Asialoglycoprotein Receptor Deficiency in Mice Lacking the Major Receptor Subunit

The 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), the removal of desialylated serum glycoproteins and apoptotic cells, clearance of lipoproteins, and the sites of entry 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 for receptor transport to the cell surface is controversial. We have previously generated HL-2-deficient mice and showed that the expression of HL-1 was significantly reduced, and the functional activity as the asialoglycoprotein receptor was virtually eliminated. However, we failed 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 is virtually abrogated, indicating that HL-1 is strictly required for the stable expression of HL-2. Although these mice are almost unable to clear asialo-orosomucoid, a high affinity ligand for asialoglycoprotein receptor, they do not accumulate desialylated glycoproteins or lipoproteins in the plasma. The 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), the removal of desialylated serum glycoproteins and apoptotic cells, clearance of lipoproteins, and the sites of entry 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 for receptor transport to the cell surface is controversial. We have previously generated HL-2-deficient mice and showed that the expression of HL-1 was significantly reduced, and the functional activity as the asialoglycoprotein receptor was virtually eliminated. However, we failed 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 is virtually abrogated, indicating that HL-1 is strictly required for the stable expression of HL-2. Although these mice are almost unable to clear asialo-orosomucoid, a high affinity ligand for asialoglycoprotein receptor, they do not accumulate desialylated glycoproteins or lipoproteins in the plasma. The asialoglycoprotein receptor (ASGPR) 1The abbreviations used are:ASGPRasialoglycoprotein receptorHLhepatic lectinMHLmouse hepatic lectinESembryonic stemASORasialo-orosomucoidHPLChigh performance liquid chromatographyLDLlow density lipoproteinHDLhigh density lipoproteinapoapolipoproteinMAAMaackia amurensis agglutininSNASambucus nigra agglutininRCARicinus communis agglutininkbkilobasekbpkilobase pair was originally identified by Ashwell and Morell as a hepatic receptor that mediates the rapid clearance of serum glycoproteins containing terminal galactose residues from the circulation (see Refs. 1Ashwell G. Morell A.G. Adv. Enzymol. Relat. Areas Mol. Biol. 1974; 41: 99-128PubMed Google Scholar, 2Spiess M. Biochemistry. 1990; 29: 10009-10018Crossref PubMed Scopus (402) Google Scholar, 3Stockert R.J. Physiol. Rev. 1995; 75: 591-609Crossref PubMed Scopus (464) Google Scholar 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 and degradation of desialylated circulating proteins. asialoglycoprotein receptor hepatic lectin mouse hepatic lectin embryonic stem asialo-orosomucoid high performance liquid chromatography low density lipoprotein high density lipoprotein apolipoprotein Maackia amurensis agglutinin Sambucus nigra agglutinin Ricinus communis agglutinin kilobase kilobase pair Nonreducing terminal of oligosaccharide moieties of glycoproteins are usually capped by sialic acid residues. When the terminal 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 (4Lodish H.F. Trends Biochem. Sci. 1991; 16: 374-377Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Both subunits contain an N-terminal cytoplasmic domain, a single transmembrane segment, a stalk domain, and a C-terminal carbohydrate recognition domain (5Halberg D.F. Wager R.E. Farrell D.C. Hildreth J., IV Quesenberry M.S. Loeb J.A. Holland E.C. Drickamer K. J. Biol. Chem. 1987; 262: 9828-9838Abstract Full Text PDF PubMed Google Scholar). ASGPR belongs to C-type animal lectins because of the requirement of Ca2+for ligand binding and disulfide bonds in carbohydrate recognition domains (6Drickamer K. J. Biol. Chem. 1988; 263: 9557-9560Abstract Full Text PDF PubMed Google Scholar). A number of diverse physiological roles have been proposed for ASGPR over the years. Among them, hepatic clearance of the desialylated and senascent serum proteins was most originally proposed (1Ashwell G. Morell A.G. Adv. Enzymol. Relat. Areas Mol. Biol. 1974; 41: 99-128PubMed Google Scholar). ASGPR was also postulated to account for the low density lipoprotein (LDL) receptor-independent clearance of lipoproteins including chylomicron remnants (7Windler E. Greeve J. Levkau B. Kolb-Bachofen V. Daerr W. Greten H. Biochem. J. 1991; 276: 79-87Crossref PubMed Scopus (52) Google Scholar, 8Ishibashi S. Perrey S. Chen Z. Osuga J. Shimada M. Ohashi K. Harada K. Yazaki Y. Yamada N. J. Biol. Chem. 1996; 271: 22422-22427Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Recently, immunoglobulin A (9Rifai A. Fadden K. Morrison S.L. Chintalacharuvu K.R. J. Exp. Med. 2000; 191: 1171-1181Crossref Scopus (136) Google Scholar) and fibronectin (10Rotundo R.F. Rebres R.A. McKeown-Longo P.J. Blumenstock F.A. Saba T.M. Hepatology. 