In vivo recognition of mannosylated proteins by hepatic
mannose receptors and mannan-binding protein
Praneet
Opanasopit,
Keiko
Shirashi,
Makiya
Nishikawa,
Fumiyoshi
Yamashita,
Yoshinobu
Takakura, and
Mitsuru
Hashida
Department of Drug Delivery Research, Graduate School of
Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
 |
ABSTRACT |
In vivo recognition of
mannosylated proteins by hepatic mannose receptors and serum
mannan-binding protein (MBP) was investigated in mice. After
intravenous administration, all three different 111In-mannosylated proteins were taken up mainly by
liver, and uptake was saturated with increasing doses.
111In-Man-superoxide dismutases and
111In-Man12- and
111In-Man16-BSA had simple dose-dependent
pharmacokinetic profiles, whereas other derivatives
(111In-Man25-, -Man35-, and
-Man46-BSA and 111In-Man-IgGs) showed slow
hepatic uptake at <1 mg/kg. Purified MBP experiments in vitro
indicated that these derivatives bind to MBP in serum after injection,
which interferes with their hepatic uptake. To quantitatively evaluate
these recognition properties in vivo, a pharmacokinetic model-based
analysis was performed for 111In-Man-BSAs, estimating some
parameters, including the Michaelis-Menten constant of the hepatic
uptake and the dissociation constant of MBP, which correlate to the
affinity of Man-BSAs for mannose receptors and MBP, respectively. The
dissociation constant of Man-BSA and MBP decreased dramatically with
increasing density of mannose, but the Michaelis-Menten constant of
hepatic uptake of Man-BSA was not so sensitive to the change in
density. This suggests that the in vivo recognition of MBP has a
stronger cluster effect than that of mannose receptors. Differences
obtained here are due to the unique arrangement of carbohydrate
recognition domains on each mannose-specific lectin available for
mannosylated ligand recognition.
mannose-specific lectin; pharmacokinetics; liver; drug
delivery
 |
INTRODUCTION |
THE MANNOSE RECEPTOR IS
A C-type lectin containing multiple carbohydrate recognition
domains (CRDs) and is expressed on Kupffer cells (16),
alveolar (42), peritoneal (40), and splenic macrophages (9), monocyte-derived dendritic cells
(4), and subsets of vascular and lymphatic endothelial
cells (44). It specifically recognizes and binds to
ligands having terminal nonreducing D-mannose,
N-acetylglucosamine, or L-fucose units in a
Ca2+-dependent manner (2, 30). These
carbohydrates are not normally displayed at the ends of carbohydrate
chains of mammalian cells but are frequently found on the surfaces of
microorganisms (1). Several in vivo functions have been
proposed for the mannose receptor on macrophages: endocytosis of
extracellular peroxidases and hydrolases during the resolution phase of
inflammation (39), phagocytosis of unopsonized pathogens
(41), and antigen capture for eventual presentation to T
cells (37).
Besides the mannose receptor, mannan-binding proteins (MBPs) also bind
to a variety of pathogens by recognizing D-mannose, N-acetylglucosamine, or L-fucose on their
surface (52). MBP is a large oligomeric serum protein of
hepatic origin, and it belongs to the family of
Ca2+-dependent collagenous lectins, most of which are
components of the innate immune system (12). MBP also
activates the complement system following binding to its ligands in an
antibody- or C1q-independent manner (15, 27). It has been
isolated from serum of several species, including human (43,
48), rabbit (20), rat (36), bovine (18), and mouse (8, 13).
Both groups of mannose-specific lectins, i.e., mannose receptors and
MBP, are believed to be involved in the in vivo recognition of
glycoproteins terminating with mannose. Taylor et al. (46) carried out a pharmacokinetic analysis of the in vivo uptake and processing of mannose-terminating glycoproteins by rat hepatic mannose
receptors. However, they did not examine the binding of the ligands to
serum lectins, such as MBP, an event that might affect their
biodistribution profiles. An in vitro experiment indicated that human
serum lectins inhibit the uptake of glycoproteins by the hepatic
mannose receptor (47). Although the recognition characteristics of both mannose-specific lectins have been extensively investigated in vitro (22, 29-30, 45, 46), it is not
yet known whether serum mannose-specific lectins affect the in vivo hepatic uptake of mannosylated ligands via mannose receptor-mediated endocytosis.
