subunit, RHL-1, represents about 70-80% of the total mass of the
receptor. The minor subunits, RHL-2 and RHL-3, are differentially
glycosylated forms of a second, homologous polypeptide. The purified
receptor is a hexamer in detergent solution, although the stoichiometry
of the different polypeptides is still unclear(4, 5) .
In contrast, the macrophage galactose receptor (MGR) contains a single
type of subunit (42 kDa) that forms homooligomers(2) . The CRDs
of MGR and RHL-1 show 77% sequence identity, whereas there is 54%
identity between RHL-2/3 and MGR. Although the CRDs of MGR and RHL-1
are quite similar to each other, the macrophage receptor has a shorter
cytoplasmic tail and an inserted segment of 24 amino acids between the
CRD and the membrane-spanning domain(2) .
Despite the
similarities in the sequences of the hepatic and macrophage receptors,
they have distinct sugar-binding properties. The hepatic receptor binds
GalNAc with higher selectivity than Gal, whereas the macrophage
receptor binds Gal and GalNAc with roughly equal affinity. For example,
in binding competition assays GalNAc competes for binding to the
hepatic receptor from rabbits between 8- and 20-fold more effectively
than Gal(6, 7) . In contrast, an assay measuring I-asialoorosomucoid uptake by macrophages in the presence
of monosaccharide inhibitors shows that GalNAc and Gal bind equally
well(8) . Sugar binding studies using the mouse macrophage
receptor show a similar pattern, although the exact results depend on
what type of assay is used. In a sugar inhibition assay, Gal and GalNAc
bind with approximately equal affinity(9) , whereas a
competitive enzyme-linked immunoabsorption assay suggests that Gal
binds with 3-fold higher selectivity than GalNAc(10) . In spite
of small variations in the different assay protocols, all of the
studies indicate that the hepatic receptor binds GalNAc far more
effectively than does the macrophage receptor.
A Gal-binding site
closely similar to that of the rat hepatic asialoglycoprotein receptor
has been created by modification of the CRD of rat serum
mannose-binding protein (MBP-A)(11) . For high affinity Gal
binding, residues Gln, Asp
, and Trp
must be inserted into the Man-binding framework. A glycine-rich
inserted sequence is necessary for the ability to exclude Man. The
crystal structure of this mutant, QPDWG, has been solved alone and in
complex with Gal and GalNAc(12) . Although these studies
provide insight into the mechanism of Gal and GalNAc binding to the
asialoglycoprotein receptor CRDs, they do not explain differential
binding of these two sugars to the hepatic receptor.
The CRDs of RHL-1 and MGR have been produced in a bacterial expression system and the sugar binding properties of these CRDs have been characterized. Amino acid residues likely to be involved in the selective binding of GalNAc to RHL-1 have been identified by analysis of chimeric and mutagenized versions of the CRDs.
Modified BamHI-HindIII fragments were inserted into
expression vector pT5T, which contains the bacteriophage T7 promotor
(see Fig. 1)(18) . The pT5T vector was altered by the
addition of the synthetic oligonucleotide shown in Fig. 1at the BamHI site in the polylinker, which causes termination of the
T7 Gene 10 product followed by reinitiation to make the CRD. The
oligonucleotides used for the liver receptor and the macrophage
receptor were identical. The polypeptide produced in each case is
extended at the NH terminus by several residues. The first
cysteine residue has been changed to serine because it would normally
be paired with a cysteine residue further toward the NH
terminus of the intact receptor polypeptide. The preceding
alanine in the macrophage receptor is changed to isoleucine as well.
Chimeric and mutant constructs were created and sequenced in vector
pGEM-3 and transferred to the pT5T vector by moving a BamHI-HindIII fragment. The expression
construct was transformed into Escherichia coli strain
BL21/DE3, in which the T7 RNA polymerase gene is under control of the lac promoter and operator(19) .
Figure 1:
Schematic diagram of
expression plasmids for CRDs of RHL-1 and MGR along with predicted
protein products. The pT5T expression vector was modified with
synthetic oligonucleotides introduced at the BamHI site in the
polylinker. The resulting sequence directs termination of the T7 Gene
10 product followed by reinitiation to make the CRD. The polypeptide
produced is extended at the NH terminus by the residues in italics. The terminal methionine residue is removed in the
bacteria, and the first cysteine residue of each CRD is changed to
serine (underlined).
