(Received for publication, May 2, 1995; and in revised form, June 28, 1995)
From the
The target site for N-linked biantennary and
triantennary oligosaccharides containing multiple terminal Le determinants was analyzed in mice. N-Linked
oligosaccharides containing a single tert-butoxycarbonyl-tyrosine attached to the reducing end were
used as synthons for human milk
-3/4-fucosyltransferase to prepare
multivalent Le
(Gal
1-4[Fuc
1-3]GlcNAc) terminated
tyrosinamide oligosaccharides. The oligosaccharides were radioiodinated
and examined for their pharmacokinetics and biodistribution in mice.
The liver was the major target site in mice at 30 min, which
accumulated 18% of the dose for Le
biantennary compared
with 6% for a nonfucosylated Gal biantennary. By comparison,
Le
- and Gal-terminated triantennary accumulated in the
liver with a targeting efficiency of 66 and 59%, respectively. The
liver targeting of Le
biantennary was partially blocked by
co-administration with either galactose or L-fucose whereas
Le
triantennary targeting was only reduced by
co-administration with galactose. In contrast to these results in mice, in vivo experiments performed in rats established that both
Le
and Gal terminated biantennary target the liver with
nearly identical efficiency (6-7%). It is concluded that the
asialoglycoprotein receptor in mice preferentially recognize Le
biantennary over Gal biantennary, whereas little or no
differentiation exists in rats. Thereby, the mouse asialoglycoprotein
receptor apparently possesses additional binding pockets that
accommodate a fucose residue when presented as Le
.
In mammals, carbohydrate/protein interactions often involve the binding of an oligosaccharide ligand to a cell surface receptor(1, 2) . The ligands are most frequently N- or O-linked oligosaccharides that are covalently attached to a glycoprotein. N-Linked oligosaccharides possess a common pentasaccharide core structure which contains a branch point resulting in two or more nonreducing end sugar residues(3) . It is often these terminal sugar residues on N-linked oligosaccharides which bind to spatially resolved binding sites on a lectin(4) .
The mammalian lectins discovered to date have
been grouped into several subcategories(5) . Several membrane
spanning lectins are known to be C-type lectins which contain a
carbohydrate recognition domain named for its calcium-dependent ligand
binding. Of the C-type lectins, the asialoglycoprotein receptor
(ASGP-R) ()found on hepatocytes has been most thoroughly
studied for its binding specificity and its intracellular routing of
ligands(6, 7, 8) . N-Linked
oligosaccharides containing multiple terminal Gal residues bind with
high affinity, although GalNAc terminated N-linked
oligosaccharides are much more potent
ligands(9, 10, 11) . A biantennary
oligosaccharide possessing only two terminal GalNAc residues is a
superior ASGP-R ligand compared with a triantennary possessing three
terminal Gal residues(12) .
In addition to the ASGP-R,
several other mammalian lectins have been isolated and characterized in
liver. Kupffer cells possess a C-type lectin that binds avidly to
Fuc-bovine serum albumin but also has shared affinity for
Le-bovine serum
albumin(13, 14, 15, 16) . A
Gal-specific lectin that binds asialyl-tetraantennary N-linked
oligosaccharides and a Fuc/GlcNAc binding lectin have been found on rat
alveolar macrophages and Kupffer cells(17, 18) . Also,
a lectin that binds GalNAc-4-sulfate on N-linked
oligosaccharides has been shown to exist on endothelial
cells(19) .
The ligand specificity of most mammalian lectins is deduced from an in vitro measurement of their relative affinity for natural glycoproteins, neoglycoproteins, or simple glycosides(20) . However, comparison of results from different binding assays using different receptor preparations contributes to the uncertainty of the identity of the natural ligand(s) for mammalian lectins(21) .