In vitro observations suggest that the configuration, number, and
density of mannose on ligands are critical determinants for their
interaction with both types of lectins (22-23, 29, 45,
46). Furthermore, since the MBP is a relatively bulky and large
lectin with several CRDs, the size of the ligands is an important
factor in determining their recognition. Therefore, to elucidate the in
vivo recognition characteristics of mannosylated ligands by hepatic
mannose receptors and MBP, we designed the following experiments. Three
proteins, recombinant human superoxide dismutase (SOD, 32 kDa), bovine
serum albumin (BSA, 67 kDa), and bovine immunoglobulin G (IgG, 150 kDa)
were selected as model ligands having different molecular
masses. Since these proteins themselves have no
mannose-terminating oligosaccharides, they were chemically modified
with mannose to obtain mannosylated glycoproteins with varying numbers
of mannose residues. Their in vivo disposition was examined in mice
after intravenous bolus injection at different doses, and their
biodistribution profiles were analyzed using a pharmacokinetic model to
obtain parameters representing the receptor affinity of both lectins.
The importance of the degree of mannosylation and the molecular mass of
ligands is discussed in relation to the molecular characteristics of
mannose receptors and MBP.
 |
MATERIALS AND METHODS |
Materials.
BSA (fraction V) and IgG were purchased from Sigma (St. Louis, MO).
Recombinant human SOD was supplied by Asahi Kasei (Tokyo, Japan).
D-Mannose was obtained from Nacalai Tesque (Kyoto, Japan). 111InCl3 was supplied by Nihon Medi-Physics
(Takarazuka, Japan). All other chemicals were reagent-grade products
obtained commercially.
Animals.
Male ddY mice (25-28 g) were obtained from the Shizuoka
Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan). All procedures were examined by the Ethics Committee on Animal
Experiment at the Kyoto University, and animal care was in accordance
with the National Institutes of Health Guidelines for Animal
Experiments and the law of the Japanese government.
Synthesis of glycosylated proteins.
Coupling of mannose moieties to protein was carried out by the method
of Lee et al. (24). Briefly, cyanomethyl
2,3,4,6-tetra-O-acetyl-1-thiomannoside was prepared from the
respective pseudothiourea derivatives and chloroacetonitrile. The
nitrile group in these cyanomethyl thioglycosides can be converted to a
methyl imidate group by treatment with sodium methoxide in dry methanol
to yield 2-imino-2-methoxyethyl 1-thioglycosides. Cyanomethyl
1-thiomannoside was treated with 0.01 M sodium methoxide at room
temperature for 24 h, and a syrup of
2-imino-2-methoxyethyl-1-thiomannoside was obtained after evaporation
of the solvent. A quantity of the resultant syrup was added to protein
in borate buffer (pH 9.5). The number of mannose residues per protein
molecule was controlled by the molar ratio of the starting reagents.
After 24 h at room temperature, the reaction mixture was dialyzed
to remove any unreacted compound and lyophilized. The physicochemical
properties of the synthetic mannosylated proteins with different sugar
densities are summarized in Table 1. The
apparent molecular mass of each mannosylated protein was estimated by
SDS-PAGE. The number of mannose residues was determined by calculating
the mannose content of the mannosylated protein solution using the
anthrone-sulfuric acid method (7). The protein content was
calculated by subtracting the weight of mannose from that of
mannosylated protein. The final number of mannose residues was obtained
by dividing the molar amount of mannose by the molar amount of protein.
111In labeling of mannosylated proteins.
Each mannosylated protein was radiolabeled with 111In using
the bifunctional chelating agent diethylenetriaminepentaacetic acid (DTPA) anhydride, according to the method of Hnatowich et al. (10). Briefly, 10 µl DTPA anhydride in dimethyl
sulfoxide was added to each mannosylated protein. The mixture was
stirred for 30 min at room temperature and purified by using a Sephadex
G-25 column to remove any unreacted DTPA. To
111InCl3 solution was added 1 M sodium acetate
(pH 6.0), followed by DTPA-coupled mannosylated protein. After 30 min,
the mixture was purified using a PD-10 column and the fractions
containing derivatives were collected and concentrated by
ultrafiltration. The protein concentration was determined by the method
of Lowry et al. (25).
In vivo distribution experiment.
Each 111In-labeled mannosylated protein in saline was
injected intravenously into mice via the lateral tail vein at a dose of 0.05, 0.1, 1, 10, or 20 mg/kg. At periods (1, 3, 5, 10, 30, and 60 min)
after injection, blood was collected from the vena cava under ether
anesthesia. The mice were then killed; the heart, lung, liver, spleen,
kidney, and muscle were removed, rinsed with saline, and weighed; and
then the radioactivity was assayed in a well-type NaI scintillation
counter (ARC-500; Aloka, Tokyo, Japan).
