Rat Hepatic Lectin Research Articles

Expression of a functional asialoglycoprotein receptor through transfection of a cloned cDNA that encodes a macrophage lectin.

The Gal/GalNAc-specific lectin on rat peritoneal macrophages (macrophage asialoglycoprotein binding protein, M-ASGP-BP) is structurally similar to rat hepatic asialoglycoprotein-binding protein (ASGP-BP) or rat hepatic lectin (RHL) and is highly homologous with the major component of RHL, RHL-1 (Ii, M, Kurata, H., Itoh, N., Yamashina, I., and Kawasaki, T. (1990) J. Biol. Chem. 265, 11295-11298). We found in this study that transfection with a cDNA clone that encodes a single polypeptide, M-ASGP-BP, was sufficient for the expression of an endocytic receptor for asialoorosomucoid (ASOR) on the COS-1 cell surface. The Kuptake value for ASOR for the transfected cells was 12.5 nM, which is similar to that for peritoneal macrophages (23 nM), and the number of ASOR bound on the cell surface was 1-8 x 10(5)/cell, this value being hundreds of times larger than that for peritoneal macrophages. 125I-ASOR bound on the surfaces of the transfected cells was rapidly internalized on incubation at 37 degrees C, and after 90 min of incubation, most of the radioactivity was recovered in acid-soluble degraded products from the medium. These results confirmed that the cDNA cloned in our previous study does in fact encode M-ASGP-BP and also that the single polypeptide chain can form a homooligomeric receptor (probably a hexamer or octamer) exhibiting high affinity for ASOR. The latter property was distinct from that of the hepatic ASGP-BP in that simultaneous transfection of two cloned cDNAs that encode RHL-1 and RHL-2/3 was required to produce an active ASOR receptor (McPhaul, M., and Berg, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8863-8867). This M-ASGP-BP expression system may serve as a simple model with which to investigate the molecular mechanisms underlying carbohydrate-mediated endocytosis.

Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages.

We previously reported that the mouse macrophage galacose and N-acetylgalactosamine-specific lectin (MMGL) may participate in the binding of the macrophages to tumor cells [Oda, S., Sato, M., Toyoshima, S., & Osawa, T. (1989) J. Biochem. 105, 1040-1043]. We now report the cloning and characterization of a cDNA encoding MMGL. The MMGL gene encoded a protein consisting of 304 amino acid residues with a molecular weight of 34,595. The deduced amino acid sequence indicated that MMGL had a single membrane-spanning region, three leucine zipper-like domains, and a carbohydrate recognition domain. Two N-glycosylation sites were found in the extracellular region of MMGL, corresponding to the heavy N-glycosylation in the native MMGL. Comparison of the amino acid sequence of MMGL with those of rat hepatic lectins revealed a high overall sequence homology. The sequence homology was especially high in the putative membrane-spanning region and carbohydrate recognition domain. There was, however, a region of 25 amino acids which did not exist on hepatic lectins. The MMGL cDNA without the region encoding the putative membrane-spanning region and intracellular region was expressed in Escherichia coli. The expressed protein had galactose-binding activity and its sugar-binding specificity was same as that of the native lectin.

Hepatocyte adhesion to carbohydrate-derivatized surfaces. II. Regulation of cytoskeletal organization and cell morphology.