Elucidating the binding specificity of liver receptors is further complicated by the unique specificity found for different animals. For example, chicken hepatic lectin fails to recognize Gal terminated oligosaccharides, but instead functions in binding GlcNAc terminated oligosaccharides(22) . The most prevalent lectin in alligator liver binds Man- and Fuc-terminated ligands(23) . Thereby, the preferred carbohydrate ligand determined using animal models may not necessarily result in comparable binding specificity for liver receptors in man.
In order to discover
the tissue location and binding specificity of mammalian lectins in
their native environment, we have initiated studies that use well
defined N-linked oligosaccharides as probes to reveal lectin
activity resulting from ligand uptake into the target organ. This
approach offers certain advantages in that only high affinity (K > micromole) binding is detected,
all receptors with serum exposure are assayed simultaneously, and the
exogenous ligand must compete with endogenous ligands, leading to
physiologically relevant results that can be directly translated into
the design of glycotargeted drug delivery systems(24) .
In
the present study, we have examined the target site for a series of
Le biantennary and triantennary oligosaccharides. The
results indicate that terminal Gal and L-Fuc residues on
biantennary oligosaccharides are simultaneously recognized by the
ASGP-R in mice but not in rats. These data further exemplify subtle
differences in carbohydrate receptor ligand specificity between closely
related species.
Sodium iodide was purchased from DuPont.
Sephadex G-10, chloramine T, sodium metabisulfite,
-monothioglycerol, alkaline phosphatase, ATP, sodium cacodylate, L-fucose, galactose, and mannose were purchased from Sigma.
Guanosine-diphosphate-L-fucose (GDP-Fuc) was purchased from
Oxford Glycosystems, Abindon, United Kingdom. Human milk was donated by
the neonatal intensive care unit of Children's Hospital,
Columbus, OH. Thin layer chromatography plates (Silica Gel, 60
F
) were from Alltech, Deerfield, IL. Ketamine
hydrochloride was purchased from Parke-Davis. Silastic catheters
(0.012-inch inner diameter
0.025-inch outer diameter) and a
Hamilton polymer PRP-1 (10 µm) reverse phase HPLC column (0.47
25 cm) were purchased from Baxter Scientific, McGaw Park, IL.
Silica C8 (5 µm) reverse phase HPLC columns (0.47
25 cm)
were purchased from Rainin, Emeryville, CA.
Analytical transferase reactions were performed by reacting 2 nmol of tyrosinamide biantennary prepared in 7.5 µl of 25 mM cacodylate enzyme buffer, pH 6.5 (containing 8 mM manganese chloride and 1.6 mM ATP), with 2 µl of enzyme, 1.5 µl (42.4 nmol) of GDP-Fuc, and 1 µl (1 unit) of alkaline phosphatase. The reaction was incubated at 37 °C and after 120 min was diluted 10-fold with water, then analyzed (200 pmol) on analytical C18 reverse phase (RP)-HPLC. The column (50 °C) was eluted isocratically at 1 ml/min with 0.1% acetic acid and 11% acetonitrile while monitoring tyrosine fluorescence at an excitation of 275 nm and an emission 305 nm. Fucosyltransferase activity was quantified from the initial rate (10 min) by integration of the biantennary oligosaccharide peak eluting at 22 min relative to the monofucosylated product eluting at 21 min. Each batch of human milk yielded approximately 5 milliunits of enzyme with specific activity of 150 microunits/mg of protein, where 1 unit is defined as the amount of enzyme to transfer 1 µmol/min of L-Fuc to a biantennary oligosaccharide. Protein concentrations were determined by the method of Bradford(28) .
Preparative fucosyltransferase reactions were performed by treating 500 nmol of biantennary with 4 milliunits of enzyme, 8 µmol of GDP-Fuc, and 5 units of alkaline phosphatase in a total volume of 500 µl of cacodylate enzyme buffer. Triantennary oligosaccharide (500 nmol) was treated with 10 milliunits of fucosyltransferase, 12 µmol of GDP-Fuc, and 5 units of alkaline phosphatase prepared in 500 µl of cacodylate enzyme buffer. The reactions were incubated at 37 °C for 48 h, which resulted in the formation of a single earlier eluting product peak when monitoring by RP-HPLC as described above.