Calculation of area under the curve and clearances.
The distribution data of compounds after intravenous injection were
analyzed in terms of the organ uptake clearance (CLorg) (33). Because of limited efflux of radioactivity from
tissues due to the characteristics of 111In-DTPA labeling,
CLorg (ml/h) can be expressed (see Ref. 34 for
details) as
|
(1)
|
where Xi (normalized to % of the dose)
represents the amount of radioactivity in tissue i after the
administration of the 111In-labeled compound,
Cp (% of dose/ml) is the concentration of radioactivity in
the plasma, and AUCt (% of
dose · h · ml
1) represents the area under
the plasma concentration-time curve from time 0 to
t, calculated by fitting a monoexponential equation to the
plasma concentration-time data of the 111In
radioactivity-time profile using the nonlinear least squares program
MULTI (53). Then CLorg can be calculated
simply from Eq. 1 at several time points after
administration. In addition, the total body clearance
(CLtotal) was calculated by dividing the dose by the AUC up
to infinity.
Pharmacokinetic analysis based on a physiological model.
The time courses of the plasma concentration and liver accumulation of
111In-mannosylated proteins were analyzed using the model
shown in Fig. 1 (33). In
this model, three compartments, i.e., the plasma pool (PP), the
sinusoidal and Disse spaces in the liver (EC), and the intracellular
space in the liver (IC), represent the body as a whole. The PP and EC
compartments have apparent volumes of distribution (Vp and
Vl, respectively). The PP compartment represents all plasma
spaces within blood vessels of all tissues except the liver; it is
connected to the EC by hepatic plasma flow (Q). The uptake of
mannosylated proteins from the EC to the IC is expressed as a saturable
process following Michaelis-Menten kinetics, with a maximum rate of
uptake (Vmax,l; nmol/h) and a Michaelis-Menten constant
(Km,l; nM). Extrahepatic elimination from the PP
is assumed to be a saturable process represented by Vmax,p
(nmol/h) and Km,p (nM). Since some derivatives
showed capacity-limited plasma protein binding, this process was
expressed as a maximum binding concentration (Bmax; nM),
and the dissociation constant (Kd; nM) was
expressed as
|
(2)
|
where Ctotal is the total concentration of
mannosylated protein in the PP and Cfree is the
concentration of free (unbound) mannosylated proteins in the PP. Only
free mannosylated protein is assumed to be involved in the hepatic
uptake and extrahepatic elimination. At time 0, the injected
mannosylated protein is assumed to be distributed in the PP and EC
compartments at the same concentration. The mass balance equation for
the concentration of mannosylated proteins in PP, EC, and IC is
expressed as
|
(3)
|
|
(4)
|
|
(5)
|
where Cp,free and Cl,free are the
concentrations of free (unbound) mannosylated proteins in the PP and
EC, respectively, Xl is the amount accumulated
in the IC, and Q is the hepatic plasma flow rate. The values of
Vp, Vl, and Q were assumed to be 1.5, 0.15, and
85 ml/h, respectively (6). To obtain the pharmacokinetic parameters, these equations were fitted to the data using the nonlinear
least squares method MULTI associated with the Runge-Kutta-Gill method,
[MULTI(RUNGE)] (damping Gauss Newton method) using an M-382 mainframe
computer located in the Kyoto University data processing center.

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Fig. 1.
Physiological pharmacokinetic model for analyzing the
disposition of mannosylated proteins. Km,p and
Km,l, Michaelis-Menten constants for plasma pool
and liver, respectively; Vmax,p and
Vmax,l, maximum rates of uptake for plasma pool
and liver, respectively; Kd, dissociation
constant for plasma protein binding; Bmax, maximum
concentration of plasma protein binding; Q, hepatic plasma flow rate;
Cbound, concentration of bound protein; Cfree,
concentration of free protein.
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Isolation of MBP from mouse serum.