Figure 2:
Oligonucleotides used to generate point
mutations in the CRD of MGR and RHL-1. The sequences of oligonucleotide
pairs used to introduce changes into regions 1, 2, 3, and 4 are
indicated along with the restriction sites at which these fragments
were inserted. Region 4 mutations were created by replacing a portion
of the cDNA for the region 3 Ser Thr mutation, replacing
nucleotides encoding residue 259 with the original serine
codon.
where MAX is the total amount of radioactivity bound in the
absence of competing monosaccharide. Mean ± standard deviation
values for at least three independent assays were used to calculate K values.
where BKG is the background binding, SLOPE is the linear
increase in nonspecific binding, MAX is the saturation level for
specific binding, and K is the concentration of
ligand at which half-maximal specific, saturable binding is achieved.
Assays were performed in duplicate. Values reported are the mean
± standard deviation for three independent assays, except in
certain cases when only a single assay was performed.
Figure 3: Purification of the CRDs from RHL-1 and MGR expressed in bacteria. The major peaks eluted from Gal-Sepharose affinity columns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie Blue. Lane 1, RHL-1. Lane 2, MGR. Molecular mass standards are indicated at left.
Solid phase competition binding assays in which
CRDs immobilized on polystyrene wells are probed with I-Gal
-BSA were used to characterize ligand
binding by the bacterially expressed CRDs. As shown in Fig. 4,
RHL-1 alone in the absence of RHL-2/3 mimics the highly
GalNAc-selective binding of the intact receptor, because GalNAc
competes for binding to RHL-1 60-fold more effectively than does Gal.
In contrast, MGR does not show this preferential binding. The absolute
affinities of the two proteins for Gal
-BSA are essentially
the same (68 ± 7 nM for RHL-1 and 69 ± 11 nM for MGR), so the difference between the two CRDs in the
competition assay reflects much higher affinity of RHL-1 for GalNAc.
These results indicate that the different selectivities of the intact
hepatic and macrophage receptors are mirrored in the properties of
isolated CRDs from RHL-1 and MGR.
Figure 4:
Solid phase binding competition assays of
the CRDs of RHL-1 and MGR. Experimental data (filled circles)
are shown for I-Gal
-BSA binding in the
presence of either Gal or GalNAc along with theoretical curves (continuous lines) fitted to the data. Left, results
for RHL-1. Right, results for MGR.
The nature of the interaction
between GalNAc and RHL-1 was also probed using a series of
galactosamine derivatives. The ability of a series of N-acyl
derivatives of galactosamine to compete with I-Gal
-BSA for binding to the CRD of RHL-1
are compared in Table 1. As the length of the side chain
increases from formyl to propionyl, the affinity for RHL-1 increases.
The propionyl derivative has the highest affinity for RHL-1 of any
monosaccharide tested, with larger chains resulting in decreased
affinity. These data suggest that there may be a hydrophobic region of
the RHL-1 CRD that accommodates small alkyl chains on the 2 substituent
of galactosamine.
Chimeric cDNAs were constructed using restriction sites that are common to both RHL-1 and MGR cDNAs (Fig. 5). The chimeric proteins expressed from these hybrid cDNAs were then tested in solid phase binding competition assays. The results summarized in Table 2reveal that all of the 60-fold selectivity for GalNAc can be accounted for in the COOH-terminal 81 residues of RHL-1 (amino acids 204-284). Although the COOH-terminal 81 residues of RHL-1 are necessary for 60-fold preferential binding of GalNAc, smaller effects are observed when shorter segments of RHL-1 are substituted into MGR. Stepwise substitution of portions of the COOH terminus of RHL-1 suggests that at least three regions contribute to high selectivity GalNAc binding. For example, incorporation of just the COOH-terminal 40 residues of RHL-1 into MGR results in a marginal increase in GalNAc binding selectivity. A more substantial 2-fold increase occurs when a further 25 residues of RHL-1 are included. The major, 20-fold increase in GalNAc selectivity occurs only when residues between 80 and 65 residues from the COOH terminus of RHL-1 are present.
Figure 5: Sequences of RHL-1/MGR chimeras and mutants of MGR in regions 1, 2, and 3. The amino acid sequence of the CRD of MGR is shown at the top. For RHL-1 and all chimeric constructs, only the amino acids that differ from MGR are indicated. Approximate locations of restriction sites in the corresponding cDNAs used in making chimeric constructs are indicated by arrows.