1998; 28: 475-485Crossref PubMed Scopus (33) Google Scholar) have emerged 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. 3Stockert R.J. Physiol. Rev. 1995; 75: 591-609Crossref PubMed Scopus (464) Google Scholar for review). 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 (11Treichel U. Meyer zum Buschenfelde K.H. Stockert R.J. Poralla T. Gerken G. J. Gen. Virol. 1994; 75: 3021-3029Crossref PubMed Scopus (113) Google Scholar), Marburg virus (12Becker S. Spiess M. Klenk H.-D. J. Gen. Virol. 1995; 76: 393-399Crossref PubMed Scopus (147) Google Scholar), and hepatitis A virus (13Dotzauer A. Gebhardt U. Bieback K. Gottke U. Kracke A. Mages J. Lemon S.M. Vallbracht A. J. Virol. 2000; 74: 10950-10957Crossref PubMed Scopus (87) Google Scholar). As an attempt to elucidate the bona fide functions of ASGPR, we have previously generated mice lacking a minor subunit of mouse ASGPR (MHL-2) (14Ishibashi S. Hammer R.H. Herz J. J. Biol. Chem. 1994; 269: 27803-27806Abstract Full Text PDF PubMed Google Scholar). As a result of disruption of MHL-2, the expression of MHL-1 was severely reduced, and the plasma clearance of asialo-orosomucoid was almost completely abrogated in theMHL-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 (14Ishibashi S. Hammer R.H. Herz J. J. Biol. Chem. 1994; 269: 27803-27806Abstract Full Text PDF PubMed Google Scholar, 15Broun J.R. Willnow T.E. Ishibashi S. Ashwall G. Herz J. J. Biol. Chem. 1996; 271: 21160-21166Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), it is still possible that the residual MHL-1 is sufficient to execute the primary task of ASGPR as suggested by in vitro transfection experiments (16Braiterman L.T. Chance S.C. Porter W.R. Lee Y.C. Townsend R.R. Hubbard A.L. J. Biol. Chem. 1989; 264: 1682-1688Abstract Full Text PDF PubMed Google Scholar, 17Geffen I. Wessels H.P. Roth J. Shia M.A. Spiess M. EMBO J. 1989; 8: 2855-2861Crossref PubMed Scopus (29) Google Scholar). In the current study we have generated mice lacking the major subunit (MHL-1) of ASGPR in mice and analyzed the resulting phenotypes. 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. (18Soukharev S. Berlin W. Hanover J.A. Bethke B. Sauer B. Gene. 2000; 241: 233-240Crossref PubMed Scopus (6) Google Scholar). 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, was replaced by the pol2neo cassette (19Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1856) Google Scholar). The short arm containing a 0.8-kb StuI/BamHI fragment containing exon 2 and the long arm containing a 9-kbXhoI/SalI fragment spanning exons 3–9 were inserted into the XhoI and NotI sites, respectively, of the vector pPol2short-neobpA-HSVTK as described previously (14Ishibashi S. Hammer R.H. Herz J. J. Biol. Chem. 1994; 269: 27803-27806Abstract Full Text PDF PubMed Google Scholar, 20Tozawa 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. Yamada N. J. Biol. Chem. 1999; 274: 30843-30848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). 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,2-fluoro-β-d-arabinofuranosyl)-5 iodouracil, were identified by polymerase chain reaction using the following primers: 5′-CTGGTCAGGGATATTTGGAGATACGG-3′ and 5′-GATTGGGAAGACAATAGCAGGCATGC-3′ (see Fig. 1). Homologous recombination was verified by Southern 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 of chimeric 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 and subsequent generations) of these animals. Total RNA was isolated from the liver by TriZOLTM reagent (Life Technologies, Inc.). 20 μg of total RNA was subjected to electrophoresis in an agarose gel and transferred to a nylon membrane (Hybond-N; Amersham Pharmacia Biotech). MHL-1 and MHL-2cDNA fragments were labeled with α-32PdCTP using a kit (Megaprime labeling kit; Amersham Pharmacia Biotech) and were used as probes for hybridization (14Ishibashi S. Hammer R.H. Herz J. J. Biol. Chem. 1994; 269: 27803-27806Abstract Full Text PDF PubMed Google Scholar). Image capture and analysis were performed with BAS 2000 (Fuji Film). Liver membrane proteins were prepared as described (14Ishibashi S. Hammer R.H. Herz J. J. Biol. Chem. 1994; 269: 27803-27806Abstract Full Text PDF PubMed Google Scholar). 50 μg 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 (14Ishibashi S. Hammer R.H. Herz J. J. Biol. Chem. 1994; 269: 27803-27806Abstract Full Text PDF PubMed Google Scholar). The antibodies were visualized by peroxidase-conjugated anti-rabbit IgG and with an ECL chemiluminescence detection kit (Amersham Pharmacia Biotech). Asialo-orosomucoid (ASOR) was prepared by incubating 100 mg of orosomucoid (Sigma) at 37 °C in 10 ml of sodium acetate buffer containing 2 mmCaCl2, pH 5, together with 1 unit of neuraminidase-type XA (Clostridium perfringens) attached to agarose beads (Sigma). After 4 h another unit of enzyme was added, and the incubation was continued overnight. Asialofetuin (Sigma) and ASOR were labeled with 125I using the IODO-GEN procedure (Pierce). Specific activities of 125I-asialofetuin and125I-ASOR were 257 and 502 cpm/ng, respectively. 