Tissue targeting of multivalent GalNAc Lex terminated N-glycans in mice

Yongsheng Yang, V.Hayden Thomas, Shouchin Man and Kevin G. Rice1

College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109–1065, USA

Received on June 6, 2000; revised on July 7, 2000; accepted on August 16, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
N-Linked biantennary and triantennary oligosaccharides containing multiple terminal GalNAc Lex (GalNAcß1–4[Fuc-{alpha}1–3]GlcNAc) determinants were radioiodinated and their pharmacokinetics, biodistribution, and hepatic cellular localization were determined in mice. Pharmacokinetic analysis revealed GalNAc Lex biantennary and triantennary oligosaccharides had a similar mean residence time and steady-state volume of distribution but differed in their total body clearance rate due a shorter {alpha} half-life for GalNAc Lex triantennary. Biodistribution and whole-body-autoradiography studies revealed that both GalNAc Lex terminated biantennary and triantennary oligosaccharides predominately targeted to the liver, which accumulated 72% and 79% of the dose 30 min after administration, respectively. Separation of mouse liver parenchymal from non-parenchymal cells demonstrated both N-glycans were almost exclusively (94%) taken up by the parenchymal cells. By comparison, GalNAc terminated biantennary and triantennary N-glycans accumulated in the liver with a targeting efficiency of 73% and 81%, respectively. It is concluded that GalNAc and GalNAc Lex terminated N-glycans are recognized in vivo with equivalent affinity by the murine hepatic asialoglycoprotein receptor.

Key words: asialoglycoprotein receptor/biodistribution/mice/N-glycans/pharmacokinetics


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
One function of the N-glycans on glycoproteins is to mediate their serum half-life by binding to lectins expressed in the liver (Ashwell and Harford, 1982). The asialoglycoprotein receptor (ASGP-R) found on hepatocytes plays a major role in removing glycoproteins from circulation by recognition of multiple terminal Gal residues that are spaced in a tight cluster on the antennae of branched N-glycans (Baenziger and Fiete, 1980Go; Lee et al., 1983Go).

The ability of the ASGP-R to recognize N-glycans with high affinity is related to the organization of its subunits such that each possesses a carbohydrate recognition domain (CRD) that clusters into a heterotrimer to position three CRDs to simultaneously accept three terminal Gal residues on a triantennary N-glycan (Lee et al., 1983Go; Townsend et al., 1986Go; Rice et al., 1990Go; Lodish, 1991Go). The receptor not only accepts N-glycans with terminal Gal but will preferentially bind oligosaccharides possessing GalNAc with higher affinity (Hudgin, 1974Go; Baenziger and Fiete, 1980Go).

Previous studies from our group attempted to characterize the in vivo specificity of the ASGP-R by studying the pharmacokinetics and biodistribution of radioiodinated tyrosinamide biantennary and triantennary N-glycans possessing terminal Gal, GalNAc, and Lex (Galß1–4[Fuc{alpha}1–3]GlcNAc) (Chiu et al., 1994Go, 1995). Analysis of Gal and GalNAc terminated N-glycans revealed that their liver targeting efficiency (% of dose recovered from liver 30 min after dosing) closely correlated with their respective binding affinity for the hepatic ASGP-R (Chiu et al., 1994Go, Rice et al., 1995Go). A GalNAc terminated biantennary N-glycan possessed a targeting efficiency of 77% relative to 18% for Lex terminated biantennary N-glycan and 7% for a Gal terminated biantennary N-glycan, suggesting that the CRD of the murine ASGP-R simultaneously recognizes both the terminal Gal and Fuc residues of Lex (Chiu et al., 1995Go).

No previous studies have examined the lectin binding properties of N-glycans possessing both terminal GalNAc and Fuc in the form of GalNAc Lex (GalNAcß1–4[Fuc{alpha}1–3]GlcNAc) N-glycans. Only a few reports have identified this unique carbohydrate epitope on glycoproteins. One study found GalNAc Lex as a major carbohydrate determinant of antigenic proteins isolated from Schistosoma mansoni (Srivatsan et al., 1992Go). Another study elucidated biantennary N-glycans possessing two GalNAc Lex determinants on recombinant human protein C expressed in human kidney 293 cells (Yan et al., 1993Go). The finding that this glycoprotein also inhibited E-selectin in vitro suggested that GalNAc Lex epitopes were capable of specific interaction with the CRD of this selectin (Grinnell et al., 1994Go).