MBP was isolated from BALB/c mouse serum (Japan Bio-supply) using a
Sepharose-mannan or Sepharose-Man-BSA column according to the procedure
reported by Kawasaki et al. (19). Briefly, mannan or
Man46-BSA was coupled to
N-hydroxysuccinimide (NHS)-activated Sepharose 4 Fast Flow (Pharmacia Biotech, Uppsala, Sweden) according to the
instructions of the manufacturer. The mouse serum was diluted with an
equal volume of a buffer consisting of 40 mM imidazol-HCl, pH 7.8, 40 mM CaCl2, and 2.5 M NaCl. The mixture was applied to the
Sepharose-mannan or Sepharose-Man-BSA column, which had been equilibrated with a loading buffer (20 mM imidazol-HCl, pH 7.8, 20 mM
CaCl2, and 1.25 M NaCl). The binding protein was eluted with an elution buffer (20 mM imidazol-HCl, pH 7.8, 1.25 M NaCl, and 2 mM EDTA). The eluate was applied to the second and third smaller
affinity columns. The final column was washed with the loading buffer
and eluted with more loading buffer containing 100 mM mannose. All of
the procedures were carried out at 4°C.
The isolated protein was subjected to SDS-PAGE (10% wt/vol acrylamide)
under reducing and nonreducing conditions by the method of Laemmli
(21). Molecular masses were estimated by comparison with
Rainbow marker proteins (Amersham Life Sciences).
In vitro assay of serum protein binding.
To deplete MBP, mouse serum was applied to a Sepharose-Man-BSA column
twice at a rate of 1 drop/15 s to obtain MBP-depleted serum.
111In-Man16-,
111In-Man25-, and
111In-Man46-BSA were incubated with mouse
serum, MBP-depleted serum, or buffer containing purified MBP
(Man-BSA:MBP; 2:1 molar ratio) for 30 min at 4°C, and then the
mixture was applied to a Sephadex G-200 column (Pharmacia) and eluted
with buffer (1.25 M NaCl, 20 mM CaCl2 and 20 mM imidazol).
In one case, 100 mM mannose or 2 mM EDTA was added to the buffer. The
111In radioactivity in each fraction was determined by
scintillation counting.
In situ liver perfusion.
To evaluate the hepatic uptake of 111In-Man-BSAs under
serum-free conditions, an in situ liver perfusion experiment was
carried out as reported previously (31). Briefly, mouse
liver was perfused in a single-pass mode at a flow rate of 2 ml/min
with Krebs-Ringer-bicarbonate buffer containing 10 mM glucose and 3%
(wt/vol) BSA. The buffer was oxygenated with 95% O2-5%
CO2, adjusted to pH 7.4, and incubated at 37°C. The liver
was perfused for 2 min to allow it to stabilize, and then 100 µl of
each 111In-Man-BSA (10 µg/ml) was administered to the
liver as a bolus through the portal cannula. Then, 3 min later, the
liver was excised and its radioactivity was counted. Statistical
analysis was performed by ANOVA. P < 0.001 was
considered statistically significant.
 |
RESULTS |
Tissue distribution of 111In-mannosylated proteins
after intravenous injection.
After intravenous injection, all 111In-mannosylated
proteins were mainly recovered in the liver. However, their
distribution patterns varied depending on the molecular mass of the
protein, the number of mannose residues, and the dose administered
(Figs. 2, 3, and 4). Figure 2 shows the
plasma concentration and liver accumulation time courses of
111In-Man-SODs after intravenous injection. At the lower
doses of 0.05 and 0.1 mg/kg, >80% of the injected dose was recovered
in the liver. The plasma elimination rate was approximately inversely related to the hepatic uptake. Increasing the dose reduced the amount
and rate of liver accumulation of both derivatives because of
saturation of the mannose receptor-mediated hepatic uptake. In such
cases, radioactivity was also detected in kidneys and urine, because
the molecular mass of the Man-SODs was less than the glomerular
filtration threshold (28).

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Fig. 2.
Time courses of plasma concentration (top) and
liver accumulation (bottom) of
111In-Man17-SOD (A) and
111In-Man21-SOD (B) after
intravenous injection into mice at doses of 0.05 ( ),
0.1 ( ), 1 ( ), 10 ( ),
and 20 ( ) mg/kg. Results are means ± SD of 3 mice.
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Fig. 3.
Time courses of plasma concentration (top) and
liver accumulation (bottom) of
111In-Man12-BSA
(A),111In-Man16-BSA
(B),111In-Man25-BSA
(C),111In-Man35-BSA (D),
and 111In-Man46-BSA (E) after
intravenous injection into mice at doses of 0.05 ( ),
0.1 ( ), 1 ( ), 10 ( ),
and 20 ( ) mg/kg. Results are means ± SD of 3 mice.
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Fig. 4.
Time courses of plasma concentration (top) and
liver accumulation (bottom) of
111In-Man32-IgG (A) and
111In-Man42-IgG (B) after
intravenous injection into mice at doses of 0.05 ( ),
0.1 ( ), 1 ( ), 10 ( ),
and 20 ( ) mg/kg. Results are means ± SD of 3 mice.
