Isolation of the SO4-4-GalNAcβ1,4GlcNAcβ1,2Manα-specific Receptor from Rat Liver
1997; Elsevier BV; Volume: 272; Issue: 23 Linguagem: Inglês
10.1074/jbc.272.23.14629
ISSN1083-351X
AutoresDorothy Fiete, Jacques Baenziger,
Tópico(s)Receptor Mechanisms and Signaling
ResumoGlycoproteins, such as the glycoprotein hormone lutropin (LH), bear oligosaccharides terminating with the sequence SO4-4GalNAcβ1,4GlcNAcβ1,2Manα (S4GGnM) and are rapidly removed from the circulation by a receptor present in hepatic endothelial cells and Kupffer cells. Rapid removal from the circulation is essential for attaining maximal hormone activity in vivo. We have isolated a protein from rat liver which has the properties expected for the S4GGnM-specific receptor (S4GGnM-R). The S4GGnM-R is closely related to the macrophage mannose receptor (Man-R) both antigenically and structurally. At least 12 peptides prepared from the S4GGnM-R have amino acid sequences that are identical to those of the Man-R. Nonetheless, the ligand binding properties of the S4GGnM-R and the Man-R differ in a number of respects. The S4GGnM-R binds to immobilized LH but not to immobilized mannose, whereas the Man-R binds to immobilized mannose but not to immobilized LH. When analyzed using a binding assay that precipitates receptor ligand complexes with polyethylene glycol, the S4GGnM-R is able to bind S4GGnM-bovine serum albumin (S4GGnM-BSA) conjugates whereas the Man-R is not. In contrast both the S4GGnM-R and the Man-R are able to bind Man-BSA. Monosaccharides that inhibit binding of Man-BSA by the Man-R enhance binding by the S4GGnM-R. Oligosaccharides terminating with S4GGnM and those terminating with Man are bound at independent sites on the S4GGnM-R. The S4GGnM-R present in hepatic endothelial cells may account for clearance of glycoproteins bearing oligosaccharides terminating with S4GGnM and glycoproteins bearing oligosaccharides terminating with either mannose, fucose, orN-acetylglucosamine. Glycoproteins, such as the glycoprotein hormone lutropin (LH), bear oligosaccharides terminating with the sequence SO4-4GalNAcβ1,4GlcNAcβ1,2Manα (S4GGnM) and are rapidly removed from the circulation by a receptor present in hepatic endothelial cells and Kupffer cells. Rapid removal from the circulation is essential for attaining maximal hormone activity in vivo. We have isolated a protein from rat liver which has the properties expected for the S4GGnM-specific receptor (S4GGnM-R). The S4GGnM-R is closely related to the macrophage mannose receptor (Man-R) both antigenically and structurally. At least 12 peptides prepared from the S4GGnM-R have amino acid sequences that are identical to those of the Man-R. Nonetheless, the ligand binding properties of the S4GGnM-R and the Man-R differ in a number of respects. The S4GGnM-R binds to immobilized LH but not to immobilized mannose, whereas the Man-R binds to immobilized mannose but not to immobilized LH. When analyzed using a binding assay that precipitates receptor ligand complexes with polyethylene glycol, the S4GGnM-R is able to bind S4GGnM-bovine serum albumin (S4GGnM-BSA) conjugates whereas the Man-R is not. In contrast both the S4GGnM-R and the Man-R are able to bind Man-BSA. Monosaccharides that inhibit binding of Man-BSA by the Man-R enhance binding by the S4GGnM-R. Oligosaccharides terminating with S4GGnM and those terminating with Man are bound at independent sites on the S4GGnM-R. The S4GGnM-R present in hepatic endothelial cells may account for clearance of glycoproteins bearing oligosaccharides terminating with S4GGnM and glycoproteins bearing oligosaccharides terminating with either mannose, fucose, orN-acetylglucosamine. Asn-linked oligosaccharides present on the glycoprotein hormones lutropin (LH) 1The