THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 16, Issue of April 20, pp. 12624 –12628, 2001
Printed in U.S.A.

Asialoglycoprotein Receptor Deficiency in Mice Lacking the Major
Receptor Subunit
ITS OBLIGATE REQUIREMENT FOR THE STABLE EXPRESSION OF OLIGOMERIC RECEPTOR*
Received for publication, December 8, 2000, and in revised form, January 11, 2001
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M011063200

Ryu-ichi Tozawa‡, Shun Ishibashi‡§, Jun-ichi Osuga‡, Kazuo Yamamoto¶, Hiroaki Yagyu‡,
Ken Ohashi‡, Yoshiaki Tamura‡, Naoya Yahagi‡, Yoko Iizuka‡, Hiroaki Okazaki‡, Kenji Harada‡,
Takanari Gotoda‡, Hitoshi Shimano储, Satoshi Kimura**, Ryozo Nagai‡‡, and Nobuhiro Yamada储
From the Departments of ‡Metabolic Diseases, ‡‡Cardiovascular Medicine, and **Infectious Diseases, Faculty of Medicine
and ¶Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8655 and 储Metabolism, Endocrinology, and Atherosclerosis, Institute of Clinical Medicine,
University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanThe asialoglycoprotein receptor is an abundant hetero-oligomeric endocytic receptor that is predominantly
expressed on the sinusoidal surface of the hepatocytes.
A number of physiological and pathophysiological functions have been ascribed to this hepatic lectin (HL), theremoval of desialylated serum glycoproteins and apoptotic cells, clearance of lipoproteins, and the sites ofentry for hepatotropic viruses. The assembly of two homologous subunits, HL-1 and HL-2, is required to form
functional, high affinity receptors on the cell surface.
However, the importance of the individual subunits forreceptor transport to the cell surface is controversial.
We have previously generated HL-2-deficient mice andshowed that the expression of HL-1 was significantly
reduced, and the functional activity as the asialoglycoprotein receptor was virtually eliminated. However, wefailed to detect phenotypic abnormalities. To explore
the significance of the major HL-1 subunit for receptor
expression and function in vivo, we have disrupted theHL-1 gene in mice. Homozygous HL-1-deficient animals
are superficially normal. HL-2 expression in the liver isvirtually abrogated, indicating that HL-1 is strictly required for the stable expression of HL-2. Although thesemice are almost unable to clear asialo-orosomucoid, ahigh affinity ligand for asialoglycoprotein receptor,
they do not accumulate desialylated glycoproteins or
lipoproteins in the plasma.

The asialoglycoprotein receptor (ASGPR)1 was originallyidentified by Ashwell and Morell as a hepatic receptor that
* This work was supported by a grant-in-aid for Scientific Researchfrom the Ministry of Education, Science and Culture, by the Promotionof Fundamental Studies in Health Science of The Organization for
Pharmaceutical Safety and Research, by Health Sciences Research
grants from the Ministry of Health and Welfare, and by Takeda Medical
Research Foundation, Ymanouchi Foundation for Research on Metabolic Disorders, and Asahi Life Foundation. The costs of publication ofthis article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-3-3815-5411(ext. 33113); Fax: 81-3-5802-2955; E-mail: ishibash-tky@umin.ac.jp.
1
The abbreviations used are: ASGPR, asialoglycoprotein receptor;
HL, hepatic lectin; MHL, mouse hepatic lectin; ES, embryonic stem;
ASOR, asialo-orosomucoid; HPLC, high performance liquid chromatography; LDL, low density lipoprotein; HDL, high density lipoprotein;
apo, apolipoprotein; MAA, Maackia amurensis agglutinin; SNA, Sambucus nigra agglutinin; RCA, Ricinus communis agglutinin; kb, kilobase; kbp, kilobase pair.