To confirm the importance of each region individually, smaller portions of RHL were used to replace corresponding parts of MGR (Fig. 5). The results of binding studies for these constructs (Table 2), combined with the single cross-over data, define three regions that contribute to GalNAc binding. The largest effect maps between residues 204 and 218 of RHL-1. Incorporation of this segment, designated region 1, into MGR increases the selectivity of MGR for GalNAc by 20-fold. Region 2, which comprises residues 219-243 of RHL-1, produces an additional 2-fold increase in GalNAc-binding affinity. Inclusion of region 3, running between residues 244 and 284 of RHL-1, causes a further slight increase in GalNAc binding selectivity.
Because incorporation of asparagine at position 230 in MGR
is sufficient for relatively high selectivity GalNAc binding, other
amino acids were tested at this position to probe the interaction that
leads to such high selectivity. All of the amino acids that were
substituted at this position are tolerated better than valine (Table 3), which is the amino acid present in MGR. Because
alanine can substitute for Asn at position 230, the presence of
substituents beyond the -carbon must not be necessary for high
affinity GalNAc binding. It is important to note that wild-type MGR,
mutant MGR/1N, and the other proteins mutants tested have nearly
identical absolute affinities for Gal
-BSA (Table 3),
indicating that the presence of Val
in wild-type MGR
affects only the extra affinity resulting from interactions with the
acetamido group of GalNAc but not other interactions common to Gal and
GalNAc.
Figure 6:
Molecular
interaction between RHL-1 and GalNAc. A, model of GalNAc bound
to RHL-1. The side chains of RHL-1 that have been shown to affect
selective binding of GalNAc have been inserted at the corresponding
positions of the crystallographically determined structure of the QPDWG
mutant of MBP-A in complex with GalNAc(12) . The substitutions
made correspond to: Asn (RHL-1) replacing Ser
(MBP-A); Arg
replacing Lys
;
Gly
replacing Asp
; His
replacing Thr
; Phe
replacing
Ile
; Thr
replacing Val
; and
Thr
replacing Asp
. Regions 1-3 are in black, whereas region 4 is in gray. The remainder of
the polypeptide is shown as an
-carbon trace only. B,
possible interactions between residues in regions 1-4 and GalNAc.
The relative importance of each side chain to the total binding
affinity is indicated the by number adjacent to the arrow, denoting fold enhancement of binding affinity for
GalNAc relative to Gal.
The
large effect on GalNAc binding resulting from changes in region 1 is
surprising because of its probable position in the CRD. In the model
shown in Fig. 6A, the shortest distance between the
side chain of Asn of RHL-1 and the N-acetyl
group of GalNAc is approximately 7 Å. This observation, together
with the fact that many amino acids at this position support high
selectivity GalNAc binding, suggests that preferential binding to
GalNAc by mutant MGR/1N is unlikely to result from direct contact with
the sugar, but the effect of the residue at position 230 could be
mediated through interactions with other amino acids in the protein.
As might be expected from the predicted
orientation of Phe toward the interior of the CRD,
substitution with isoleucine (mutant MGR/1N/4F
I) results in only
a slight loss of selectivity for GalNAc (Table 4). Replacement of
Thr
with alanine (mutant MGR/1N/4T
A) has only a
2-fold effect on GalNAc binding. However, substitution of His
with alanine results in an almost total loss of GalNAc
selectivity. Given the apparent importance of histidine at position
278, it can be suggested that the decrease in GalNAc selectivity in the
alanine mutant at positions 280 may result from changes in the position
of the histidine residue. To probe the nature of the interaction of
His
with GalNAc, individual changes were made at this
position. None of the amino acids substituted support GalNAc-binding
selectivity comparable with that seen when His
is present (Table 4).
The importance of histidine in region 4 was further
substantiated by incorporating the change His
Ala
in the CRD of RHL-1 rather than in the MGR background. As shown in Table 4, this change results in 25-fold loss of relative affinity
for GalNAc without detectable change in the absolute affinity for
Gal
-BSA. This finding confirms the importance of
His
in RHL-1 and verifies that the high affinity
GalNAc-binding site created in the context of MGR accurately models the
natural site in RHL-1. In all cases, the dramatic effects of changing
the histidine residue in region 4 on binding of GalNAc compared with
Gal are not accompanied by substantial changes in the absolute affinity
for Gal
-BSA, demonstrating that although this histidine
plays a critical role in establishing the high affinity of RHL-1 for
GalNAc, it is does not form part of the binding site for Gal.