abbreviations used are: LH, lutropin; TSH, thyrotropin; CG, chorionic gonadotropin; PEG, polyethylene glycol; BSA, bovine serum albumin; S4GGnM, SO4-4-GalNAcβ1,4GlcNAcβ1,2Manα; S4GGnM-R, S4GGnM-specific receptor; S3GGnM, SO4-3-GalNAcβ1,4GlcNAcβ1, 2Manα; Man-R, macrophage mannose-specific receptor; PAGE, polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin; Cys-R, cysteine-rich; Fn-II, fibronectin type II; CRD, carbohydrate recognition domain; ASGP-R, asialoglycoprotein receptor; bLH, bovine lutropin; CAPS, 3-(cyclohexylamino)propanesulfonic acid. and thyrotropin (TSH) terminate with the sequence SO4-4GalNAcβ1,4GlcNAcβ1,2Manα (S4GGnM), whereas those on follitropin and chorionic gonadotropin (CG) terminate with the sequence Siaα2,3/6Galβ1,4GlcNAcβ1, 2Manα (1Green E.D. Baenziger J.U. J. Biol. Chem. 1988; 263: 25-35Google Scholar, 2Green E.D. Baenziger J.U. J. Biol. Chem. 1988; 263: 36-44Google Scholar, 3Baenziger J.U. Green E.D. Ginsberg V. Robbins P.W. Biology of Carbohydrates.JAI Press Ltd. 1991; 3: 1-46Google Scholar, 4Stockell Hartree A. Renwick A.G.C. Biochem. J. 1992; 287: 665-679Google Scholar, 5Endo Y. Yamashita K. Tachibana Y. Tojo S. Kobata A. J. Biochem. ( Tokyo ). 1979; 85: 669-679Google Scholar). We have proposed that the sulfated oligosaccharides present on LH and TSH are critical for the expression of full biologic function by these hormones (6Baenziger J.U. Endocrinology. 1996; 137: 1520-1522Google Scholar, 7Baenziger J.U. Kumar S. Brodbeck R.M. Smith P.L. Beranek M.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 334-338Google Scholar, 8Dharmesh S.M. Baenziger J.U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11127-11131Google Scholar, 9Manzella S.M. Dharmesh S.M. Beranek M.C. Swanson P. Baenziger J.U. J. Biol. Chem. 1995; 270: 21665-21671Google Scholar). Terminal GalNAc-4-SO4 does not influence binding to or activation of the LH/CG receptor itself (10Smith P.L. Kaetzel D. Nilson J. Baenziger J.U. J. Biol. Chem. 1990; 265: 874-881Google Scholar) but does have a marked impact on the circulatory half-life of LH following secretion (7Baenziger J.U. Kumar S. Brodbeck R.M. Smith P.L. Beranek M.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 334-338Google Scholar, 11Smith P.L. Bousfield G.R. Kumar S. Fiete D. Baenziger J.U. J. Biol. Chem. 1993; 268: 795-802Google Scholar) due to recognition of the sulfated oligosaccharides by a receptor expressed at the surface of hepatic endothelial cells and Kupffer cells (11Smith P.L. Bousfield G.R. Kumar S. Fiete D. Baenziger J.U. J. Biol. Chem. 1993; 268: 795-802Google Scholar, 12Fiete D. Srivastava V. Hindsgaul O. Baenziger J.U. Cell. 1991; 67: 1103-1110Google Scholar). The rapid removal of LH from the circulation in conjunction with its release from granules in response to gonadotropin releasing hormone accounts for the episodic rise and fall in hormone levels seen in the circulation. Since the LH/CG receptor is a G-protein-coupled receptor, which rapidly becomes refractory to further stimulation following ligand binding (13Segaloff D.L. Ascoli M. Endocr. Rev. 1993; 14: 324-347Google Scholar, 14Lefkowitz R.J. Cell. 1993; 74: 409-412Google Scholar, 15Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Google Scholar), episodic stimulation may provide for maximal activation during the preovulatory surge in circulating LH levels. TSH shows similar properties with respect to half-life and receptor activation (16Leitolf H. Szkudlinski M.W. Hoang-Vu C. Thotakura N.R. von zur Muhlen A. Brabant G. Weintraub B.D. Horm. Metab. Res. 1995; 27: 173-178Google Scholar, 17Szkudlinski M.W. Thotakura N.R. Tropea J.E. Grossmann M. Weintraub B.D. Endocrinology. 