mediates the rapid clearance of serum glycoproteins containing
terminal galactose residues from the circulation (see Refs. 1–3
for review). ASGPR is abundantly expressed on the sinusoidal
surface of the parenchymal cells of the liver. Its primary physiological function has been considered to be the removal anddegradation of desialylated circulating proteins.
Nonreducing terminal of oligosaccharide moieties of glycoproteins are usually capped by sialic acid residues. When theterminal sialic acid residues are removed by neuraminidases,
penultimate galactose residues are exposed and recognized by
ASGPR. High affinity binding requires the receptor to be assembled as a hetero-oligomer consisting of two highly homologous subunits termed hepatic lectin (HL) 1 and 2 (4). Bothsubunits contain an N-terminal cytoplasmic domain, a single
transmembrane segment, a stalk domain, and a C-terminal
carbohydrate recognition domain (5). ASGPR belongs to C-type
animal lectins because of the requirement of Ca2⫹ for ligand
binding and disulfide bonds in carbohydrate recognition domains (6).
A number of diverse physiological roles have been proposed
for ASGPR over the years. Among them, hepatic clearance ofthe desialylated and senascent serum proteins was most originally proposed (1). ASGPR was also postulated to account for
the low density lipoprotein (LDL) receptor-independent clearance of lipoproteins including chylomicron remnants (7, 8).
Recently, immunoglobulin A (9) and fibronectin (10) haveemerged as likely candidates of natural ligands for ASGPR.
The clearance of apoptotic cells or a subpopulation of lymphocytes in the liver has also attributed to ASGPR (See Ref. 3 forreview). It is particularly noteworthy that ASGPR has also
been proposed to be utilized as entry sites into hepatocytes by
several hepatotropic viruses including hepatitis B virus (11),Marburg virus (12), and hepatitis A virus (13).
As an attempt to elucidate the bona fide functions of ASGPR,
we have previously generated mice lacking a minor subunit ofmouse ASGPR (MHL-2) (14). As a result of disruption of
MHL-2, the expression of MHL-1 was severely reduced, and theplasma clearance of asialo-orosomucoid was almost completely
abrogated in the MHL-2⫺/⫺ mice. However, the MHL-2⫺/⫺
mice were apparently normal and showed no detectable abnormalities even in the metabolism of remnant lipoproteins. Because MHL-2⫺/⫺ liver expresses small but significant
amounts of MHL-1 (14, 15), it is still possible that the residual
MHL-1 is sufficient to execute the primary task of ASGPR assuggested by in vitro transfection experiments (16, 17). In thecurrent study we have generated mice lacking the major sub-12624

This paper is available on line at http://www.jbc.org

Asialoglycoprotein Receptor Knockout Miceunit (MHL-1) of ASGPR in mice and analyzed the resulting
phenotypes.
EXPERIMENTAL PROCEDURES

Generation of MHL-1 Knockout Mice—The MHL-1 gene was cloned
from the 129/Sv mouse genomic library. The genomic organization of
the MHL-1 gene was essentially the same as recently reported by
Soukharev et al. (18). A replacement-type targeting vector was constructed so that the genomic fragment containing exons 2–3, which
encoded the ATG initiation codon and transmembrane domain, wasreplaced by the pol2neo cassette (19). The short arm containing a 0.8-kb
StuI/BamHI fragment containing exon 2 and the long arm containing a
9-kb XhoI/SalI fragment spanning exons 3–9 were inserted into the
XhoI and NotI sites, respectively, of the vector pPol2short-neobpAHSVTK as described previously (14, 20).
After linearization by digestion with SalI, the vector was electroporated into JH-1 embryonic stem (ES) cells (a gift from Dr. Herz at the
University of Texas Southwestern Medical Center). Targeted clones,
which had been selected in the presence of G418 and 1-(2-deoxy,2fluoro- ␤ - D -arabinofuranosyl)-5 iodouracil, were identified bypolymerase chain reaction using the following primers: 5⬘-CTGGTCAGGGATATTTGGAGATACGG-3⬘ and 5⬘-GATTGGGAAGACAATAGCAGGCATGC-3⬘ (see Fig. 1). Homologous recombination was verified bySouthern blot analysis after digesting the genomic DNA with EcoRI
using a 0.7-kbp StuI fragment as a probe (see Fig. 1). Targeted ES
clones were injected into the C57BL/6 blastocysts, yielding 14 lines ofchimeric mice that transmitted the disrupted allele through the germline from four independent ES cell clones. All experiments reported
here were performed with 129/Sv-C57BL6 hybrid descendants (F1 andsubsequent generations) of these animals.
Northern Blot Analysis—Total RNA was isolated from the liver by
TRIZOLTM reagent (Life Technologies, Inc.). 20 ␮g of total RNA was
subjected to electrophoresis in an agarose gel and transferred to a nylon
membrane (Hybond-N; Amersham Pharmacia Biotech). MHL-1 andMHL-2 cDNA fragments were labeled with [␣-32P]dCTP using a kit(Megaprime labeling kit; Amersham Pharmacia Biotech) and were usedas probes for hybridization (14). Image capture and analysis were12625