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Figure 3 shows the results for the
111In-Man-BSAs. All 111In-Man-BSAs accumulated
mainly in the liver, and there was no significant accumulation of
radioactivity in any other tissue or urine. Increasing the dose above 1 mg/kg resulted in a reduced hepatic uptake, as observed for the
111In-Man-SODs. At the lower doses of 0.05 and 0.1 mg/kg,
111In-Man16-BSA showed the fastest hepatic
uptake of all the Man-BSAs, and other BSA derivatives having more
mannose residues were retained in plasma for a prolonged period.
111In-Man-IgGs also exhibited distribution profiles similar
to those of 111In-Man25-,
111In-Man34- and
111In-Man46-BSA (Fig.
4). The hepatic uptake was fastest at the
dose of 1 mg/kg. Compared with 111In-Man46-BSA,
which showed the most prolonged plasma circulation of all of the
111In-Man-BSAs, 111In-Man-IgGs exhibited a
slower hepatic uptake and a longer retention in plasma at the low doses
of 0.05 and 0.1 mg/kg.
In vitro interaction of 111In-mannosylated proteins
with serum-type MBP.
Under reducing conditions, a single band (~30 kDa) could be detected
on the SDS-PAGE of the protein(s) purified from mouse serum using an
affinity column (Fig. 5). Several
high-molecular-mass bands were seen under nonreducing conditions. These
results agreed with those of previous papers (8, 13,
19-20), indicating that the purified protein is serum-type
MBP. In the following experiments, MBP and MBP-depleted serum were
prepared by this affinity column procedure as described in
MATERIALS AND METHODS.

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Fig. 5.
SDS-PAGE of isolated mannose binding protein. Purified
binding protein was subjected to 10% polyacrylamide gel
electrophoresis under reducing conditions ( -mercaptoethanol).
Lane 1, molecular mass marker; line 2, mannose
binding protein using a Man-BSA-Sepharose affinity column; lane
3, mannose binding protein using a Mannan-Sepharose affinity
column. The binding protein band has an apparent molecular mass of
~30 kDa.
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Fig. 6 shows the gel filtration patterns
of 111In-Man16-,
111In-Man25-, and
111In-Man46-BSA, with or without preincubation
in mouse serum, under various conditions. Without any preincubation,
111In-Man16-,
111In-Man25-, and
111In-Man46-BSA were eluted at 30 ml. When
incubated with serum, 111In-Man46-BSA (Fig.
6A) had a peak at 15 ml in addition to the original one,
suggesting an interaction with some serum components. In the presence
of EDTA or excess mannose, the elution profile of
111In-Man46-BSA incubated with serum was
superimposable on that of intact, unbound
111In-Man46-BSA.
111In-Man25-BSA (Fig. 6C) also gave
similar results to 111In-Man46-BSA. These
results indicated that mannose residues, as well as divalent cations,
play an important role in the binding of mannosylated proteins to serum
components. Preincubation of 111In-Man46-BSA
with purified MBP gave a similar elution profile to that with serum,
whereas preincubation with MBP-depleted serum did not (Fig.
6B). These data suggest that serum MBP is the component that
is bound to Man-BSAs. However, 111In-Man16-BSA
exhibited no significant changes in elution profile following various
treatments (Fig. 6D).

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Fig. 6.
Gel filtration (Sephadex G-200) patterns of
111In-Man-BSAs. A:
111In-Man46-BSA alone incubated with buffer
( ), incubated with serum ( ), eluted
with buffer containing mannose 100 mM ( ), and eluted
with buffer containing EDTA 2 mM ( ). B:
111In-Man46-BSA incubated with serum-depleted
mannan-binding protein (MBP) ( ) or incubated with
purified MBP ( ) (Man-BSA:MBP; 2:1 mol ratio).
C: 111In-Man25-BSA incubated with
buffer ( ) or incubated with serum ( ).
D: 111In-Man16-BSA incubated with
buffer ( ) or incubated with serum ( )
for 30 min at 4°C and eluted with buffer (1.25 M NaCl, 20 mM imidazol
and 20 mM CaCl2).
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Calculation of AUC and clearances.
Table 2 summarizes the AUC,
CLtotal, hepatic uptake clearance (CLliver),
and tissue uptake rate index (tissue uptake clearance per unit tissue
weight) for representative tissues of 111In-mannosylated
proteins after intravenous injection at various doses. As for all
111In-mannosylated proteins, the CLliver made
the major contribution to the CLtotal. The
CLliver of 111In-Man-SODs as well as
111In-Man12- and
111In-Man16-BSA decreased on increasing the
dose, whereas the CLliver of other derivatives peaked at a
dose of 1 mg/kg.