1995; 136: 3325-3330Google Scholar, 18Grossmann M. Szkudlinski M.W. Tropea J.E. Bishop L.A. Thotakura N.R. Schofield P.R. Weintraub B.D. J. Biol. Chem. 1995; 270: 29378-29385Google Scholar, 19Szkudlinski M.W. Thotakura N.R. Weintraub B.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9062-9066Google Scholar). Glycoproteins bound by the S4GGnM-specific receptor are subsequently transported to lysosomes and degraded. There are roughly 600,000 S4GGnM-specific binding sites at the cell surface of hepatic endothelial cells, which bind LH through its sulfated oligosaccharides with an apparent Kd of 2.7 × 10−7m. Binding is pH-dependent, requiring a pH > 5.0, but does not require Ca2+ (12Fiete D. Srivastava V. Hindsgaul O. Baenziger J.U. Cell. 1991; 67: 1103-1110Google Scholar). The location of the sulfate in the 4-position is critical since glycoconjugates bearing oligosaccharides terminating with the sequence SO4-3GalNAcβ1,4GlcNAcβ1,2Manα (S3GGnM) are not bound by hepatic endothelial cells. We have now identified and isolated a glycoprotein from rat liver that has the properties expected for the receptor, which mediates removal of LH from the circulation on the basis of its sulfated oligosaccharides. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad) or ISS Protein-Gold (Integrated Separation Systems). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed according to Laemmli (20Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar). Following separation by SDS-PAGE on 5% or 7.5% acrylamide gels, proteins were transferred electrophoretically to polyvinylidene difluoride membranes using CAPS buffer as described by Matsudaira (21Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Google Scholar) for amino-terminal sequence determination, peptide mapping, and detection with specific antisera. Proteins detected by antisera were developed using 125I-F(ab′)2 goat anti-rabbit IgG. WGA (Sigma) was dissolved in 100 mm NaHCO3, 100 mmGlcNAc, 200 mm NaCl, pH 8.4, at a concentration of 2.0 mg/ml and added to cyanogen bromide-activated Sepharose 4B (Sigma) at a ratio of 5.0 mg/ml Sepharose 4B. Following 2 h of rotation at room temperature, >95% of the WGA was coupled. Remaining active sites were quenched by incubation overnight at 4 °C in 200 mmglycine, pH 8.0. The WGA-Sepharose was washed successively with 0.1m sodium acetate, 1.0 m NaCl, pH 4.0, and 0.1m borate, 1.0 m NaCl, pH 8.0. WGA-Sepharose was stored in 20 mm Tris-HCl, 0.15 m NaCl, 2.0 mm CaCl2, pH 7.8, containing 0.2% NaN3. bLH-Sepharose was prepared in the same fashion with the following modifications. bLH was dissolved in 100 mm NaHCO3, 200 mm NaCl, pH 8.4, at a concentration of 3.8 mg/ml and added to cyanogen bromide-activated Sepharose 4B at a ratio of 6.5 mg/ml Sepharose. The coupling efficiency was >95% after 2 h at room temperature. Remaining active sites were quenched by incubation at 4 °C overnight in 1.0 methanolamine, pH 8.3. bLH-Sepharose was stored in 20 mmTris HCl, 0.15 m NaCl, 2.0 mmCaCl2, pH 7.8, containing 0.2% NaN3. SO4-4GalNAcβ1,4GlcNAcβ1,2Manα-(CH2)8COO-bovine serum albumin (S4GGnM-BSA), SO4-3GalNAcβ1,4GlcNAcβ1,2Manα-(CH2)8COO-bovine serum albumin (S3GGnM-BSA), Mannose-bovine serum albumin (Man-BSA), or purified S4GGnM receptor, 5–10 μg, were dissolved in 50 μl of 20 mm Tris-HCl, 0.15 m NaCl, pH 7.4, and incubated for 15 min on ice with a single IODOBEAD (Pierce) and 2.5 μCi of125I (Amersham, IMS30). Labeled proteins were separated from reaction products by gel filtration on Sephadex G-10 (Pharmacia Biotech Inc.) in 20 mm Tris-HCl, 0.15 m NaCl, pH 7.8, containing 1 mg/ml bovine serum albumin. The fractions containing 125I-labeled product were pooled and stored at −20 °C for no longer than 2 months. F(ab′) goat anti-rabbit IgG (250 μg) was iodinated in the same fashion using 500 μCi of125I in 500 μl of 20 mm