performed with BAS 2000 (Fuji Film).
Immunoblot Analysis—Liver membrane proteins were prepared as
described (14). 50 ␮g of the proteins was separated by 5–20% SDS
polyacrylamide gel electrophoresis under a nonreducing condition. Proteins were transferred to nitrocellulose membrane, and immunoblot
analyses were performed using specific rabbit polyclonal anti-peptide
antibodies for MHL-1 and MHL-2 (14). The antibodies were visualized
by peroxidase-conjugated anti-rabbit IgG and with an ECL chemiluminescence detection kit (Amersham Pharmacia Biotech).
Plasma Turnover Experiments—Asialo-orosomucoid (ASOR) was
prepared by incubating 100 mg of orosomucoid (Sigma) at 37 °C in 10 mlof sodium acetate buffer containing 2 mM CaCl2, pH 5, together with 1
unit of neuraminidase-type XA (Clostridium perfringens) attached toagarose beads (Sigma). After 4 h another unit of enzyme was added, andthe incubation was continued overnight. Asialofetuin (Sigma) and
ASOR were labeled with 125I using the IODO-GEN procedure (Pierce).
Specific activities of 125I-asialofetuin and 125I-ASOR were 257 and 502
cpm/ng, respectively. 10 ␮g of iodinated protein in 200 ␮l of saline
containing 2 mg/ml bovine serum albumin were injected intravenously
into the jugular vein of anesthetized male mice (n ⫽ 3) that werewild-type and homozygous for MHL-1 gene disruption. Blood was collected at the indicated intervals from the retroorbital venous plexus.
After the labeled proteins in 20 ␮l of plasma were precipitated withtrichloroacetic acid, their radioactivities were determined.
Analyses of Mouse Plasma Lipoprotein Profile—Plasma lipoprotein
analyses were performed as previously described (21). Briefly, after
mice were bled from the retroorbital venous plexus, the blood wascollected into tubes containing EDTA. Total cholesterol and triglyceride
levels in the plasma were determined enzymatically using kits (Determiner TC555 and Determiner TG555; Kyowa Medex). 5 ␮l of plasma
was diluted to 100 ␮l with saline and subjected to high performanceliquid chromatography (HPLC) analyses using four columns of TSK gel
Lipopropak XL (TOSOH, Tokyo, Japan) connected in tandem.
Lectin Blot Analyses of Plasma Protein—1 ␮l of plasma was separated by 3–15% SDS polyacrylamide gel electrophoresis under a reducing condition and transferred to polyvinylidene difluoride membranes.
Lectin blotting was done using Maackia amurensis agglutinin (MAA;

FIG. 1. Targeted disruption of the MHL-1 gene. A, a targeting vector of the replacement type was constructed by replacing the exons
encoding the ATG initiation codon and transmembrane domain by the pol2neo cassette (Neor). Two copies of herpes simplex virus thymidine kinase(HSV-TK) were attached to the 5⬘ side of the vector. E, EcoRI; B, BamHI. The probe for Southern blot analysis is indicated by the shaded box. B,
genotypes of offspring from matings of MHL-1⫹/⫺ mice. Tail DNA was digested with EcoRI. After the transfer, the membranes were hybridized
with 32P-labeled probe (a 0.7-kbp StuI fragment). The positions of migration of disrupted (5.4 kb) and of wild-type alleles (16 kb) are indicated.