Le oligosaccharides were
purified from the enzyme reaction from a mixed bed ion exchange column
(1
37 cm; top: AG50WX2 acid form, bottom: AG1-X2 acetate form)
eluted with water while detecting A
. The
oligosaccharide peak eluting at the void of the column (14 ml) was
collected and freeze-dried, then purified to homogeneity by RP-HPLC.
Multiple 100-nmol injections were performed on an analytical PRP-1
column eluted at 1 ml/min with 0.1% acetic acid and 11% acetonitrile
while detecting A
0.05 absorbance units at
full scale. The peak eluting at 20-25 min was collected and
freeze-dried, reconstituted in water, and a yield of 70% was determined
by A
(
= 1330 M
).
Monosaccharide analysis of Le biantennary and triantennary oligosaccharides was performed
following trifluoroacetic acid hydrolysis according to method of the
Hardy et al.(29) . Oligosaccharides were prepared for
500-MHz proton NMR spectroscopy by repeatedly freeze-drying
0.4-0.5 µmol in D
O. The sample was prepared in
0.5 ml of 99.98% D
O containing 0.01% acetone as an internal
standard and analyzed on a Bruker 500-MHz NMR spectrometer operating at
23 °C. All spectra were processed utilizing resolution enhancement
parameters supplied by the Felix software package (Hare Research,
Eugene, OR).
Each oligosaccharide was analyzed by FAB-MS by
preparing the sample (5 nmol) in 10 µl of water containing 1 µl
of -monothioglycerol. The water was removed by speed vacuum, and
the 1-µl sample was applied to the probe of the Finigan Matt 900
FAB-MS operated in the positive ion mode.
The pharmacokinetics of
Le-terminated biantennary and triantennary oligosaccharides
were analyzed as described for other
tyrosinamide-oligosaccharides(12) . Serial blood time points
were analyzed by direct
counting, after which the oligosaccharide
was extracted and analyzed by TLC/autoradiography(12) .
Pharmacokinetic parameters were derived from direct blood counts versus time for three data sets for each oligosaccharide.
Iterative nonlinear least squares fits for individual data sets were
obtained with PCNONLIN (SCI Software, Lexington, KY) using a
two-compartment open model as described previously(12) .
In vivo inhibition experiments were performed in mice by jugular vein dosing 10 pmol (1.8 µCi) of oligosaccharide containing 0.1, 0.2, or 0.3 mmol of Man, L-Fuc, or Gal prepared in 200 µl of sterile water. The mice were sacrificed 30-min postadministration and the tissue distribution analyzed as described above.
In vivo saturation experiments were performed by increasing the oligosaccharide dose from 10 pmol to 100 nmol while keeping the radioactive dose (1.8 µCi) constant. Oligosaccharide biodistribution was analyzed at 30-min postadministration as described above.
Asialyl biantennary, with and without core Fuc, and a triantennary oligosaccharide were isolated from porcine fibrinogen and bovine fetuin as described previously(25, 26) . Prior to purification, the oligosaccharides were derivatized at the reducing end to form an oligosaccharide-glycosylamine which coupled to tert-butoxycarbonyl-tyrosine results in tyrosinamide-oligosaccharides(30) . This aglycone was radioiodinated prior to biodistribution experiments, providing oligosaccharide probes that have greater metabolic stability compared with natural glycopeptides, which are rapidly metabolized and exocytosed from liver (12) .
tert-Butoxycarbonyl-tyrosinamide-oligosaccharides were used
as substrates to generate Le-terminated oligosaccharides.