NaPO4, 0.15 m NaCl, pH 7.5, and incubated 1 h at room temp. Binding assays (total volume of 150 μl) contained 3–5 ng of S4GGnM receptor 125I-S4GGnM-BSA (2–3 × 105 dpm), and 90 μg of hyaluronic acid and/or 90 μg of fucoidin in 20 mm Tris-HCl, 0.15 m NaCl, 2 mmCaCl2, 1% (w/v) Triton X-100, pH 7.8. Hyaluronic acid is a weak inhibitor of S4GGnM-BSA binding, whereas fucoidin is a potent inhibitor (12Fiete D. Srivastava V. Hindsgaul O. Baenziger J.U. Cell. 1991; 67: 1103-1110Google Scholar). Incubations were performed in a 10 × 75-mm glass tube at room temperature for 30 min. The reactions were terminated by adding 1.5 ml of ice-cold 10% (w/v) PEG 8000 (Sigma) in 20 mm Tris-HCl, 0.15 m NaCl, 2 mmCaCl2 and mixing. After 30 min on ice, precipitated125I-S4GGnM-BSA·S4GGnM receptor complexes were collected by vacuum filtration on Whatman GF/C filter discs, which had been soaked in 20 mm Tris-HCl, 0.15 m NaCl, 2 mm CaC12, 5 mg/ml bovine serum albumin. The filters were washed twice with 1.5 ml of ice-cold 20 mmTris-HCl, 0.15 m NaCl, 2 mm CaCl2, 10% (w/v) PEG 8000, and the amount of 125I determined by counting the filter in a γ-counter. In the absence of added S4GGnM receptor, <5% of the added 125I-S4GGnM-BSA was captured on the filter. One unit of activity is defined as the amount of S4GGnM receptor that is able to precipitate 1 ng of S4GGnM-BSA in the presence of hyaluronic acid above that precipitated in the presence of both hyaluronic acid and fucoidin. Harlan Sprague Dawley rats, 150–200 g each, were anesthetized and heparinized and their livers perfused with ice-cold 20 mm PO4, 0.15 m NaCl, pH 7.5, through the portal vein. Each liver was suspended in 20 ml of 0.25 mm EDTA, 0.02% NaN3 (w/v) brought to pH 7.8 with solid NaHCO3 and containing 50 units/ml aprotinin. The suspension was homogenized with three 20-s bursts of a Polytron homogenizer (Brinkman) at a setting of 5. Alternatively, 50 frozen rat livers weighing 300 g (Pel-Freez) were ground while frozen using a meat grinder and suspended in 400 ml of 0.25 mm EDTA, 0.02% NaN3 (w/v) brought to pH 7.8 with solid NaHCO3and containing 50 units/ml aprotinin. The suspension was homogenized with four 15-s bursts of a Polytron homogenizer (Brinkman) at a setting of 8. Sufficient 25% (w/v) Triton X-100 (Boehringer Mannheim) was added to bring the concentration of the homogenate to 10%, and the pH was adjusted to 7.7 with 1.0m Tris-HCl, pH 7.8. After stirring for 1 h at 4 °C, the Triton extract was passed through cheesecloth to remove connective tissue and sedimented at 7100 × g for 20 min. Solid PEG 8000 (Sigma) was added to the supernatant to a final concentration of 10% (w/v). The extract was stirred for 15 min at 4 °C and then allowed to stand for 30 min. Precipitated proteins were collected by sedimentation at 7100 ×g for 90 min. The supernatant was discarded. The PEG precipitate was resuspended by vigorous stirring for 30 min in 20 mm Tris-HCl, 0.2 m NaCl, 0.05 mNaN3, 3 mm CaCl2, 1% (w/v) Triton X-100, pH 7.8, 20 ml/liver. Solubilized proteins from the PEG precipitate were incubated with WGA-Sepharose (1–2 ml of WGA-Sepharose/liver) overnight at 4 °C. Unbound proteins were removed by washing on a sintered glass funnel with 20 mmTris-HCl, pH 7.8, 150 mm NaCl2, 1% Triton X-100. Bound glycoproteins were subsequently eluted using the same buffer containing 300 mm GlcNAc. The WGA-Sepharose eluate was incubated with 0.5 ml of bLH-Sepharose/liver overnight at 4 °C with rotation. Unbound material was removed by washing on a sintered glass funnel with 20 mm Tris-HCl, pH 7.8, 150 mmNaCl, 2 mm CaCl2, 1% Triton X-100. The bLH-Sepharose was then eluted with 50 mm sodium acetate, pH 4.0, 0.1% Triton X-100. The eluate was immediately adjusted to pH 7.0 by addition of 1.0 m