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Table 2.
AUC, clearances, and tissue uptake rate index of
111In-Man-BSAs after intravenous injection in mice at
various doses
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To clarify the relationship between the physicochemical properties of
mannosylated proteins and their serum protein binding at low doses, the
AUC of 111In-mannosylated proteins at a dose of 0.05 mg/kg
was plotted against their molecular masses and mannose content (Fig.
7). Both 111In-Man-SODs had
very small AUCs in spite of their high mannose content. Although
111In-Man-IgGs have a lower mannose content than
111In-Man46-BSA, their AUC values were higher
than that of 111In-Man46-BSA. These results
suggest that the molecular mass of mannosylated proteins is a very
important factor in determining their interaction with MBP. Comparing
111In-Man-BSAs with different numbers of mannose units, the
mannose content (number) also seems to affect the interaction.

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Fig. 7.
AUC of mannosylated compounds at a dose of 0.05 mg/kg
plotted against molecular mass and mannose content (wt/wt%)
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Pharmacokinetic analysis based on a physiological model.
On the basis of the pharmacokinetic analysis carried out so far, it is
obvious that both the molecular mass and mannose content of the
mannosylated proteins play crucial roles in determining the interaction
with plasma MBP. To achieve a more precise and quantitative
analysis of the processes of hepatic uptake and plasma protein binding
of mannosylated proteins, the biodistribution profiles of
111In-Man-BSAs were analyzed using the physiological
pharmacokinetic model shown in Fig. 1. Differential Eqs.
2-4 were simultaneously fitted to the plasma concentration
and liver accumulation data for each 111In-mannosylated
protein at five doses, and the pharmacokinetic parameters were
estimated (Table 3) and plotted against
the number of mannose units per BSA (Fig.
8). Of these parameters, only
Km,l and Kd, which
correspond to the affinity of mannosylated proteins for the hepatic
mannose receptors and MBP, respectively, varied depending on the number
of mannose units. The Km,l values were all
fairly similar (34-68 nM) except for
111In-Man12-BSA (300 nM). On the other hand,
the Kd of 111In-mannosylated
proteins increased exponentially on increasing the number of mannose
residues from 3,000 nM for 111In-Man12-BSA to
0.27-0.3 nM for 111In-Man35-BSA and
111In-Man46-BSA.

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Fig. 8.
Effect of the number of mannose residues on the
pharmacokinetic parameters of Man-BSAs: Km,l
(A), representing the affinity for the hepatic receptor, and
Kd (B), representing the plasma
protein binding, respectively. The parameters obtained by fitting
Eqs. 3-5 to the experimental data were plotted as
means ± SD against the number of mannose residues for each
protein.
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In situ liver perfusion.
Fig. 9 shows the hepatic recovery
(%dose/g liver) of 111In-Man-BSAs in perfused mouse liver
after bolus administration to the liver via the portal vein. There were
no significant differences in recovery between
111In-Man16-,
111In-Man25-,
111In-Man35- and
111In-Man46-BSAs (11-14% of the dose/g
tissue), but that of 111In-Man12-BSA was
significantly lower than those of other 111In-Man-BSAs
(6.5% of dose/g tissue).

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Fig. 9.
Percentage recovery of radioactivity in liver of
111In-Man-BSAs after bolus injection via the portal vein in
the mouse liver perfusion experiment. Three minutes after injection,
the liver was excised and radioactivity was measured. Results are
expressed as the means ± SD of 5 mice.
#P < 0.001 vs. other Man-BSAs.
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 |
DISCUSSION |
Although various types of cells have mannose receptors on their
surface, ligands with mannose are largely taken up by liver nonparenchymal cells after intravenous injection. Therefore, it has
been demonstrated that naturally or chemically mannosylated ligands are
promising candidates for drug carriers targeting liver nonparenchymal
cells via mannose receptors (3, 17, 38). In one case, the
in vivo targeting efficiency of Man-BSA to the whole liver exceeded
80% of the injected dose (35, 46), and the endothelial
and Kupffer cells contribute about 66% and 21% of the uptake,
respectively, after intravenous injection (35). On the
other hand, Kawasaki et al. (19) have reported that MBP, another mannose-specific lectin, circulates in serum and recognizes pathogens that have mannose units on their surface. Although the in
vitro binding specificity and characteristics of hepatic mannose receptors and serum MBP have been studied in detail (22,
29-30, 45, 46), little information is available on their in
vivo recognition characteristics (46). Furthermore, little
attention has been paid to the in vivo interaction between serum MBP
and mannosylated ligands. Therefore, in the present study, we tried to
obtain information about the in vivo recognition characteristics of
these mannose-specific lectins by analyzing the biodistribution of
mannosylated proteins with different physicochemical properties.