Partially purified human milk fucosyltransferase catalyzed the transfer
of L-Fuc from GDP-Fuc to the 3-hydroxyl group on the
subterminal GlcNAc of each of the antennae of a biantennary or
triantennary oligosaccharide. Optimal reaction conditions were
determined by varying the concentration of GDP-Fuc, fucosyltransferase,
and oligosaccharide substrate until transfer was complete (48 h). The
transferase reactions were analyzed using RP-HPLC while monitoring the
fluorescence of the tyrosinamide group in order to detect earlier
eluting peaks resulting from successive transfer of L-Fuc to
the substrate. Biantennary oligosaccharides produced one intermediate
and a final product (Fig. 1), whereas triantennary resulted in
two intermediates and a single product.
Figure 1: Analysis of fucosyltransferase activity on biantennary by RP-HPLC. A biantennary tyrosinamide-oligosaccharide substrate (200 pmol) (S) is chromatographed on an analytical RP-HPLC column eluted isocratically at 1 ml/min with 0.1% acetic acid and 11% acetonitrile while detecting the fluorescence of the tyrosine group attached to the reducing end (A). After 180-min reaction with fucosyltransferase and GDP-Fuc as described under ``Materials and Methods,'' two early eluting peaks are detected which correspond to monofucosylated (M) and difucosylated (D) oligosaccharides (B). At reaction completion (15 h) only the difucosylated biantennary oligosaccharide is detected (C).
Le oligosaccharides I, II, and III were
prepared in 500-nmol quantities using biantennary and triantennary
substrates (Fig. 2, structures IV and V) and
purified using a polymeric RP-HPLC column. Each purified
oligosaccharide demonstrated the presence of either two or three Fuc
residues by monosaccharides analysis. The detailed structure of
Le
oligosaccharides was elucidated using a combination of
proton NMR and FAB-MS. Mass spectral analysis provided a dominant
molecular ion at m/z of 2363.7, 2217.5, or 2729.8
corresponding to within 0.8 mass unit of the anticipated mass (M +
23) for oligosaccharides I, II, and III,
respectively.
Figure 2:
Structure of Le- and
Gal-terminated biantennary and triantennary
tyrosinamide-oligosaccharides. Oligosaccharides I and II represent
Le
biantennary oligosaccharides, whereas III is a novel
Le
triantennary. Oligosaccharide IV and V were used as
synthons to prepare II and III.
Proton NMR analysis of oligosaccharides I, II, and III resulted in signal patterns consistent with
multivalent Le oligosaccharides (Fig. 3) (29) . Biantennary oligosaccharides I and II had
characteristic anomeric protons for Man-4,4` (5.107-5.109 ppm)
and GlcNAc-1 (4.913-4.917 ppm) but also demonstrated two new
anomeric protons for Fuc A and B (5.123-5.133 ppm). The presence
of two new L-Fuc residues was also observed from the C6 methyl
protons at 1.17-1.18 ppm.
Figure 3:
Partial 500-MHz proton NMR spectra of
LeN-linked oligosaccharides. The Man, Fuc, and
GlcNAc-1 anomeric signals (4.80-5.20 ppm), Gal and GlcNAc
anomeric signals (4.40-4.65 ppm), GlcNAc N-acetyl
protons (1.94-2.12 ppm), and methyl protons of Fuc
(1.12-1.24 ppm) are shown for oligosaccharides I, II, and III.
The structural reporter group signal for I and II were assigned by
comparison with a previous report (31) . The assignments made
to III are tentative. See Fig. 2for structure and residue
nomenclature and Table 1for chemical shift
data.
Triantennary oligosaccharide III represents a novel structure not reported previously. The NMR spectra for III contained anomeric (5.110-5.131 ppm) and methyl resonances (1.173-1.176 ppm) assigned to L-Fuc residues A, B, and C. The chemical shifts for the structural reporter group signals for oligosaccharides I-III are included in Table 1.
Le oligosaccharides I-III were radioiodinated and analyzed for their pharmacokinetics and
biodistribution in mice. Following jugular vein dosing, pharmacokinetic
analysis verified that the oligosaccharides were rapidly cleared from
the blood within 1 h with biphasic kinetics (Fig. 4). Extraction
of oligosaccharides from blood followed by TLC separation and
autoradiography demonstrated that the oligosaccharides were cleared
without the formation of detectable metabolites (Fig. 4).