Tris base and the volume reduced using a Centriprep-10 (Amicon). The bLH-Sepharose eluate was incubated with either mannan-Sepharose (0.05 ml/liver) or mannose-Sepharose (0.05 ml/liver) and the unbound fraction taken. Bound proteins were eluted from the mannan-Sepharose and mannose-Sepharose by successive incubation with 50 mmgalactose in 20 mm Tris, 150 mm NaCl, 0.05% Triton X-100, and 0.2 mm Pefabloc SC (Boehringer Mannheim) adjusted to pH 7.5 and 200 mm mannose and 5 mmEDTA in the same buffer. Polyclonal antisera to the S4GGnM receptor were raised in New Zealand White rabbits by immunization with the 180-kDa protein band isolated from an SDS-polyacrylamide gel. The protein band was excised, and, after extracting the gel pieces with 95% ethanol to reduce the SDS content, the gel was lyophilized. The dried gel was pulverized using a mortar/pestle and then emulsified with saline by passing through a series of successively smaller needles ranging from 18 to 23 gauge. The emulsified gel containing 5–10 μg of protein was added to TDM-emulsion (RIBI) and injected intramuscularly into the hind leg and subcutaneously at four separate sites. The rabbit was boosted in the same manner 3 weeks later and sera obtained 8–10 days later. The rabbit was subsequently boosted with antigen as required to maintain the titer of the antisera. The affinity-purified S4GGnM receptor was labeled with125I as described above. An antibody saturation curve was established using a constant amount of radiolabeled receptor (5–10 ng, 3 × 105 cpm) and increasing amounts of antisera. Following an overnight incubation at 4 °C, protein A-Sepharose antigen-antibody complexes were washed twice with 20 mmphosphate-buffered saline, pH 7.5, containing 0.1% BSA (w/v) and counted in a γ counter. Standard inhibition curves were constructed using a constant amount of radiolabeled receptor, sufficient antisera to precipitate 50–70% of the 125I-S4GGnM-R added, and increasing amounts of unlabeled receptor that had been quantitated by amino acid analysis. The amount of receptor in "unknown" samples was then determined by comparison to the standard inhibition curve. We previously identified a receptor in rat liver that can account for the rapid removal of native LH bearing Asn-linked oligosaccharides terminating with the sequence S4GGnM from the circulation (12Fiete D. Srivastava V. Hindsgaul O. Baenziger J.U. Cell. 1991; 67: 1103-1110Google Scholar). The S4GGnM-R is located predominantly in hepatic endothelial cells and Kupffer cells and displays a high degree of specificity, recognizing S4GGnM-BSA but not S3GGnM-BSA. Fucoidin, a sulfated polysaccharide, inhibits binding of S4GGnM-BSA and LH by the receptor, whereas other sulfated and anionic polysaccharides do not inhibit binding or require much higher concentrations to do so. Glycoproteins bound to the S4GGnM-R are internalized, transported to lysosomes, and degraded. Binding is pH-dependent, requiring a pH above 5–6, and is not dependent on divalent cations even though divalent cations do enhance binding. We established conditions that allowed us to detect and solubilize a binding activity with the properties we had described for the S4GGnM-R. Parenchymal cells (hepatocytes) and endothelial/Kupffer cells, prepared by collagenase perfusion as described previously (12Fiete D. Srivastava V. Hindsgaul O. Baenziger J.U. Cell. 1991; 67: 1103-1110Google Scholar, 22Baenziger J.U. Fiete D. Cell. 1980; 22: 611-620Google Scholar), were disrupted by Dounce homogenization, and the nuclei and unbroken cells were collected by centrifugation. Soluble and total membrane fractions were obtained by sedimentation onto a 65% sucrose cushion. Fractions were brought to a final concentration of 1% (w/v) Triton X-100 and