10 μg of iodinated protein in 200 μl of saline containing 2 mg/ml bovine serum albumin were injected intravenously into the jugular vein of anesthetized male mice (n = 3) that were wild-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 μl of plasma were precipitated with trichloroacetic acid, their radioactivities were determined. Plasma lipoprotein analyses were performed as previously described (21Yagyu 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. Yamada N. J. Lipid Res. 1999; 40: 1677-1685Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, after mice were bled from the retroorbital venous plexus, the blood was collected 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 μl of plasma was diluted to 100 μl with saline and subjected to high performance liquid chromatography (HPLC) analyses using four columns of TSK gel Lipopropak XL (TOSOH, Tokyo, Japan) connected in tandem. 1 μl 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 amurensisagglutinin (MAA; 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 phosphatase-labeled anti-biotin antibody (Roche Molecular Biochemicals), respectively. The MHL-1 gene was cloned by hybridization screening of a mouse genomic library using a mouse cDNA probe. A gene replacement vector was constructed so that the initiation codon and transmembrane domain were interrupted by the pol2neo cassette (Fig.1 A). Following electroporation of the linearized targeting vector into JH-1 ES cells, targeted clones were obtained. mice were generated from targeted clones using a independent ES cell cloned total of 14 chimeric were to wild-type C57BL/6 mice. (F1 were with and to mice wild-type or homozygous for the allele in with = = = (Fig.1 Homozygous mice were and no under The animals to have a normal blot analysis showed that mice wild-type MHL-1 completely A). mice expressed an of the MHL-1 A of MHL-1 was expressed in mice the disrupted MHL-1 allele or the were no in the and amounts of The disruption of the MHL-1 gene in of the protein in 2 mice showed expression of MHL-1 protein as with the wild-type indicating the gene on the expressed protein levels of In wild-type and four that were with the antibody were and Because the with were not under a reducing condition not of these of MHL-2 protein in for for and for with the antibody were in mice. To the disruption of the MHL-1 gene in the of the hepatic clearance of we injected the iodinated or ASOR into and mice and the radioactivities in the plasma The plasma clearance was not in mice A). after the plasma 125I-asialofetuin was to and of the in and respectively. mice with The plasma clearance was severely mice after the plasma was to and of the in and respectively. mice with To the expression of the sialic and serum plasma from the wild-type mice were subjected to lectin blot analysis The following lectins were used to detect terminal a lectin specific for on a lectin specific for on and a lectin specific for were no in the of the or in their and mice. To ASGPR is in chylomicron remnant clearance by the we analyzed the plasma levels of and mice that also the receptor The plasma levels of the mice were from of animals wild-type in the or presence of functional The lipoprotein by analyses failed to in lipoprotein wild-type and MHL-1 mice levels in mice in was collected from mice after an Total cholesterol and triglyceride in the plasma were determined and expressed as of was to the the and MHL-1 low density lipoprotein receptor and mouse hepatic respectively. in a lipoproteins in mice in was collected from mice after an Plasma lipoproteins were analyzed by high performance liquid The were expressed as of was to the the and low density lipoproteins, low density lipoproteins, and high density lipoproteins, respectively. in a Blood was collected from mice after an Total cholesterol and triglyceride in the plasma were determined and expressed as of was to the the and MHL-1 low density lipoprotein receptor and mouse hepatic respectively. Blood was collected from mice after an Plasma lipoproteins were analyzed by high performance liquid The were expressed as of was to the the and low density lipoproteins, low density lipoproteins, and high density lipoproteins, respectively. We have generated mice lacking functional asialoglycoprotein receptors by the gene for the major MHL-1 receptor subunit by homologous recombination in embryonic stem mice no as long as they were under the condition. Although liver expressed a of a no with the antibody was indicating that mice were virtually for the MHL-1 In the amounts of and protein of MHL-1 were indicating the of the was by the in the amounts of MHL-2 even the levels were not MHL-2 protein was that MHL-1 is required for the stable expression of is with the in vitro in cells that the minor subunit is in the of of the major subunit M.A. H. Sci. U. S. A. 1989; PubMed Scopus Google Scholar, H.