Therefore, blood time points were directly
-counted and fit by
nonlinear least squares analysis to derive pharmacokinetic constants as
described previously(12) . Oligosaccharides I and II had a total body clearance (Cl
) and steady
state volume of distribution (Vd
) that was
comparable with that reported previously for a Gal biantennary (Table 2)(12) . Also, the presence of a core Fuc residue
on II did not influence the clearance rate of this
oligosaccharide. Oligosaccharide III showed a much more rapid
elimination from the blood as compared with I and II (Fig. 4), resulting in a further elevation of Cl
and Vd
(Table 2).
The mean resonance time, which approximates the serum half-life, was
14-17 min for all three oligosaccharides.
Figure 4:
Pharmacokinetic analysis of Le biantennary and triantennary oligosaccharides in mice. The
concentration of oligosaccharide determined from direct
counting
of blood time points is plotted versus time for
oligosaccharides I, II, and III. The fitted line is the
nonlinear least squares fit of the data determined by PCNONLIN
resulting in parameters reported in Table 2(12) . The right panels illustrate the autoradiograph of TLC analysis of
each time point, demonstrating the lack of significant metabolites. The
rapid clearance of oligosaccharide III is evident from both the direct
counts and the TLC analysis.
Whole body
autoradiography established that at 30-min postadministration,
oligosaccharides I, II, and III each targeted the
liver as the major site with only a minor percentage of the dose
(1-4%) targeting kidney and small intestine (Fig. 5).
Other tissues possessed only background counts, whereas the urine was
the major route of elimination. counting of the major organs
established that biantennary oligosaccharides I and II each targeted the liver with similar efficiency (17-18% of
the dose at 30-min postadministration), whereas triantennary
oligosaccharide III targeted liver with approximately 3-fold
higher (66%) efficiency (Fig. 5). In contrast, studies in rats
revealed the liver targeting for either Le
(II) or
Gal (IV) biantennary was approximately 5-6% (Table 2).
Figure 5:
Biodistribution of Le
biantennary and triantennary in mice. Whole body autoradiography was
used to determine the major target sites for oligosaccharide I, II, and
III (see insets). Quantitative biodistribution was performed
by direct
counting of the dissected tissues (bars). The
targeting efficiency (percent of dose in the target organ at 30-min
postadministration) was 17% for I, 18.5% for II, and 66% for III. The
kidney and small intestine were the only other organs with
radioactivity in excess of 1% of dose. Each oligosaccharide was
analyzed in triplicate.
The elimination rate of biantennary I and II from mouse liver was analyzed to determine if core
fucosylation would slow metabolism as was previously noted for
GalNAc-terminated biantennary oligosaccharide(12) . At times
ranging from 30- to 180-min postadministration, both I and II were eliminated from liver with a similar half-life and appeared in
the small intestine, suggesting that the ligands were internalized into
the target cells and excreted into the bile (Fig. 6). However,
the elimination rate for Le oligosaccharides was
comparatively slower (t =2.5 h) to that determined
previously for GalNAc biantennary (t = 1
h)(12) .
Figure 6:
Elimination rate of biantennary
oligosaccharides I and II from liver. The influence of core
fucosylation on elimination from the target site was examined by
comparing the percent of dose in liver (solid lines) and small
intestine (dashed lines) for biantennary I (,
) and
II (
,
). The results established that core fucosylation
did not influence clearance rate as had been previously detected for
GalNAc biantennary(12) . However, the clearance rate is much
slower (t = 2.5 h) for Le
biantennary versus GalNAc biantennary oligosaccharide. Each data point was
determined from dosing three different
mice.
In vivo inhibition studies were performed by co-administering Man, L-Fuc, or Gal with iodinated II, III, or V in mice. Preliminary experiments established that 0.1-0.3 mmol was adequate to compete for receptor binding, whereas higher concentration resulted in increased mortality of the mice. Also, the viscosity of the dosing solution in these experiments resulted in slightly higher (3-5%) targeting efficiencies compared to dosing in saline.