assayed for S4GGnM-BSA binding using the PEG precipitation assay. Binding activity was confined to the membrane fraction and the pellet containing nuclei and unbroken cells. None was found in the soluble fraction. As much as 80% of the binding activity was recovered in the membrane fraction after Dounce homogenization. The majority of the S4GGnM-BSA-specific binding activity, 64% of the total, was found in the endothelial and Kupffer cells, while 36% was in hepatocytes. We examined the ability of Triton X-100 to solubilize the binding activity from membranes. Membranes were incubated with125I-S4GGnM-BSA in the presence of increasing amounts of Triton X-100 (Fig. 1). Membranes were either collected by sedimentation in an Airfuge at 190,000 × g(Beckman) in the absence of added PEG or by filtration on GF/C filters following addition of PEG. 125I-S4GGnM-BSA was found in the membrane pellet (Fig. 1, −PEG) following addition of 0.05% Triton but not 0.01%, 0.5%, or 1.0% Triton. In contrast,125I-S4GGnM-BSA was bound in the presence of 0.05% Triton as well as 0.5% and 1.0% Triton when complexes were collected by precipitation with PEG (+PEG). At a Triton concentration of 0.05%, membrane vesicles become sufficiently permeable for the S4GGnM-R to be accessible to the 125I-S4GGnM-BSA; however, the S4GGnM-R is not solubilized at this Triton concentration and can be sedimented in the absence of added PEG. At Triton concentrations above 0.5%, the S4GGnM-R is fully solubilized and requires PEG for precipitation of complexes. Using both the sedimentation assay in the presence of 0.05% Triton and the PEG precipitation assay in the presence of 1% Triton, we determined that: 1) 3-fold more125I-S4GGnM-BSA is bound at pH 7.5 than at pH 5.0 or below; 2) EDTA does not abolish binding of S4GGnM-BSA but does reduce it to 62% of that seen in the presence of 4 mm Ca2+; and 3) fucoidin is a significantly more potent inhibitor of binding than other sulfated or anionic polysaccharides such as hyaluronic acid, heparin, chondroitin sulfate, and dextran sulfate. Thus, a binding activity with the properties expected for the S4GGnM-R could be detected in rat liver membranes and solubilized with Triton X-100. We therefore developed the isolation scheme summarized in TableI.Table IIsolation of the S4GGnM-specific receptor from rat liverStepProteinBindingTotal binding units/rat liverRIATotal receptor by RIAmgunits × 106%μg%1.HomogenateND1-aND, the amount of protein determined to be in the homogenate and the PEG pellet resuspended in Triton X-100 was not considered accurate and is not included.2.Triton X-1003556210033.31003.PEG Pellet180508113.2404.Triton X-100ND1-aND, the amount of protein determined to be in the homogenate and the PEG pellet resuspended in Triton X-100 was not considered accurate and is not included.30485.WGA-Sepharose bound6.46.3109.9306.bLH-Sepharose bound0.05–0.21.151.96.7207.Man-Sepharose unbound0.05–0.21.081.7Protein values were determined using the Bio-Rad protein assay kit or the ISS Protein-Gold kit. Binding units were determined using125I-S4GGnM-BSA and the PEG precipitation assay. The amount of receptor protein present was determined by a RIA in which unlabeled S4GGnM-R was used to inhibit binding of 125I-S4GGnM-R by a rabbit antibody raised to the purified S4GGnM-R.1-a ND, the amount of protein determined to be in the homogenate and the PEG pellet resuspended in Triton X-100 was not considered accurate and is not included. Open table in a new tab Protein values were determined using the Bio-Rad protein assay kit or the ISS Protein-Gold kit. Binding units were determined using125I-S4GGnM-BSA and the PEG precipitation assay. The amount of receptor protein present was determined by a RIA in which unlabeled