12626

Asialoglycoprotein Receptor Knockout Mice

FIG. 2. Northern and Western blot analysis of ASGPR subunits
in mouse liver. A, Northern blot analysis of MHL-1 (lanes 1– 6) andMHL-2 (lanes 7–12) in the liver of 6-week-old mice wild-type (⫹/⫹; lanes
1, 2, 7, and 8), heterozygous (⫹/⫺; lanes 3, 4, 9, and 10), and homozygous (⫺/⫺; lanes 5, 6, 11, and 12) for MHL-1 gene disruption. 15 ␮g oftotal RNA was used for Northern blot analysis. 1.3-kb MHL-1 and1.4-kb MHL-2 wild-type transcripts are indicated. B, Western blot
analysis of MHL-1 (lanes 1– 6) and MHL-2 (lanes 7–12) in the liver of
6-week-old mice wild-type (⫹/⫹; lanes 1, 2, 7, and 8), heterozygous(⫹/⫺; lanes 3, 4, 9, and 10), and homozygous (⫺/⫺; lanes 5, 6, 11, and 12)
for the MHL-1 gene disruption. The size of the molecular mass markers
are indicated.
Roche Molecular Biochemicals) and Sambucus nigra agglutinin (SNA;
Roche Molecular Biochemicals), which were conjugated with digoxigenin, and Ricinus communis agglutinin (RCA120; Sigma), which was
conjugated with biotin. Digoxigenin and biotin were detected by alkaline phosphatase-labeled anti-digoxigenin antibody (Digoxigenin detection kit; Roche Molecular Biochemicals) and alkaline phosphataselabeled anti-biotin antibody (Roche Molecular Biochemicals),
respectively.
RESULTS

The MHL-1 gene was cloned by hybridization screening of amouse genomic library using a mouse cDNA probe. A genereplacement vector was constructed so that the initiation codon
and transmembrane domain were interrupted by the pol2neo
cassette (Fig. 1A). Following electroporation of the linearizedtargeting vector into JH-1 ES cells, targeted clones were obtained. Chimeric mice were generated from six independently
targeted clones using a standard procedure. Four independent
ES cell cloned yielded total of 14 germ line chimeric males.
They were bred to wild-type female C57BL/6 mice. Heterozygous offspring (F1 generation) were crossed with each otherand gave rise to mice wild-type (⫹/⫹), heterozygous (⫾), or
homozygous (⫺/⫺) for the disrupted MHL-1 allele in accordance with Mendelian law (⫹/⫹:⫾:⫺/⫺ ⫽ 29:65:43; ␹2 ⫽ 1.51;
p ⫽ 0.47) (Fig. 1B). Homozygous MHL-1-deficient mice wereviable and displayed no obvious phenotype under laboratory
housing conditions. The animals appeared to have a normal life
span.