The liver targeting of
Le biantennary II was not inhibited by
co-administration with Man but was partially inhibited by L-Fuc or Gal (Fig. 7). The most potent inhibitor was
Gal which reduced liver targeting in a dose-dependent fashion to 3%
when 0.3 mmol was used. L-Fuc was a weaker inhibitor of II, only reducing the targeting to 12% at 0.3 mmol (Fig. 7). The liver targeting of Le
triantennary III was also partially inhibited by Gal, resulting in 43%
targeting at a dose of 0.3 mmol of Gal (Fig. 7), but was not
affected by co-administration with Man or L-Fuc. This is in
contrast to triantennary V, which was substantially inhibited to
13% by co-administration with 0.3 mmol of Gal but not with Man or L-Fuc (Fig. 7).
Figure 7:
In
vivo inhibition of Le oligosaccharide liver targeting using
monosaccharides. The targeting efficiency for oligosaccharide II, III,
and V co-administered with either 0.1, 0.2, or 0.3 mmol of Man (open columns), Fuc (striped columns), or Gal (solid columns) are presented. The results indicated that 0.3
mmol of Man failed to inhibit the liver targeting of Le
biantennary II, whereas the targeting was partially inhibited in
a dose-dependent fashion by L-Fuc and significantly inhibited
with Gal. The targeting of Le
triantennary III could not be
inhibited with either Man or L-Fuc, but was partially
inhibited with Gal. Triantennary V was also not inhibited by Man or L-Fuc but was significantly inhibited by Gal. Each bar represents the average of three biodistribution experiments in
mice.
To further substantiate that
Le oligosaccharides underwent receptor-mediated
internalization in mice, 0.3 mmol of L-Fuc or Gal was
administered at 26-min postinjection of radioiodinated II or V. Pharmacokinetic analysis was used to monitor the reappearance
of the radiolabeled ligand in the blood which established that neither
oligosaccharide could be released from the cell surface receptor by
monosaccharide ligand.
To confirm that the liver targeting efficiency was not a function of dose, the targeting efficiency was measured for oligosaccharides dosed in the range of 10 pmol to 100 nmol (Fig. 8). The results indicate that receptor saturation is reached in the range of 10-30 nmol when dosing with either II, III, or V. This established that the targeting efficiency is insensitive to dose in the 10-50-pmol range normally used. The results also suggest that the number of liver receptors that bind oligosaccharides II, III, and V are roughly equivalent.
Figure 8:
Influence of dose on targeting efficiency.
Oligosaccharides II (), III (
), and V (
) were dosed
in mice via vial jugular vein injection in a constant volume of 50
µl containing 10 pmol to 100 nmol of oligosaccharide (1.8 µCi).
At 30 min the liver was removed and the amount of radioactivity
determined by direct
counting. Receptor saturation was only
evident at doses of 10 nmol or greater.
The biodistribution pattern of complex oligosaccharides in mice can be used to search for the existence of new carbohydrate receptors and elucidate the carbohydrate binding specificity of known receptors (12, 32) . Utilizing structurally well defined N-linked oligosaccharides eliminates heterogeneity often encountered in glycoproteins or neoglycoproteins and allows the determination of pharmacokinetic and biodistribution parameters on oligosaccharides that resemble natural ligands found in animal glycoproteins. Thereby, comparison of the bioactivity of closely related oligosaccharides provides the opportunity to study structure activity relationships for N-linked oligosaccharides.
Enzymatic remodeling of the outer antenna of N-linked
oligosaccharides is a powerful approach to improve on the structural
diversity of oligosaccharides obtained from common glycoprotein
sources. The synthesis of multivalent LeN-linked
oligosaccharides reported here, and GalNAc-terminated oligosaccharides
reported previously(12) , are examples of the utility of this
approach to produce rare or completely novel N-linked
oligosaccharides. Although Le
biantennary oligosaccharides
have been previously identified in animal
glycoproteins(33, 34) , these represent rare sources
which would not allow the isolation of micromole quantities of
oligosaccharide necessary to perform detailed in vivo analysis. Likewise, Le
triantennary represents a novel
structure which may exist in nature but most probably in quantities
which would make its detection difficult.