Rat hepatic lectins mediate adhesion of isolated rat hepatocytes to synthetic surfaces derivatized with galactosides. Initial weak adhesion is followed by rapid adhesion strengthening. After hepatocytes contact galactose-derivatized gels, the hepatic lectins move rapidly into an inaccessible patch at the adhesive surface (Weisz, O. A., and R. L. Schnaar. 1991. J. Cell Biol. 115:485-493). Hepatic lectin patching, which occurs both at 37 degrees C and 4 degrees C, is not responsible for adhesion strengthening, which does not occur at 4 degrees C. Of various cytoskeletal and metabolic perturbants tested, only a combination of hyperosmotic medium, colchicine, and cytochalasin caused a marked (72%) reduction of adhesion strengthening (without reducing weak cell adhesion). Clathrin and actin were readily detected in the adhesive patch by immunofluorescence microscopy. Rat hepatocytes also adhered avidly to surfaces derivatized with asialofetuin, a high-affinity ligand for the rat hepatic lectins. However, hepatic lectin molecules did not migrate into a patch on the asialofetuin-derivatized surface, suggesting that hepatic lectin-asialofetuin binding may have resulted in the rapid formation of a ring of essentially irreversibly adherent receptors that prevented diffusion of additional lectin molecules into the contact site. The cells were unable to increase their adhesive contact area by flattening onto the derivatized surface. Treatment of cells with cytochalasin, however, did result in an increase in the size of the contact area. Cells adhering to surfaces derivatized with an adhesion-promoting peptide (containing an arg-gly-asp sequence) had larger contact areas than those adhering to galactoside-derivatized surfaces. A model is proposed in which carbohydrate-mediated adhesion causes specific reorganization of cytoskeletal components, leading to strengthened adhesion and a characteristic spherical cell morphology.

Hepatocyte adhesion to carbohydrate-derivatized surfaces. I. Surface topography of the rat hepatic lectin.

The rat hepatic lectins, galactose- and N-acetylgalactosamine-binding proteins found on the hepatocyte cell surface, mediate adhesion of isolated primary rat hepatocytes to artificial galactose-derivatized polyacrylamide gels. Biochemical and immunohistochemical techniques were used to examine the topographical redistribution of the rat hepatic lectins in response to galactose-mediated cell adhesion. Hepatocytes isolated from rat liver by collagenase perfusion had an average of 7 x 10(5) cell surface lectin molecules per cell, representing 30-50% of the total lectin molecules per cell, the remainder residing in intracellular pools. Hepatocytes incubated on galactose-derivatized surfaces, whether at 0-4 degrees C or 37 degrees C, rapidly lost greater than 80% of their accessible cell surface lectin binding sites into an adhesive patch of characteristic morphology. The kinetics of rat hepatic lectin disappearance were used to estimate a lateral diffusion coefficient greater than 9 x 10(-9) cm2/s at 37 degrees C, suggesting rapid and unimpeded lectin diffusion in the plane of the membrane. Indirect immunofluorescence labeling of adherent cells using antihepatic lectin antibody revealed a structured ring of receptors surrounding an area of exclusion (patch) of reproducible size and shape which represented approximately 8% of the hepatocyte cell surface. Notably, adherent cells, which had lost greater than 80% of their accessible surface binding sites, still endocytosed soluble galactose-terminated radioligand at greater than 50% of the rate of nonadherent control cells. No net movement of rat hepatic lectin from intracellular pools to the cell surface was found on cells recovered after adhesion to galactose-derivatized surfaces at 37 degrees C, suggesting that the physical size and/or lectin density of the patch was restricted by kinetic or topological constraints.

Ligand-binding characteristics of rat serum-type mannose-binding protein (MBP-A). Homology of binding site architecture with mammalian and chicken hepatic lectins.

Sugar-binding characteristics of rat serum mannose-binding protein (MBP) were studied using the carbohydrate-recognition domain of this protein expressed from a cloned cDNA. To assess the binding affinity of various test compounds, they were added as inhibitors in a binding assay in which 125I-MBP was incubated with yeast cells and the extent of binding was estimated from the radioactivity associated with the pelleted cells. The results of such inhibition assays suggest that MBP has a small binding site which is probably of the trough-type. The 3- and 4-OH of the target sugar are indispensable, while the 6-OH is not required. These characteristics are shared by the rat hepatic lectin and chicken hepatic lectin, both of which are C-type lectins containing carbohydrate-recognition domains highly homologous to that of MBP. Apparently, the related primary structures of these lectins give rise to similar gross architecture of their binding sites, despite the fact that each exhibits different sugar binding specificities.