Northern blot analysis showed that MHL-1⫺/⫺ mice lacked
1.3-kb wild-type MHL-1 transcript completely (Fig. 2A). MHL1⫹/⫺ mice expressed an intermediate amount of the MHL-1
transcript. A reduced amount of truncated MHL-1 transcript
was expressed in mice having the disrupted MHL-1 allele(MHL-1⫹/⫺ or MHL-1⫺/⫺). On the other hand, there were nodifferences in the size and amounts of MHL-2 mRNA. The
disruption of the MHL-1 gene resulted in complete absence ofthe 40-kDa-encoded protein in MHL-1⫺/⫺ animals (Fig. 2B).
MHL-1⫹/⫺ mice showed reduced expression of MHL-1 protein
as compared with the wild-type mice, indicating the gene dosage effect on the expressed protein levels of MHL-1. In wildtype and MHL-1⫹/⫺ mice, four distinct bands that were immunoreactive with the anti-MHL-2 antibody were observed(36, 47, 80, and 192 kDa). Because the bands with higher
molecular mass were not observed under a reducing condition(data not shown), three of these multiple bands may represent
oligomers of MHL-2 protein in different degrees (monomer for
36 kDa, dimer for 80 kDa, and tetramer for 192 kDa). No bands
immunoreactive with the anti-MHL-2 antibody were visible inMHL-1⫺/⫺ mice.
To examine whether the disruption of the MHL-1 gene resulted in the impairment of the hepatic clearance of asialoglycoproteins, we injected the iodinated asialofetuin or ASOR into⫹/⫹, ⫹/⫺, and ⫺/⫺ mice and compared the radioactivities
remaining in the plasma (Fig. 3). The plasma clearance of
125
I-asialofetuin was severely, albeit not completely, impairedin MHL-1⫺/⫺ mice (Fig. 3A). At 30 min after the injection,
plasma 125I-asialofetuin was decreased to 4, 8, and 40% of theinitial dose in ⫹/⫹, ⫹/⫺, and ⫺/⫺ mice, respectively. Heterozygous mice cleared 125I-asialofertuin with only partially reduced
efficiency. The plasma clearance of 125I-ASOR was more severely impaired in MHL-1⫺/⫺ mice (Fig. 3B). At 30 min after
the injection, plasma 125I-ASOR was decreased to 3, 24, and
65% of the initial dose in ⫹/⫹, ⫹/⫺, and ⫺/⫺ mice, respectively.
Heterozygous mice cleared 125I-ASOR with intermediate
efficiency.
To examine the expression of the sialic acid- and galactoseterminated serum glycoproteins, plasma from the wild-type
and MHL-1⫺/⫺ mice were subjected to lectin blot analysis(Fig. 4). The following lectins were used to detect terminal
sugars: MAA, a lectin specific for NeuNAc(␣2–3)Gal on Nlinked carbohydrate; SNA, a lectin specific for NeuNAc(␣2–6)Gal on N-linked carbohydrate; and RCA120, a lectin specificfor ␤-galactose. There were no consistent differences either inthe pattern of the bands or in their intensities between MHL1⫹/⫹ and MHL-1⫺/⫺ mice.
To test whether ASGPR is involved in chylomicron remnant
clearance by the liver, we analyzed the plasma lipid levels of
MHL-1⫺/⫺ mice and MHL-1⫺/⫺ mice that also lacked the
LDL receptor (Table I). The plasma lipid levels of the MHL1⫺/⫺ mice were indistinguishable from those of control animals expressing wild-type MHL-1, in the absence or presence offunctional LDL receptors. The lipoprotein profiles evaluated by
HPLC analyses failed to reveal any differences in lipoprotein
fractions between wild-type and MHL-1 ⫺/⫺ mice (Table II).
DISCUSSION

We have generated mice lacking functional asialoglycoprotein receptors by disrupting the gene for the major MHL-1
receptor subunit by homologous recombination in embryonicstem cells. Homozygous MHL-1-deficient mice displayed no
obvious phenotype as long as they were maintained under the
standard laboratory housing condition.
Although MHL-1⫺/⫺ liver expressed a reduced amount of atruncated transcript, no band immunoreactive with the antiMHL-1 antibody was detected, indicating that MHL-1⫺/⫺

Asialoglycoprotein Receptor Knockout Mice12627

FIG. 3. Disappearance of 125I-labeled glycoproteins from the plasma.
125
I-labeled asialofetuin (Panel A) or
asialo-orosomucoid (Panel B) was injected
into 12-week-old female mice wild-type(●), heterozygous (E), and homozygous(X) for the MHL-1 gene disruption. Values
shown are means of triplicates of trichloroacetic acid-insoluble radioactivity remaining in plasma at the indicated time
points and are calculated as a percentage
of radioactivity present 1 min after injection of the label.

FIG. 4. Lectin blot of plasma proteins. Blood was collected from 32-weekold male mice wild-type (lanes 1–5) or homozygous (lanes 6 –10) for the MHL-1
disruption. Lectin blotting was done using MAA, SNA that were conjugated withdigoxygenin, and RCA120 that was conjugated with biotin. Digoxigenin and biotin
were detected by alkaline phosphataselabeled anti-digoxigenin antibody and alkaline phosphatase-labeled anti-biotin
antibody, respectively.

TABLE I
Plasma lipid levels in mice deficient in MHL-1
Blood was collected from mice aged 8 –12 weeks after an overnight
fast. Total cholesterol (TC) and triglyceride (TC) concentrations in theplasma were determined and expressed as means ⫾ S.D. Analysis ofvariance was employed to compare the means between the genotypes.
LDLR and MHL-1 denote low density lipoprotein receptor and mouse
hepatic lectin-1, respectively.
LDLR