Tyrosinamide derivatization of N-linked oligosaccharides was developed as a means to produce glycoconjugates that could be used to study the pharmacokinetics and biodistribution of N-linked oligosaccharides(12, 32) . However, in addition to radioiodination, the tyrosinamide group aids both in providing enhanced chromatographic resolution on RP-HPLC and in allowing sensitive (100-200 pmol) monitoring via fluorescence(35) . In the present study, the fluorescence of tyrosinamide-oligosaccharides allowed the optimization of the fucosyltransferase reaction using partially purified enzyme obtained from human milk (Fig. 1). This enzymatic assay is able to resolve partially fucosylated intermediates which may find utility when analyzing the enzyme kinetics of fucosyltransferases acting on multivalent oligosaccharides. Additional attributes of tyrosinamide-oligosaccharides, such as their reversibility to form reducing oligosaccharides and their utility in glycopeptide synthesis, have been reported recently(35, 36) .
This study examined the
influence of terminal fucosylation on N-linked
oligosaccharides with respect to target site in mice. The
pharmacokinetic and biodistribution data both indicate that Le biantennary and triantennary oligosaccharides are rapidly cleared
from the blood (t = 14-17 min) and primarily
target the liver (Fig. 5). These experiments do not discern
which of the liver receptors are involved in binding of these ligands,
since the hepatocyte ASGP-R and Kupffer cell fucose receptor of mouse
liver both reportedly bind Le
-terminated neoglycoproteins
to varying degrees(13, 14, 15, 16) .
But given that the number of mouse liver receptors that bind Le
or Gal triantennary and Le
biantennary is roughly
equivalent (Fig. 8), and that both types of ligand are
endocytosed by the liver and eliminated via the bile to the small
intestine, it is assumed in the following discussion that the ASGP-R is
primarily responsible for the liver binding activity being measured in
mice.
The primary specificity of Le oligosaccharide
targeting to liver is related to terminal Gal residues since the
targeting of both II and III were partially inhibitable
by co-administration with Gal but not Man (Fig. 7). However,
several criteria indicate that L-Fuc on I, II,
and III is also recognized by the liver receptor.
Oligosaccharide I and II each possess two Le
determinants resulting in a liver targeting efficiency of
17-18%, whereas Gal biantennary IV targets liver with only
7% efficiency (12) (Table 2). Therefore, either L-Fuc influences the conformation of oligosaccharide I and II to enhance terminal Gal recognition or contributes to
the binding affinity directly by interacting with the liver receptor.
The finding that the liver targeting of Le biantennary II is partially inhibited by co-administration with L-Fuc supports the latter hypothesis. High concentrations
(0.1-0.3 mmol) of L-Fuc were necessary to inhibit the
targeting of II, since monosaccharides are weak ligands for
mammalian lectins compared with multivalent
oligosaccharides(20) . Thereby, this result suggests that the
multivalency of Le
determinants on biantennary contributes
to the tight binding to the mouse liver receptor.
However, the
location of L-Fuc in the oligosaccharide must also be
important, because comparison of I and II indicates that
the presence or absence of core fucosylation on Le biantennary oligosaccharide results in the same degree of liver
targeting activity in mice. Thus, the simultaneous presence of a
terminal Gal and L-Fuc as exists in Le
appears to
be responsible for the enhanced targeting efficiency of biantennary
oligosaccharides. Although L-Fuc is clearly involved in the
binding of Le
biantennary to the liver receptor, its
contribution to the overall affinity is minor relative to Gal which was
found to be a more potent antagonist.