To further extend our investigation of the ASGP-R specificity in vivo and to probe for possible unique biodistribution sites we enzymatically remodeled Gal terminated biantennary and triantennary N-glycans into GalNAc Lex terminated N-glycans (Thomas et al., 1998Go). In the present study we compare the pharmacokinetics and hepatic targeting efficiency of GalNAc Lex N-glycans relative to GalNAc terminated N-glycans in mice. The results not only enhance our understanding of the specificity of the ASGP-R for recognition of different determinants but also shed light on the possible in vivo fate of glycoproteins or organisms that possess these unique oligosaccharides.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The pharmacokinetic and biodistribution analysis of GalNAc Lex biantennary and triantennary N-glycans (I and II) were compared to GalNAc biantennary and triantennary (III and IV) in mice (Figure 1). Each oligosaccharide was radioiodinated then dosed i.v. in mice. Following jugular vein dosing, pharmacokinetic analysis verified that I and II were rapidly cleared from the blood within 1 h with biphasic kinetics demonstrating a rapid distribution phase (t1/2{alpha} = ~1–2 min) followed by a prolonged elimination phase (Figure 2). Extraction of N-glycan I or II from blood followed by TLC and autoradiography demonstrated their clearance without the formation of detectable metabolites (Figure 2, inset). Therefore, blood time points were directly {gamma} counted and fit by nonlinear least squares analysis to derive pharmacokinetic constants (Chiu et al., 1994Go) (Table I). N-Glycan I and II had a MRT of ~32–38 min that was comparable to that reported previously (20 min) for N-glycans III and IV (Table I) (Chiu et al., 1994Go). The Vdss was not significantly different for I versus II but, the shorter {alpha} half-life of II resulted in a significantly faster Cltb of 1.7 ml/min relative to 0.8 ml/min for I (Table I).



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Fig. 1. Structure of GalNAc and GalNAc-Lex terminated biantennary and triantennary tyrosinamide N-glycans. Oligosaccharides I and II represent GalNAc Lex biantennary and triantennary N-glycans, whereas III and IV represent GalNAc terminated biantennary and triantennary N-glycans.

 


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Fig. 2. Pharmacokinetic analysis of GalNAc Lex biantennary and triantennary N-glycans in mice. The concentration of oligosaccharide determined from direct {gamma}-counting of blood time points is plotted versus time for oligosaccharides I (circles) and II (squares). The fitted line is the non-linear least squares fit of the data determined by PCNONLIN resulting in parameters reported in Table I. The inset demonstrates TLC evidence of the recovery of unmetabolized II from blood at times ranging from 1–15 min.

 

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Table I. Pharmacokinetic analysisa of N-glycans
 
A biodistribution time of 30 min was selected to analyze I and II since pharmacokinetic studies determined that less than 6% of the dose remained in the blood (Figure 2). Whole-body-autoradiographic analysis allowed screening of all tissues to identify the major biodistribution sites (Figure 3, inset). Direct {gamma}-counting of dissected tissues confirmed results predicted by whole-body-autoradiography indicating the liver as the predominant target site for both oligosaccharides I and II whereas the kidney and intestine were the only other reference organs that contained radioactivity in excess of 1% (Figure 3). Quantitative biodistribution analysis determined liver targeting efficiencies of 72 ± 1.9% and 79 ± 2.3% for I and II, respectively (p < 0.02). To establish if the liver targeting efficiency for I and II were significantly different from GalNAc biantennary and triantennary, the biodistribution of N-glycans III and IV were examined under identical conditions resulting in targeting efficiencies of 73 ± 3.7% and 81 ± 1.7%, respectively.



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Fig. 3. Biodistribution of 125I labeled N-glycans. Quantitative biodistribution was performed by direct {gamma}-counting of the dissected tissues. The liver targeting efficiency (% of dose in the target organ at 30 min post-administration) was 72% (I), 79% (II), 73% (III), and 81% (IV). The kidney, small intestine, and large intestine were the only other organs with radioactivity in excess of 1% of dose. Each oligosaccharide was analyzed in triplicate mice. *, p < 0.02; **, p < 0.03.