MHL-1

n

TC

⫹/⫹
⫹/⫹
⫹/⫹
⫺/⫺
⫺/⫺
⫺/⫺

⫹/⫹
⫹/⫺
⫺/⫺
⫹/⫹
⫹/⫺
⫺/⫺

4
4
4
5
9
7

83 ⫾ 22105 ⫾ 1189 ⫾ 18328 ⫾ 70327 ⫾ 21279 ⫾ 19TG
mg/dl

89 ⫾ 699 ⫾ 17102 ⫾ 38277 ⫾ 33
268 ⫾ 29233 ⫾ 27

TABLE II
Plasma lipoproteins in mice deficient in MHL-1
Blood was collected from 4 –5 mice aged 12 weeks after an overnight
fast. Plasma lipoproteins were analyzed by high performance liquid
chromatography. The results were expressed as means ⫾ S.D. Analysis
of variance was employed to compare the means between the genotypes.
CM, VLDL, LDL, and HDL denote chylomicron; very low density lipoproteins, low density lipoproteins, and high density lipoproteins,respectively.
Cholesterol

Wild-type

⫹/⫺

⫺/⫺

mg/dl

Total
CM
VLDL
LDL
HDL

85.7 ⫾ 2.5
0.2 ⫾ 0.2
1.9 ⫾ 1.2
7.7 ⫾ 1.3
78.0 ⫾ 3.4

79.1 ⫾ 13.0
0.1 ⫾ 0.1
1.7 ⫾ 1.0
5.4 ⫾ 2.4
72.0 ⫾ 12.0

72.2 ⫾ 8.4
0.3 ⫾ 0.4
1.5 ⫾ 1.4
4.8 ⫾ 2.2
65.5 ⫾ 7.6

mice were virtually null for the MHL-1 gene. In MHL-1⫹/⫺
mice, the amounts of both mRNA and protein of MHL-1 werereduced 2-fold, indicating the gene-dosage effects of the inactivation. This reduction was accompanied by the reduction in theamounts of MHL-2 protein, even if the mRNA levels were notaffected. In MHL-1⫺/⫺ mice, MHL-2 protein was undetectable. These results strongly indicate that MHL-1 is obligatorily

required for the stable expression of MHL-2. This is consistent
with the in vitro results in transfected cells that the minor
subunit is unstable in the absence of coexpression of the major
subunit (22–24). Without HL-1, HL-2 may succumb to degradation within endoplasmic reticulum. It is interesting to compare this to MHL-2⫺/⫺ mice in which substantially reduced
but still significant amounts of MHL-1 were expressed. Together these results indicate that both subunits are required
for the stable expression of oligomeric receptor and that HL-1 ismore strictly required than HL-2.
As was observed in MHL-2⫺/⫺ mice, the plasma clearanceof asialoglycoproteins was severely impaired in MHL-1⫺/⫺
mice (Fig. 3). It is interesting to note that the impairment of theclearance of ASOR appeared milder than that of asialofetuin in
the heterozygotes. This is probably because some other pathway also contributes to the plasma clearance of asialofetuin but
not to that of ASOR. For example, a macrophage lectin that isconceivably expressed in hepatic Kupffer cells may mediate
this uptake, because it recognizes both galactose and N-acetylgalactosamine (25, 26). The plasma clearance curve of ASOR
was indistinguishable from that of orosomucoid (data notshown), suggesting that MHL-1⫺/⫺ mice clear ASOR via apathway(s) that is irrelevant to the galactose-recognition system. Although ASGPR function was severely impaired in MHL1⫺/⫺ mice, the plasma glycoproteins levels were not significantly increased (Fig. 4). Together with our previous
observations in MHL-2 knockout mice, ASGPR is unlikely to be
essential for the homeostasis of the major plasma glycoproteinsas has been frequently discussed. In support of this, Kido et al.
(27) have recently reported that serum glycoprotein levels weremaintained in mice lacking ␤-1,4-galactosyltransferase I. Although nearly 90% of the serum glycoproteins lacked ␤-1,4galactose in the knockout mice, their serum protein concentrations were similar to those in wild-type mice. This does notnecessarily rule out the possible role of ASGPR in the regulation of minor serum glycoproteins (28). In this context, Rotundo
et al. (10) have proposed that ASGPR is responsible for the
disposal of cellular fibronectin from the plasma and/or in the