Residue
His was changed to alanine in mutant
MGR/1N/2A
R,2K
G, resulting in the loss of high selectivity
GalNAc binding as expected. However, the effect on GalNAc binding seen
in the presence of the region 2 changes alone is still evident, because
mutant MGR/1N/2A
R,2K
G/4H
A binds GalNAc with at least
2-fold higher selectivity than does wild-type MGR CRD (Table 5).
These data suggest that the effects of the residues in region 2 are
independent of the interactions of His
in RHL-1.
The data presented indicate that substitution of four amino acids, asparagine at position 230, arginine at position 258, glycine at position 260, and threonine at position 281 of MGR, is sufficient to give GalNAc binding that is comparable with that of native RHL-1. Using the structure of the QPDWG mutant of MBP-A as a scaffold, the relative positions of the amino acid side chains known to be required for high affinity binding of GalNAc to RHL-1 can be modeled as shown in Fig. 6A. Although the conformations of the side chains are hypothetical, a binding surface can be formed around the atoms on the acetamido substituent of the sugar, with the relative importance of specific interactions shown schematically in Fig. 6B.
The most important interactions documented in the present studies
are with His of RHL-1, which is predicted to be
positioned approximately halfway between the terminal methyl group of
the 2 substituent of GalNAc and the side chain of Asn
.
The proximity of Asn
to His
(under 4
Å in the model) is consistent with the hypothesis that the
presence of valine at position 208 decreases the affinity of the CRD
for GalNAc indirectly by altering the disposition of the histidine
residue. The fact that many other side chains are tolerated at position
208 is also consistent with this suggestion.
The approximate
distance from the imidazole ring of His to the terminal
methyl group of the N-acetyl moiety in GalNAc is also under 4
Å in the model, suggesting that there may be a direct interaction
between His
and the 2 substituent of GalNAc. Such an
interaction would be consistent with the observed importance of this
amino acid side chain for the binding of GalNAc but not of Gal. From
the enhancement in GalNAc binding affinity, it can be calculated that
the interaction with His
contributes approximately 8
kJ/mol to the free energy of binding GalNAc. The exact nature of the
interaction is not obvious from the data available. Because asparagine
and glutamine might be expected to form hydrogen bonds similar to those
made by the imidazole ring of histidine, the failure of these amino
acids to support tight binding of GalNAc argues against hydrogen
bonding. Conversely, packing of the methyl group of the N-acetyl moiety against the imidazole ring might be important,
but the inability of tyrosine to sustain tight binding does not support
this suggestion. These results must be interpreted with caution,
because it is possible that some of the changes may result in
structural changes in the adjacent region of the CRD, so that
asparagine, glutamine, and tyrosine side chains might be positioned
differently from the imidazole ring of histidine and thus be
unavailable for making the same types of interactions. The only other
obvious possible direct contacts are in region 2, because Arg
has the potential to form a hydrogen bond with the carbonyl
oxygen on the acetyl portion of the sugar, which is located less than 4
Å away. The interaction with Arg
contributes
approximately 2 kJ/mol to the total free energy of GalNAc binding to
RHL-1.
Although these studies do not allow a detailed description of the nature of the interactions between these amino acids and the 2 substituent of GalNAc, they are certainly consistent with the probable orientation of the sugar ligand predicted from the three-dimensional structure of the QPDWG mutant of MBP-A complexed with GalNAc. The amino acids required for selective high affinity binding of GalNAc more or less surround the acetamido group. It will be of interest to determine how this pocket is able to accommodate the various acyl groups with longer alkyl chains (Table 1).
It is noteworthy that the GalNAc-binding site involves residues from several different portions of the RHL-1 CRD, in contrast with the relatively local region of the MBP-A CRD that is involved in Man binding. As noted previously, the minor subunit of hepatic asialoglycoprotein receptor, RHL-2/3, shows equal affinity for Gal and GalNAc and thus resembles MGR in its ligand-binding properties. Examination of the RHL-2/3 sequence reveals that of the critical residues in regions 1-4 of RHL-1, none are present. From an evolutionary perspective, the need for amino acid substitutions in at least four regions of the polypeptide to achieve tight binding to GalNAc suggests that this specificity has been selected in RHL-1 to serve an important biological function, although there has been no such selective pressure on RHL-2/3 or MGR. Evaluation of potential naturally occurring ligands for each of these receptors, possibly bearing distinct patterns of terminal GalNAc and Gal residues, will be of interest.