Using a metabolizable 125I labeling, Taylor et al.
(46) studied the uptake and metabolic fate of mannosylated
ligands after systemic administration. Instead, we applied an
111In labeling combined with DTPA anhydride, since
111In labeling prepared by this method is metabolically
stable in the bloodstream and is locked within cells that take up the
labeled compounds even after intracellular degradation
(10). These characteristics of labeling enabled us to
quantitatively trace the plasma profile and tissue uptake of
mannosylated proteins by simply counting radioactivity.
Because of the limited number of mannose receptors, increasing the dose
of ligands saturates the receptors followed by a decrease in hepatic
uptake when normalized with the administration dose. This is the case
for 111In-Man-SODs as well as
111In-Man12- and
111In-Man16-BSA; their hepatic uptake simply
decreased with increasing dose, as observed with galactosylated
proteins (32). Although uptake clearances by other tissues
decreased on the increasing the dose in some cases, the absolute values
were very small compared with those by the liver (Table 2). Therefore,
the mannose receptor-mediated hepatic uptake seems to be the only
mannose-specific and capacity-limited process determining the
biodistribution of these Man-SODs and Man-BSAs with fewer mannose units.
When mannosylated ligands interact with serum lectins like MBP, their
biodistribution becomes more complicated. Incubated in mouse serum in
vitro, 111In-Man25-BSA and
111In-Man46-BSA showed an interaction with a
serum component (Fig. 6) that was identified as MBP by determining its
molecular size and in vitro binding characteristics. Martinez-Pomares
et al. (26) found in mouse serum the presence of soluble
mannose receptors that interact with carbohydrate chains in a mannose-
or fucose-dependent manner. However, we could not detect any other band
on SDS-PAGE except for 30 kDa (which corresponds to MBP), indicating
that the level of soluble mannose receptors was too low to be detected in normal serum or that they were so unstable that we could not separate them by this procedure.
MBP is known to activate the complement system on binding to its
ligands, but the in vivo fate of MBP/mannosylated ligand complex is
hardly understood. Several studies showed that MBP is recognized by
collectin receptors expressed on macrophages, monocytes, and
neutrophils (5, 12, 49-50). However, on the basis of
the biodistribution of 111In-Man-BSAs and
111In-Man-IgGs, MBP-bound mannosylated ligands do not seem
to be rapidly removed by hepatic mannose receptors or by collectin
receptors. This is also supported by our preliminary observation that
the uptake of 111In-Man46-BSA by cultured
peritoneal macrophages is not assisted by complex formation with MBP.
In addition, 111In-Man46-BSA preincubated with
purified MBP (1:1 molar ratio) showed a slower hepatic uptake than
111In-Man46-BSA itself after intravenous
injection in mice (data not shown). Therefore, it is reasonable to
assume that MBP/mannosylated protein complex is not taken up by liver
nonparenchymal cells via mannose receptor-mediated endocytosis. On the
basis of this assumption, the AUC value at the lowest dose of 0.05 mg/kg is a good index for the semiquantitative comparison of the
binding strength of each mannosylated protein to MBP in vivo: a large AUC indicates strong binding to MBP, whereas a small AUC means weak or no binding (Fig. 7). 111In-Man-SODs had very
small AUCs in spite of their high mannose content (wt/wt).
Although the 111In-Man-IgGs used in this study have a lower
mannose content than 111In-Man46-BSA, their
AUCs were larger than that of 111In-Man46-BSA.
These results suggest that the molecular mass of mannosylated proteins
is very important for their interaction with MBP. When the molecular
mass is identical, the number of mannose moieties also has a
significant effect on recognition by MBP.
MBP-mediated retardation of hepatic uptake was only obvious at the low
doses of 0.05 and 0.1 mg/kg. This phenomenon could be explained by the
limited amount of MBP in serum; its concentration in mouse serum was
reported to be from 5 to 80 µg/ml (8). Increasing the
dose will saturate the binding of ligands to MBP, and excess ligands,
existing in the free form in the circulation, will be recognized by
hepatic mannose receptors. At >1 mg/kg, the mannose receptors were
also saturated with the ligands and the hepatic uptake was retarded.