Carbohydrate-recognition domains as tools for rapid purification of recombinant eukaryotic proteins

Methods have been developed for expression and purification of eukaryotic proteins by creating fusions with the carbohydrate-recognition domain (CRD) of the galactose-specific rat hepatic lectin. In order to generate the fusion proteins, vectors have been constructed so that cDNAs for passenger proteins can be inserted in any reading frame following a segment of DNA encoding the CRD. The feasibility of using this approach as an aid to protein purification has been demonstrated using human placental alkaline phosphatase. Following expression in either of two different eukaryotic expression systems, the CRD-phosphatase fusion protein can be isolated by one step of affinity chromatography on galactose-Sepharose under mild, non-denaturing conditions. Incorporation of a proteinase-sensitive linker allows cleavage of the CRD from the passenger protein. Immobilised proteinase could be rapidly separated from the cleavage products and the released, active phosphatase was purified away from the CRD by re-chromatography on galactose-Sepharose. These methods provide a means of isolating correctly folded recombinant eukaryotic proteins when cDNAs are available, but the properties of the encoded proteins are unknown.

Topography of binding sites of animal lectins: ligands’ view

Abstract

Expression and compartmentalization of integral plasma membrane proteins by hepatocytes and their progenitors in the rat pancreas

A combination of Western blotting, Northern blotting and immunofluorescence was used to examine the expression and compartmentalization of plasma membrane proteins by those hepatocyte-like cells that arise in the pancreases of rats subjected to sequential dietary copper depletion and repletion. The pancreatic hepatocytes were found to: (1) express several integral membrane proteins known to be concentrated within the apical, lateral or basolateral domains of the plasma membranes of hepatocytes in liver; and (2) compartmentalize the membrane proteins to equivalent plasma membrane domains, despite the organization of these cells into clusters instead of highly vascularized plates. The apical plasma membrane proteins dipeptidylpeptidase IV and HA 4 were found to line bile canaliculus-like openings between adjacent pancreatic hepatocytes; these openings were shown to be continuous with the pancreatic exocrine duct by India ink infusion. In contrast, the basolateral plasma membrane protein rat hepatic lectin-1 and lateral plasma membrane protein HA 321 were detected elsewhere about the surfaces of the pancreatic hepatocytes: by analogy to their respective localizations on hepatocytes in liver, rat hepatic lectin-1 was concentrated on those surfaces exposed to the pancreatic matrix at the periphery of the hepatocyte clusters (the basal surface equivalent), whereas HA 321 was concentrated on those surfaces exposed to adjacent hepatocytes within the clusters. The hepatocyte plasma membrane proteins were found to be expressed in the pancreas at different times during the copper depletion/repletion protocol: for example, rat hepatic lectin-1 and the bulk of the HA 4 were expressed relatively late in the protocol, only after large numbers of pancreatic hepatocytes had appeared; whereas dipeptidylpeptidase IV was induced greater than 10-fold early in the protocol and proved to be an apical-specific marker for those ductular epithelial cells that are believed to be the progenitors of the pancreatic hepatocytes.

Structure of the gene for a carbohydrate-binding receptor unique to rat kupffer cells

The gene encoding the rat Kupffer cell receptor for fucose and galactose (Lehrman, M. A., and Hill, R. L. (1986) J. Biol. Chem. 261, 7419-7425 and Lehrman, M. A., Haltiwanger, R. S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7426-7432) has been isolated and characterized. The nucleotide sequence of the 9.7-kilobase gene and of 1 kilobase of 5'-flanking sequence was determined. The 5' end of the receptor coding sequence was determined by primer extension and S1 mapping of the receptor transcript. Knowledge of the transcription start site and the 5'-flanking sequence allowed potential promoter elements to be identified. The Kupffer cell receptor coding sequence is encoded by seven exons that are split by six introns. The divisions among domains in the receptor can be correlated with divisions among exons in the gene. The carbohydrate recognition domain is encoded by three exons similar to those in the rat hepatic lectin, human Fc receptor for IgE, and rat cartilage proteoglycan core protein genes. A model for the evolution of the vertebrate lectin genes that have been analyzed is presented.