 
The cellular targeting of I and II was established from the ratio of the radioactive dose in liver nonparenchymal and parenchymal cells. The results established that over 94% of the liver radioactivity accumulated in parenchymal cells for both I and II (Table II), suggesting that the terminal GalNAc Lex residues contribute to the cell-type-specific targeting through binding to the ASGP-R and not a Fuc receptor reported on Kupffer cells (Lehrman et al., 1986Go).


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Table II. N-Glycan distribution in parenchymal and nonparenchymal cells
 
The results of this study indicate that i.v. dosed GalNAc Lex biantennary and triantennary N-glycans primarily target to parenchymal cells in mice. The difference in the liver targeting efficiency between I (72%) and II (79%) is statistically significant (p < 0.02) indicating that the third GalNAc Lex on II contributes to the binding affinity to the ASGP-R. Likewise, a significant difference (p < 0.03) in the targeting efficiency of III (73%) and IV (81%), demonstrates the same degree of binding affinity contribution for the third GalNAc residue on triantennary N-glycan. However, comparison of the targeting efficiency of biantennary I with III or triantennary II with IV failed to establish any significant difference (p > 0.1), indicating that the Fuc residues attached to GalNAc Lex N-glycans I or II neither contribute nor interfere with binding to the mouse ASGP-R.

These results are in contrast to similar studies that compared the liver targeting efficiency of N-glycans possessing terminal Gal versus Lex determinants (Chiu et al., 1994Go, 1995). The liver targeting efficiency for an Lex terminated biantennary N-glycan was 18% compared to 7% for a Gal terminated biantennary. Likewise, a significant difference also exist between the liver targeting efficiency of Lex terminated triantennary (66%) relative to Gal terminated triantennary (59%).

Comparison of these earlier results with those in the present study suggests that the mouse ASGP-R only possesses weak binding affinity for Fuc residues attached in an Lex or GalNAc Lex configuration. The high affinity binding of GalNAc terminated N-glycans to the ASGP-R apparently masks the weaker binding contribution provided by Fuc in GalNAc Lex. This hypothesis is supported by the finding that the mouse ASGP-R does not distinguish between II and IV, suggesting that the three Fuc residues in II, which are positioned in close proximity to the CRD, do not create steric crowding or inhibit binding. This result is consistent with a hypothesis invoking a preexisting binding pocket on each of the three CRDs of the mouse ASGP-R that can accommodate the Fuc residues without significantly increasing the binding affinity for a GalNAc Lex N-glycan.

This hypothesis is also consistent with the recent proposal that mouse E-selectin binds to a sialyl Lewisx (SLex) biantennary N-glycan in vivo (Thomas et al., 1999Go). Since E-selectin and the ASGP-R are evolutionarily related (Drickamer, 1999Go), it is conceivable that the architecture of E-selectin’s CRD has been enhanced from the more primitive ASGP-R CRD to increase the binding affinity for Fuc residues, making these essential for E-selectin recognition.

The results of this study suggest that glycoproteins possessing the GalNAc Lex determinants are likely to be rapidly taken up by the hepatic ASGP-R. However, it is difficult to extrapolate result across species, since we previously noted that the rat ASGP-R does not differentiate between Lex and Gal terminated N-glycans (Chiu et al., 1995Go). Still it is tempting to speculate that one function of the GalNAc Lex terminated N-glycans on Schistosoma mansomi is to mediate rapid uptake by the liver of the infected individual (Srivatsan et al., 1992Go). It is also evident that glycoproteins possessing GalNAc Lex terminated N-glycans would be very weak inhibitors of E-selectin in vivo due to their rapid clearance by the ASGP-R.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sodium 125iodide was purchased from DuPont, Boston MA. Sephadex G-10, chloramine T, sodium metabisulfite, heparin, bovine serum albumin, HEPES, collagenase from Clostridium histolyticum type IV (lot number 47H6865), and carbonyl iron were purchased from Sigma, St. Louis, MO. Thin layer chromatography (TLC) plates (Silica Gel-60 F-254) were purchased from Alltech, Deerfield, IL. Ketamine hydrochloride was purchased from Fort Dodge Laboratories, Fort Dodge, IA. Xylazine hydrochloride was purchased from Miles Inc., Shawnee Mission, KS. Silastic catheters (0.305 mm inner diameter x 0.635 mm outer diameter) were purchased from Baxter, Obetz, OH. ICR mice (30–35 g) were purchased from Harlan, Indianapolis, IN, and housed in cages located in a limited access area maintaining a 12 h light–dark cycle and controlled temperature (26–28°C). Mice had free access to standard laboratory mouse food (Charles River Inc.) and water.