On the basis of the same model as this study, we concluded that the in
vivo affinity of galactosylated proteins with the asialoglycoprotein receptor is simply increased on increasing density of galactose on the
ligand surface (32). In the present study, a
capacity-limited binding in plasma was also assumed due to the presence
of MBP in serum. We also assumed that the bound form of the ligand does not participate in the hepatic uptake on the basis of the
biodistribution experiments.
Increasing the number of mannose units from 12 (Man12-BSA) to 16 (Man16-BSA) greatly reduced
the Km,l, indicating that the hepatic mannose
receptors require at least 16 mannose residues on BSA for efficient
uptake in vivo. The binding affinity of ligands for mannose receptors
has been reported to depend on the clustering and geometric
organization of the mannose residues on a branched sugar chain
(30). Man-BSA, having a larger number of mannose residues,
could take part in a multivalent interaction with mannose receptors,
followed by rapid uptake. On the basis of an in vitro binding study
using alveolar macrophages, Hoppe and Lee (14) reported
that the binding affinity of Man-BSA increased on increasing the number
of mannose units from 5 to 43. In the present study, however, the
Km,l of 111In-Man-BSAs, except for
111In-Man12-BSA, remained relatively constant
regardless of the number of mannose units. To confirm the results
obtained by the analysis, the in situ hepatic uptake of
111In-Man-BSAs was examined in perfused mouse livers where
no blood components were involved. All 111In-Man-BSAs,
except for 111In-Man12-BSA, showed a similar
level of hepatic uptake, supporting the parameters obtained by the
pharmacokinetic analysis. This finding, that the affinity does not
increase on increasing the number of mannose units above 16 per BSA, is
not consistent with the previous report (14). This
discrepancy could be due to differences in the experimental conditions:
cultured cells vs. whole animals, and/or alveolar macrophages vs.
hepatic nonparenchymal cells. The results obtained here were based on
the in vivo disposition of mannosylated proteins, so they reflect the
in vivo recognition characteristics of the mannose receptors more
accurately than those obtained in vitro.
In contrast to the affinity with hepatic mannose receptors, the
Kd on the protein binding in serum, which
correlates with the affinity of 111In-Man-BSAs to MBP, fell
markedly on increasing the number of mannose residues from 3,000 to
0.27-0.3 nM. This discrepancy between the two types of
mannose-specific lectins in the characteristics of ligand recognition
can be explained by the difference in the molecular structures of the
hepatic mannose receptors and MBP. The mannose receptor is a type I
transmembrane protein with eight different C-type CRDs in a single
polypeptide. On the other hand, the monomer of MBP contains only one
CRD at the COOH-terminus, three monomers are held together by
interaction with the
-helical neck region, and two to six sets of
the trimer form the bouquet-like structure of MBP (52).
These structural characteristics of MBP could account for the strong
binding of highly mannosylated BSAs (11). The relationship
between the density of the sugar units and the
Kd was similar to that between the density of
galactose units on proteins and the Km,l of
their hepatic uptake (32). These findings suggest that MBP
exhibits a significant cluster effect in its recognition of ligands
larger than BSA, as is the case with the asialoglycoprotein receptors
(51).
In summary, serum MBP only recognized ligands with a larger molecular
mass, and its binding affinity significantly increases on increasing
the molecular mass of the ligands and the density of mannose units. It
was quantitatively shown to have a strong cluster effect. In contrast,
recognition of the hepatic mannose receptors was hardly affected by the
ligand size and depended only on the density of the mannose units on
ligands. The differences in the recognition of mannosylated ligands by
hepatic mannose receptors and serum MBP could be explained by the fact
that the hepatic mannose receptors contain multiple different CRDs in
the single polypeptide, whereas serum MBP is composed of six or more monomers with only a single CRD. These findings will prove useful not
only for understanding the physiological roles of these lectins in host
defense but also for designing drug carriers targeting liver
nonparenchymal cells.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Toshisuke Kawasaki and Dr. Kazuhide Uemura for their
helpful advice on the separation of serum mannan-binding protein.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: M. Hashida, Dept. of Drug Delivery Research, Graduate School of
Pharmaceutical Sciences, Kyoto Univ., Sakyo-ku, Kyoto 606-8501, Japan
(E-mail: hashidam{at}pharm.kyoto-u.ac.jp).
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.
Received 26 June 2000; accepted in final form 13 November 2000.
 |
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