Determination of the minimum carbohydrate-recognition domain in two C-type animal lectins

Comparison of the primary structures of numerous Ca(2+)-dependent animal lectins reveals the presence of a common sequence motif which has been suggested to form the carbohydrate-recognition domain in these proteins. The extent of the functional carbohydrate-recognition domains in two rat C-type lectins, mannose-binding protein A and the major subunit of the asialoglycoprotein receptor (rat hepatic lectin 1), has been defined by expressing truncated fragments of the proteins in an in vitro transcription and translation system. The shortest fully functional fragments constitute the COOH-terminal 120 amino acids of mannose-binding protein A and 135 amino acids of rat hepatic lectin 1. These segments correspond closely to protease-resistant protein cores which can be isolated from the native lectins. The NH2-terminal boundary of each minimum carbohydrate-recognition domain falls near the site of an intron in the corresponding gene.

Defined geometry of binding between triantennary glycopeptide and the asialoglycoprotein receptor of rat heptocytes.

Three derivatives of a triantennary glycopeptide, each containing a single uniquely located 6-amino-galactose residue at either position 6', 6, or 8, were modified at the 6-amino group by attachment of a photolyzable reagent and radiolabeled by iodination of tyrosine. These were allowed to bind to the asialoglycoprotein receptor of isolated rat hepatocytes and photolyzed for affinity labeling. (formula; see text) Each probe specifically labeled either the major (RHL1) or minor (RHL2/3) subunits which comprise the receptor. A photolyzable group attached to galactose residue 6 6' specifically radiolabeled RHL1, whereas a photolyzable group attached to galactose 8 specifically labeled RHL2/3. Photoaffinity labeling of a soluble rat hepatic lectin preparation demonstrated that the minor subunits (RHL2/3) were no longer labeled by the triantennary probe with a photolyzable group at galactose 8. The inhibitory potency of a variety of complex glycopeptides against radiolabeled ligand binding to both rat hepatocytes and soluble lectin are in agreement with photoaffinity results that galactose 8 of triantennary glycopeptide is of unique importance by binding solely to the minor subunits (RHL2/3) of the asialoglycoprotein receptor on hepatocytes. Conversely, galactose residues 6 and 6' bind specifically to the major subunit (RHL1), indicating a precise binding geometry between the trivalent ligand and lectin.

Surface and internal galactosyl receptors are heterooligomers and retain this structure after ligand internalization or receptor modulation

We have developed a specific chemical affinity reagent for the hepatic galactosyl receptor (GalR) by derivatizing asialoorosomucoid (ASOR) with the homobifunctional N-hydroxysuccinimide (NHS) ester cross-linker disuccinimidyl suberate [Herzig, M. C. S., & Weigel, P. H. (1989) Biochemistry 28, 600]. NHS-ASOR cross-links with 30-50% efficiency to the three GalR subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Here, we examined the subunit structure of both surface and internal receptors of two functionally distinct GalR subpopulations, designated state 1 or state 2 GalR. Freshly isolated cells, referred to as state 1 cells, kept at 4 degrees C express only active state 1 GalR on their surface. When these cells are equilibrated at 37 degrees C, they then express both state 1 GalR and state 2 GalR on their surface. These cells are referred to as state 1,2 cells. After incubation at 4 degrees C with NHS-125I-ASOR, surface or internal GalR of state 1 cells or of state 1,2 cells incorporated 125I-ASOR into all three RHL subunits. As analyzed by autoradiography of SDS-PAGE, radiolabeling was identical for all conditions and was in a ratio of 1:1:1 for RHL 1:2:3. Native GalR structure was also examined by first cross-linking nonradiolabeled NHS-ASOR at 4 degrees C to surface or internal receptors of state 1 or state 1,2 hepatocytes. These cells were then washed with EGTA, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid antibody, and subjected to Western blot analysis. Antisera specific for RHL 1 or RHL 2/3 detected cross-linked complexes of Mr congruent to 85K or congruent to 90K-115K, respectively, as well as un-cross-linked native subunits. In all four cases, the ratio of free to cross-linked subunits was greater than or equal to 5:1 for RHL 1 and less than or equal to 0.5:1 for RHL 2/3. Internalized GalR had the same ratio of free to cross-linked subunits as noninternalized GalR. Depletion of ATP either before or after cross-linking GalR to NHS/ASOR also did not alter the ratio of free cross-linked RHL subunits. We conclude that the surface and internal GalR of the two functionally distinct GalR populations have the same heterooligomeric subunit composition and that this GalR structure persists following endocytosis or ATP depletion.