Radiolabeling N-glycans
Tyrosinamide N-glycans possessing terminal GalNAc Lex were enzymatically synthesized from Gal terminated biantennary and triantennary oligosaccharides as described previously (Thomas et al., 1998Go). Tyrosinamide N-glycans were iodinated using a modification of the chloramine T method (Chiu et al., 1994Go). Briefly, tyrosinamide N-glycans (1 nmol in 30 µl of 0.5 M sodium phosphate buffer pH 7.0) were added to 0.25 mCi of sodium 125I in 12.5 µl of 0.1 M sodium hydroxide. Chloramine T (10 µl of 10 mM in phosphate buffer) was added and allowed to react for 3 min followed by the addition of sodium metabisulfite (40 µl of 10 mM in phosphate buffer) in order to quench the reaction. Radioiodinated oligosaccharides were chromatographed on a Sephadex G-10 column (0.8 x 25 cm) eluted with 0.15 M sodium chloride (pH 7.0) during collection of 0.5 ml fractions. The oligosaccharides eluting between 4 and 5 ml had a specific activity of 110 µCi/nmol, assuming quantitative recovery.

The purity of each iodinated oligosaccharide was analyzed by spotting 1 µl (2 nCi) at the origin of a TLC plate developed with ethyl acetate:acetic acid:pyridine:water at a ratio optimized for each oligosaccharide (I and II, 4:3:2:2.5; III, 4.5:3:1.5:2.5; IV, 4:3:1.5:2.5). Quantitative densitometry was performed on a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA) following 12 h autoradiographic exposure at room temperature. ImageQuant software (Molecular Dynamics, Sunnyvale, CA) was used to integrate the densitometry trace and established >95% purity for each iodinated oligosaccharide.

Analysis of oligosaccharides stability in vitro
Each radioiodinated oligosaccharide (1.5 µl, 75 nCi) was added to 100 µl of heparinized whole mouse blood and incubated at 37°C. Time points (10 µl) were removed at 10, 20, and 30 min and 1, 2, 3, 4, 5, and 6 h, and N-glycans were extracted from blood as described below and analyzed using TLC and quantitative autoradiography as described above.

Pharmacokinetic analysis of N-glycans
The pharmacokinetics of N-glycans was performed in three to four mice. Mice were anesthetized and a dual jugular vein cannulation was performed. Oligosaccharides were dosed in the left vein while blood time points were taken at 1, 3, 6, 10, 15, 20, 30, 40, and 60 min from the right vein. Serial blood time points were analyzed by direct {gamma}-counting, after which, the oligosaccharide was extracted from blood by adding 60 µl of water and 200 µl of acetonitrile. Proteins were precipitated by centrifugation for 10 min (13,000 x g) and the pellet was washed twice with 50 µl of 80 v/v% acetonitrile, resulting in recovery of 80% of the radioactivity. Extracts were combined and evaporated to dryness on a Centra-Vap under reduced pressure and reconstituted in 3 µl of water. Each time point was analyzed by spotting 1 µl onto a TLC plate which was developed and autoradiographed as described above.

Pharmacokinetic parameters were derived from direct blood counts versus time for triplicate data sets of each oligosaccharide then averaged to obtain the mean and standard deviation. Iterative non-linear least-squares fits for individual data sets were obtained with PCNONLIN (SCI Software, Lexington, KY) using a two-compartment open model described by the integrated equation 1:

Eq. 1 Cb=Ae{alpha}t + Beßt

where Cb is the concentration of oligosaccharide in blood. A and B are constants, and {alpha} and ß are hybrid first-order rate constants that characterize the slopes of the fast and slow phases of decline in a plasma concentration versus time profile. The mean residence time (MRT) was calculated according to equation 2:

Eq. 2 MRT =

which is the average time that the oligosaccharide was in the mouse (Riegelman and Collier, 1980Go). The total body clearance (Cltb) was calculated using equation 3:

Eq. 3 Cltb =

and the volume of distribution at steady-state (Vdss) was calculated according to equation 4 (Benet and Galeazzi, 1979Go).