12628

Asialoglycoprotein Receptor Knockout Miceliver. Cellular fibronectin contains large amounts of terminal
galactose residues, and intravenous infusion of excess asialofetuin caused retention of labeled cellular fibronectin in the liver(29). However, in multiple experiments not shown here, byWestern blot analyses in the liver membrane we failed to findthe abnormal accumulation of fibronectin in the liver of MHL1⫺/⫺ mice. Furthermore, we failed to find significant morphological changes in the liver of MHL-1⫺/⫺ mice. With regard to
IgA metabolism, Rifai et al. (9) have recently reported the
impaired clearance of the IgA2 isoform in MHL-2⫺/⫺ mice.
However, its physiological relevance is currently unknown.
Windler et al. (7) proposed a possible function of ASGPR in
the hepatic lipoproteins metabolism. ApoB-100 and apo(a) areheavily glycosylated giant proteins. Therefore it is reasonable
to consider that the lipoproteins containing these apolipoproteins is cleared by ASGPR when the LDL receptor system isdysfunctional. To test this hypothesis, we crossed MHL-1⫺/⫺
mice to the LDL receptor-deficient mice to generate mice lacking both ASGPR and the LDL receptor. As was the case in themice lacking both MHL-2 and the LDL receptor, we failed todetect the elevation of plasma lipoproteins as compared with
the LDL receptor knockout mice (Tables I and II). Accumulating evidence indicates that LDL receptor-related protein is
involved in the plasma clearance of apoE-rich remnant lipoproteins (30). Therefore, the role of ASGPR in the plasma clearance of lipoproteins is minimal if present.
ASGPR is a member of animal C-type lectins (6). Because
most of C-type lectins appear to be involved in host defense,
ASGPR may have been evolved as a molecule to protect mammals from viral or bacterial insults (31), probably because
exposed galactose residues may be harmful to the vascularsystem (32). Avian and reptiles have a similar hepatic lectinthat binds to N-acetylglucosamine. The ASGPR system may
protect vertebrates from pathogenic organisms that take advantage of neuraminidase to invade the hosts. A significant
decrease in the expression of ASGPR accompanied by accumulation of serum asialoglycoproteins is observed in the patientswith advanced liver diseases such as liver cirrhosis (33, 34).
They frequently develop serious infectious diseases such as
spontaneous bacterial peritonitis and sepsis. It is tempting to
speculate that ASGPR is relevant to these complications associated with liver diseases. Besides hepatocytes, macrophages
express a lectin specific for both galactose and N-acetylgalactosamine as mentioned above (25, 26). Its presence may havemasked the phenotype in ASGPR-deficient animals. Further
studies are absolutely needed to prove these possibilities. In
this context, it is intriguing that some hepatotropic virus infects the hepatocytes through ASGPR (11–13).
In summary, ASGPR functions were more completely abrogated in MHL-1-deficient mice as compared with in MHL-2deficient mice. Probably, requirement of HL-1 for stable expression of functional ASGPR is stricter than that of HL-2.