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H.F. J. Biol. Chem. Scholar). HL-2 to degradation It is to this to in which but still significant amounts of MHL-1 were these that subunits are required for the stable expression of receptor and that HL-1 is strictly required HL-2. As was in MHL-2−/− the plasma clearance of was severely in mice It is to that the of the clearance of ASOR that of in the is because also to the plasma clearance of but not to that of a lectin that is expressed in hepatic cells this because it galactose and S. M. S. T. J. Biochem. 1988; PubMed Scopus Google Scholar, M. H. N. I. T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). The plasma clearance of ASOR was from that of orosomucoid not that clear ASOR a that is to the Although ASGPR function was severely in the plasma glycoproteins levels were not significantly with in MHL-2 ASGPR is to be for the of the major plasma glycoproteins as has been In of et M. M. Y. M. K. K. 1999; PubMed Scopus Google Scholar) have recently reported that serum levels were in mice lacking I. Although of the serum glycoproteins in the their serum protein were to in wild-type mice. not the possible of ASGPR in the of minor serum glycoproteins K. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). In this et al. (10Rotundo R.F. Rebres R.A. McKeown-Longo P.J. Blumenstock F.A. Saba T.M. Hepatology. 1998; 28: 475-485Crossref PubMed Scopus (33) Google Scholar) have proposed that ASGPR is for the of fibronectin from the plasma in the liver. fibronectin amounts of terminal galactose and of of labeled fibronectin in the liver R.F. McKeown-Longo P.J. Blumenstock F.A. Saba T.M. J. Physiol. 1999; Google Scholar). However, in experiments not by blot analyses in the liver membrane we failed to the of fibronectin in the liver of mice. we failed to significant in the liver of mice. to et al. (9Rifai A. Fadden K. Morrison S.L. Chintalacharuvu K.R. J. Exp. Med. 2000; 191: 1171-1181Crossref Scopus (136) Google Scholar) have recently reported the clearance of the in MHL-2−/− mice. However, physiological is et al. (7Windler E. Greeve J. Levkau B. Kolb-Bachofen V. Daerr W. Greten H. Biochem. J. 1991; 276: 79-87Crossref PubMed Scopus (52) Google Scholar) proposed a possible function of ASGPR in the hepatic lipoproteins and are proteins. it is to that the lipoproteins containing these is by ASGPR the receptor is To this we mice to the mice to mice lacking ASGPR and the As was the in the mice lacking MHL-2 and the receptor, we failed to detect the of plasma lipoproteins as with the receptor mice and that protein is in the plasma clearance of remnant lipoproteins A. M. Hammer R.E. Herz J. J. 1998; PubMed Scopus Google Scholar). the of ASGPR in the plasma clearance of lipoproteins is ASGPR is a of animal C-type lectins (6Drickamer K. J. Biol. Chem. 1988; 263: 9557-9560Abstract Full Text PDF PubMed Google Scholar). Because most of C-type lectins to be in ASGPR have been as a to from or R. M. P. R. PubMed Scopus Google Scholar), because exposed galactose residues be to the 1994; 16: PubMed Scopus Google Scholar). and have a hepatic lectin that to The ASGPR from that of to the A significant in the expression of ASGPR by of serum is in the with liver as liver J. S. A. J. 1974; PubMed Scopus Google Scholar, T. S. Y. Y. H. Y. Full Text PDF PubMed Scopus (87) Google Scholar). as and It is to that ASGPR is to these with liver a lectin specific for galactose as S. M. S. T. J. Biochem. 1988; PubMed Scopus Google Scholar, M. H. N. I. T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). Its presence have the in animals. are to these In this it is that hepatotropic virus the hepatocytes through ASGPR (11Treichel U. Meyer zum Buschenfelde K.H. Stockert R.J. Poralla T. Gerken G. J. Gen. Virol. 1994; 75: 3021-3029Crossref PubMed Scopus (113) Google Scholar, S. Spiess M. Klenk H.-D. J. Gen. Virol. 1995; 76: 393-399Crossref PubMed Scopus (147) Google Scholar, A. Gebhardt U. Bieback K. Gottke U. Kracke A. Mages J. Lemon S.M. Vallbracht A. J. Virol. 2000; 74: 10950-10957Crossref PubMed Scopus (87) Google Scholar). In ASGPR functions were completely abrogated mice as with mice. requirement of HL-1 for stable expression of functional ASGPR is that of HL-2. of ASGPR we failed to detect physiological for several postulated functions that have been ascribed to ASGPR. mice the for the of this