S4GGnM-R was used to inhibit binding of 125I-S4GGnM-R by a rabbit antibody raised to the purified S4GGnM-R. The S4GGnM-R was solubilized using 10% Triton X-100, concentrated by precipitation with PEG 8000, and solubilized in 10% Triton X-100 prior to incubation with WGA-Sepharose. The S4GGnM-R bound to WGA-Sepharose and was eluted with 300 mm GlcNAc. The WGA-Sepharose eluate containing the S4GGnM-R was incubated with bLH-Sepharose. S4GGnM-R that bound to bLH-Sepharose was eluted by reducing the pH to 4.0 with acetate buffer. When this material was examined by SDS-PAGE, a major band was found to be present, which had an Mr of 180,000 (Fig. 2 A). Additional proteins with apparent molecular weights of 75–80,000, 60,000, 43,000, and 35,000 were also present. Following electrophoretic transfer to Immobilon-P, a ligand blot was performed using 125I-S4GGnM-BSA, which demonstrated that only the protein with an Mr of 180,000 was reactive (Fig. 2 B). An NH2-terminal sequence of LK(Y)S(Q)YQFLIYNE was obtained for the protein with an Mr of 180,000, suggesting it was closely related to the murine macrophage mannose receptor (Man-R), which has an NH2-terminal sequence of LLDARQFLIYNE (23Harris N. Super M. Rits M. Chang G. Ezekowitz R.A.B. Blood. 1992; 80: 2363-2373Google Scholar). S4GGnM-R that had been eluted from bLH-Sepharose was incubated with mannan-Sepharose or mannose-Sepharose. Neither the S4GGnM-BSA-specific binding activity nor the protein with an Mr of 180,000 was bound by immobilized mannan or mannose, whereas the other proteins in the eluate from bLH-Sepharose were bound to either mannan-Sepharose or mannose-Sepharose and removed. The mannose-Sepharose unbound fraction was homogeneous and consisted of a single band, migrating with an Mr of 180,000 when analyzed by SDS-PAGE (see Fig. 4), which was designated the S4GGnM-R. The NH2-terminal sequence of the S4GGnM-R suggested a close relationship to the macrophage Man-R. However, the inability of the S4GGnM-R to bind to either immobilized mannan or mannose, which are used for affinity-based purification of the Man-R from lung (24Lennartz M.R. Wileman T.E. Stahl P.D. Biochem. J. 1987; 245: 705-711Google Scholar), placenta (25Lennartz M.R. Cole F.S. Shepherd V.L. Wileman T.E. Stahl P.D. J. Biol. Chem. 1987; 262: 9942-9944Google Scholar), and macrophages (26Blum J.S. Stahl P.D. Diaz R. Fiani M.L. Carbohydr. Res. 1991; 213: 145-153Google Scholar, 27Ii M. Wada M. Kawasaki T. Yamashina I. J. Biochem. ( Tokyo ). 1988; 104: 587-590Google Scholar), indicated the S4GGnM-R is distinct from the Man-R. We examined the potential relationship between the S4GGnM-R and the Man-R by isolating the macrophage Man-R from rat lung by affinity chromatography on Mannose-Sepharose as described by Lennartz et al. (24Lennartz M.R. Wileman T.E. Stahl P.D. Biochem. J. 1987; 245: 705-711Google Scholar) for direct comparison with the S4GGnM-R isolated from rat liver. The Man-R isolated from lung by this procedure is homogeneous and has an Mr of 180,000 when examined by SDS-PAGE (see Fig. 4). As will be presented in greater detail below, the S4GGnM-R and Man-R have a number of features that indicate they are distinct and other features that indicate they are closely related. For example the following features indicate that the receptors differ. 1) The S4GGnM-R will bind to immobilized ligands containing terminal GalNAc-4-SO4 but not ligands with terminal Man, while the Man-R will bind to immobilized ligands containing terminal Man but not those containing terminal GalNAc-4-SO4. 2) The S4GGnM-R is able to bind soluble ligands terminating with S4GGnM as well as ligands with terminal Man or Fuc in the PEG precipitation assay, whereas the Man-R will bind ligands with terminal Man or Fuc but not those with terminal S4GGnM in the same assay. Features indicating the receptors are structurally related include the following observations. 