Despite such absolute deficiency of ASGPR function, we failed
to detect physiological evidence for several postulated functions
that have been ascribed to ASGPR. These MHL-1-deficient
mice should provide the basis for understanding the physiologyof this receptor.
REFERENCES
1. Ashwell, G., and Morell, A. G. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41,
99 –128
2. Spiess, M. (1990) Biochemistry 29, 10009 –10018
3. Stockert, R. J. (1995) Physiol. Rev. 75, 591– 609
4. Lodish, H. F. (1991) Trends Biochem. Sci. 16, 374 –377
5. Halberg, D. F., Wager, R. E., Farrell, D. C., Hildreth, J., IV, Quesenberry,
M. S., Loeb, J. A., Holland, E. C., and Drickamer, K. (1987) J. Biol. Chem.
262, 9828 –9838
6. Drickamer, K. (1988) J. Biol. Chem. 263, 9557–9560
7. Windler, E., Greeve, J., Levkau, B., Kolb-Bachofen, V., Daerr, W., and Greten,
H. (1991) Biochem. J. 276, 79 – 87
8. Ishibashi, S., Perrey, S., Chen, Z., Osuga, J., Shimada, M., Ohashi, K., Harada,
K., Yazaki, Y., and Yamada, N. (1996) J. Biol. Chem. 271, 22422–22427
9. Rifai, A., Fadden, K., Morrison, S. L., and Chintalacharuvu, K. R. (2000) J.
Exp. Med. 191, 1171–1181
10. Rotundo, R. F., Rebres, R. A., McKeown-Longo, P. J., Blumenstock, F. A., andSaba, T. M. (1998) Hepatology 28, 475– 485
11. Treichel, U., Meyer zum Buschenfelde, K. H., Stockert, R. J., Poralla, T., andGerken, G. (1994) J. Gen. Virol. 75, 3021–3029
12. Becker, S., Spiess, M., and Klenk, H.-D. (1995) J. Gen. Virol. 76, 393–399
13. Dotzauer, A., Gebhardt, U., Bieback, K., Gottke, U., Kracke, A., Mages, J.,
Lemon, S. M., and Vallbracht, A. (2000) J. Virol. 74, 10950 –10957
14. Ishibashi, S., Hammer, R. H., and Herz, J. (1994) J. Biol. Chem. 269,
27803–27806
15. Broun, J. R., Willnow, T. E., Ishibashi, S., Ashwall, G., and Herz, J. (1996)
J. Biol. Chem. 271, 21160 –21166
16. Braiterman, L. T., Chance, S. C., Porter, W. R., Lee, Y. C., Townsend, R. R.,
and Hubbard, A. L. (1989) J. Biol. Chem. 264, 1682–1688
17. Geffen, I., Wessels, H. P., Roth, J., Shia, M. A., and Spiess, M. (1989) EMBO
J. 8, 2855–2861
18. Soukharev, S., Berlin, W., Hanover, J. A., Bethke, B., and Sauer, B. (2000)
Gene 241, 233–240
19. Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991) Cell 64,
693–702
20. Tozawa, R., Ishibashi, S., Osuga, J., Yagyu, H., Oka, T., Chen, Z., Ohashi, K.,
Perrey, S., Shionoiri, F., Yahagi, N., Harada, K., Gotoda, T., Yazaki, Y., andYamada, N. (1999) J. Biol. Chem. 274, 30843–30848
21. Yagyu, H., Ishibashi, S., Chen, Z., Osuga, J., Okazaki, M., Perrey, S.,
Kitamine, T., Shimada, M., Ohashi, K., Harada, K., Shionoiri, F., Yahagi,N., Gotoda, T., Yazaki, Y., and Yamada, N. (1999) J. Lipid Res. 40,
1677–1685
22. Shia, M. A., and Lodish, H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,
1158 –1162
23. Wikstrom, L., and Lodish, H. F. (1992) J. Biol. Chem. 267, 5– 8
24. Wikstrom, L., and Lodish, H. F. (1993) J. Biol. Chem. 168, 14412–14416
25. Oda, S., Sato, M., Toyoshima, S., and Osawa, T. (1988) J. Biochem. 104,
600 – 605
26. Ii, M., Kurata, H., Itoh, N., Yamashina, I., and Kawasaki, T. (1990) J. Biol.
Chem. 265, 11295–11298
27. Kido, M., Asano, M., Iwakura, Y., Ichinose, M., Miki, K., and Furukawa, K.
(1999) FEBS Lett. 464, 75–79
28. Drickamer, K. (1991) Cell 67, 1029 –1032
29. Rutondo, R. F., Vincent, P. A., McKeown-Longo, P. J., Blumenstock, F. A., andSaba, T. M. (1999) Am. J. Physiol. 277, G1189 –G1199
30. Rohmann, A., Gotthardt, M., Hammer, R. E., and Herz, J. (1998) J. Clin.
Invest. 101, 689 – 695
31. Schauer, R., Sander-Wewer, M., Gutschker-Gdaniec, G. H., Roggentin, P.,
Randow, E. A., and Hobrecht, R. (1985) Clin. Chim. Acta 146, 119 –127
32. Weigel, P. H. (1994) Bioessays 16, 519 –524
33. Marshall, J. S., Green, A. M., Pensky, J., Williams, S., Zinn, A., and Carlson,
D. M. (1974) J. Clin. Invest. 54, 555–562
34. Sawamura, T., Kawasato, S., Shiozaki, Y., Sameshima, Y., Nakada, H., andTashiro, Y. (1981) Gastroenterology 81, 527–533