Eq. 4 Vdss = CLtb * MRT

Whole-body autoradiography
Mice were anesthetized by i.p. injection of ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (10 mg/kg). A single silastic catheter was inserted into the right jugular vein and an i.v. bolus dose of oligosaccharide (60 µl, 7 µCi, in saline) was administered after which the catheter was removed and the vein was ligated. After 30 min, mice were euthanized by lethal injection of phenobarbital (100 mg/kg). Immediately after sacrifice, the mice were immersed in a hexane dry ice bath (–70°C) for 5 min and mounted in a 4% (w/v) carboxymethylcellulose block which was then cooled to –20°C. Longitudinal sections of 25 µm were cut near the midline of the mice at a temperature of –15°C on a cryo-microtome (LKB 2250, Sweden). The sections were collected on adhesive tape (Scotch 810, 3M Co., Minneapolis, MN), dehydrated at –15°C for 24 h, and then autoradiographed for 48 h using a Phosphor Imager.

Biodistribution analysis of N-glycans
Mice were anesthetized, as described above, followed by insertion of a single cannula into the right jugular vein. Oligosaccharides (15 µl, 2 µCi, in saline) were dosed i.v. and allowed to biodistribute 30 min after which mice were sacrificed by cervical dislocation. The major organs (liver, lungs, spleen, stomach, kidneys, heart, large intestine, and small intestine) were harvested, rinsed with saline, and measured by direct {gamma}-counting for total radioactivity.

Isolation of liver parenchymal and nonparenchymal cells
Mice were dosed with 20 mg of carbonyl iron in 0.2 ml of saline via tail vein injection and then anesthetized prior to insertion of a single catheter into the right jugular vein. After 1 h, an oligosaccharide (2 µCi, 50 µl saline) was dosed i.v. and allowed to biodistribute for 30 min at which time the portal vein was cannulated and used to administer 0.2 ml of heparin (100 U/ml), followed immediately by the perfusion buffers. The liver was first perfused for 2 min at 5 ml/min with oxygenated (95% O2 and 5% CO2) pre-perfusion buffer (Ca2+- and Mg2+-free Hepes solution, pH 7.45, 37°C), and then for an additional 3 min at a rate of 3 ml/min.

The liver was digested during a 16–20 min perfusion at 3 ml/min with oxygenated Seglen’s Buffer (pH 7.45, 37°C) containing 0.058% (w/v) collagenase type IV. At the start of the perfusion the vena cava and aorta were cut, and at completion the liver was excised and placed in a petri dish (4°C) and cut into small pieces. Cells were dislodged and dispersed in ice-cold Hanks’ solution (containing 10 mM Hepes, pH 7.45, 0.1% BSA) and then incubated at 37°C for 20 min with shaking (30 rev/min). The dispersed cells were filtered through a 73 µm mesh filter, then transferred to a 35 ml glass tube. The iron-filled nonparenchymal cells were attracted to the wall of the tube with a magnet while the parenchymal cells were decanted off. The procedure was repeated three times and the nonparenchymal cells were combined and resuspended in 0.8 ml Hanks’ Hepes Buffer. The remaining suspension was centrifuged at 50 x g for 1 min and the supernatant was discarded. The pelleted parenchymal cells were washed two times with ice-cold Hanks’-Hepes buffer followed by centrifuging at 50 x g for 1 min. The cells were resuspended in 2 ml of Hanks’-Hepes buffer and the cell purity (>90%) and number (6 x 106 PC and 4.8 x 105 NPC) were determined using a hemocytometer and cell viability (>90%) was determined by the trypan blue exclusion. The radioactivity of each cell fraction was measured by direct {gamma}-counting.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The authors acknowledge financial support from NIH AI33189 and NIH GM48049.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ASGP-R, asialoglycoprotein receptor; Boc-Tyr, tert-butoxycarbonyl-D-tyrosine; TLC, thin layer chromatography; PC, parenchymal cell; NPC, nonparenchymal cell


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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