1) The S4GGnM-R and the Man-R both react with 125I-Man-BSA in ligand blots. 2) Antibodies raised to either the purified S4GGnM-R from liver or the Man-R from lung react with either receptor in Western blots. 3) The receptors have similar peptide maps, and multiple peptides prepared from the S4GGnM-R have sequences that are identical to those of the murine macrophage Man-R (23Harris N. Super M. Rits M. Chang G. Ezekowitz R.A.B. Blood. 1992; 80: 2363-2373Google Scholar). The S4GGnM-R (200 μg) and the Man-R (150 μg) were subjected to electrophoretic separation on 5% polyacrylamide gels and electrophoretically transferred to Immobilon (Millipore) in CAPS buffer. After staining with Ponceau Red, the regions containing the transferred protein were excised for analysis. Peptides were released by digestion with LysC or trypsin in the presence of reduced Triton X-100 and separated by reverse phase chromatography. The separations are shown in Fig. 3 for peptides released by LysC digestion of the S4GGnM-R and the Man-R. The profiles were nearly identical. Only peaks 57 and 75 of the S4GGnM-R were not also present in the Man-R. Peaks 45, 62, and 91 of the S4GGnM-R appeared to be identical to peaks 37, 54, and 84 of the Man-R, respectively. Peaks that appeared to be identical and peaks that appeared to differ between the S4GGnM-R and the Man-R were analyzed. The results of these analyses are summarized in Table II.Table IIComparison of peptides obtained from the S4GGnM-R and the Man-RSource of PeptideResidues from Man-R or ASGP-R sequenceRegion of Man-RPeptide sequenceS4GGnM-RMan-RPK-45PK-37Man-R:447–458CRD 2WLPGEPSHENNRPK-62PK-54Man-R:327–335Stalk 2WENLECVQKPK-62PK-54Man-R:178–187Fibronectin type IIWYADCTSAGRPK-91PK-84Man-R:132–151Cysteine-richGSGLWSRWKVYGTTDDLCSRPK-57No equivalent peakMan-R:24–33Cysteine-richQFLIYNEDHKPK-57No equivalent peakASGP-R:211–222WVDGTDYETGFKPK-78No equivalent peakASGP-R:223–238NLRPGQPDDWYGHILGPK-41 (tryptic map)Man-R:34–53Cysteine-richR-VDALSAISVQTAT-NPEAPK-46 (tryptic map)Man-R:1320–1341CRD 8TGDPSGE(R)ND-VVLSSSS(G)L-NPK-61 (tryptic map)Man-R:573–596CRD 3GTFRWTVDEQVFTH-NADMPGRKPK-75 (tryptic map)Man-R:829–848CRD 5KNFGDLATIKSESEKKFLWKPeptides were released from the S4GGnM-R or the Man-R by LysC digestion and fractionated by reverse phase chromatography as shown in Fig. 3. The peptides in the peaks indicated had their amino acid sequences determined and were found to be present in either the macrophage mannose receptor or the heptocyte asialoglycoprotein receptor (ASGPP-R). The sequences and their locations within these receptors are indicated. PK-41, PK-46, and PK-61 were obtained from an earlier analysis using a different preparation of the S4GGnM-R and release by digestion with trypsin. Open table in a new tab Peptides were released from the S4GGnM-R or the Man-R by LysC digestion and fractionated by reverse phase chromatography as shown in Fig. 3. The peptides in the peaks indicated had their amino acid sequences determined and were found to be present in either the macrophage mannose receptor or the heptocyte asialoglycoprotein receptor (ASGPP-R). The sequences and their locations within these receptors are indicated. PK-41, PK-46, and PK-61 were obtained from an earlier analysis using a different preparation of the S4GGnM-R and release by digestion with trypsin. Sequence was obtained for 12 peptides from three different S4GGnM-R preparations. Nine of the peptide sequences obtained from the S4GGnM-R were identical to peptide sequences predicted to be present in the macrophage Man-R (23Ha
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