These experiments are
contrasted with those performed in rats which established that both
Le (II) and Gal (IV) terminated biantennary
oligosaccharides target liver equally (5-6%) (Table 2). The
results for Le
biantennary are therefore markedly different
for mice and rats, which strongly suggests this is related to a
specificity difference for the ASGP-R between these two species as
suggested previously(13) .
One of the most potent
Gal-terminated ligands for the ASGP-R is triantennary oligosaccharide V, which has a measured K of 4 nM for binding to rat hepatocytes and a targeting efficiency of 59%
for mouse liver(12, 37) . Le
triantennary III differs from V by the addition of three terminal L-Fuc residues. Surprisingly, the liver targeting of
oligosaccharide III is only 7% higher than V despite this
major structural difference. Therefore, compared with the liver
targeting activity of a GalNAc-terminated triantennary (85%), III displays only a modest enhancement in receptor affinity over V. This may arise from the failure of all three Fuc residues to
participate in binding to the receptor when presented as Le
triantennary III.
Le (III) and Gal (V) terminated triantennary display a nearly indistinguishable
(66 ± 4% versus 59 ± 4%) targeting efficiency in
mice. However, when co-administered with 0.3 mmol of Gal, the liver
targeting of III is inhibited to 43%, while V was
inhibited to 13% (Fig. 8). While it is assumed that triantennary
V is a selective ligand for the ASGP-R, the L-Fuc residues on
Le
triantennary III cause this ligand to retain high
affinity for the liver, even when administered with 0.3 mmol of Gal.
Furthermore, even the highest dose (0.3 mmol) of L-Fuc was
incapable of causing inhibition of the liver targeting of III.
This result could be due to the inability of monovalent L-Fuc
to compete for receptor binding with an oligosaccharides possessing
multiple (three Fuc and Gal residues) determinants. Therefore, the
partial inhibition observed for 0.3 mmol of Gal reflects an enhanced
receptor affinity for III, which most likely occurs as the
result of binding of some of the L-Fuc residues on Le
triantennary.
The presence or absence of a core Fuc did not
affect the elimination rate for Le biantennary I and II from the liver. This could relate to the finding that
Le
oligosaccharides are refractory to digestion with bovine
testes
-galactosidase. Thereby, metabolism may depend on
-fucosidase to act first before exoglycosidase trimming can
proceed. If this enzyme is rate-limiting, it could explain why the
liver elimination rate of biantennary I and II were
identical and much slower than GalNAc biantennary oligosaccharides as
reported previously (12) . Nonetheless, the finding that
Le
biantennary II appears in the bile and could not
be displaced from the liver by postadministration at 26 min with either
0.3 mmol of Gal or L-Fuc suggests this ligand is internalized
into its target cell, which is a property associated with the ASGP-R
but not the fucose receptor(14, 15, 16) .
The dose is an important parameter that must be considered when
designing experiments that attempt to reveal lectin activity in animals (12, 36) . Liver targeting efficiencies in mice were
constant up to a dose of 5 nmol. At doses of 10-100 nmol the
percent of dose bound to the liver decreased. Apparently this occurs as
the receptor becomes saturated, allowing the excess ligand to escape
from the circulation by rapid renal filtration. This suggests that the
number of receptors that bind oligosaccharides III and V are nearly identical and further implicates the involvement of the
ASGP-R, since V is a potent and selective ligand for this
receptor system. However, it is also evident that while the binding
affinity and targeting efficiency of biantennary II is less than
either III or V, the dose that saturates the receptor is
nearly equivalent (Fig. 8). This evidence supports the proposal
that Le biantennary I and II both target the
mouse ASGP-R.
The present study has supplied new information on the
biodistribution of Le containing N-linked
oligosaccharides. Differences in the targeting efficiency for
biantennary ligands in mice and rat liver point to subtle differences
in receptor specificity. It will be important to determine if the human
ASGP-R also preferentially binds Le
- over Gal-terminated N-linked oligosaccharides and if so how L-Fuc orients
in the binding pocket.