Molecular cloning and sequence analysis of cDNA encoding the macrophage lectin specific for galactose and N-acetylgalactosamine.

The primary structure of the macrophage lectin specific for galactose and N-acetylgalactosamine (macrophage asialoglycoprotein-binding protein, M-ASGP-BP) has been deduced from its cDNA sequence. The M-ASGP-BP cDNA encoded a protein consisting of 306 amino acid residues with a molecular mass of 34,242 daltons. The sequence was highly homologous with that of the rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL), particularly that of RHL-1 (the major form of RHL), throughout its whole length, and especially so in its putative membrane-spanning region and carbohydrate recognition domain. There were two N-glycosylation sites in M-ASGP-BP, the location of which were identical to those in RHL-1. However, M-ASGP-BP was characteristic in having a shorter cytoplasmic tail, and an inserted segment of 24 amino acids containing an Arg-Gly-Asp sequence between the membrane-spanning region and carbohydrate recognition domain.

Polarized expression of functional rat liver asialoglycoprotein receptor in transfected Madin-Darby canine kidney cells.

The rat liver asialoglycoprotein receptor or rat hepatic lectin (RHL) consists of two polypeptide species, a major one designated RHL-1 and a minor one designated RHL-2/3, which exists in two differentially glycosylated forms. We have studied the biosynthesis, targeting, and function of the different forms after transfection of their cDNAs into the polarized Madin-Darby canine kidney cell line. In cells expressing only RHL-1, newly synthesized protein undergoes rapid intracellular degradation and is not detected at the cell surface. In contrast, RHL-2/3 when transfected alone is much more stable and is expressed at the basolateral surface of fiber-grown cells. When both forms are expressed together, newly synthesized RHL-1 escapes rapid degradation and is detected at the basolateral surface. In double transfectants a functional receptor is formed that specifically endocytoses and degrades ligand at the basolateral side.

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

We have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degrees C. Specific binding of these 125I-labeled derivatives was approximately 90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/125I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. Cross-linking proceeded rapidly at 4 degrees C as a first-order process (k = 0.25 min-1, t1/2 = 2.8 min). After being cross-linked with any of the NHS/125I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDS-PAGE and autoradiography. Major bands were observed at Mr congruent to 84K, 93K, and 105K corresponding to the expected size of 1:1 adducts between NHS/ASOR (Mr congruent to 41.3K) and the three subunits of the receptor, Mr congruent to 43K, 50K, and 60K. The three subunits, rat hepatic lectin (RHL) 1, 2, and 3, were labeled in the ratio of about 1.0:1.2:1.0, respectively. After cross-linking, a polyclonal goat antibody to the receptor immunoprecipitated up to 100% of the specifically cross-linked NHS/125I-ASOR. Preimmune IgG immunoprecipitated less than 1% of the radiolabeled ligand. Cell surface receptors were cross-linked to NHS-ASOR, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid-Sepharose, and subjected to Western blot analysis. By use of anti-sera specific for RHL 1 or RHL 2/3 (from K. Drickamer), cross-linked complexes of Mr congruent to 85K or approximately 90-115K, respectively, were detected as were un-cross-linked native subunits. The ratio of free to cross-linked subunits was approximately 10:1 for RHL 1 and approximately 0.5:1 for RHL 2/3. We conclude that all three receptor subunits can cross-link to ligand. We propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.

The Major Subunit of the Rat Asialoglycoprotein Receptor Can Function Alone as a Receptor

Mammalian hepatic asialoglycoprotein receptors (ASGP-R) are composed of two unique, but closely related polypeptides, which in the rat are designated rat hepatic lectins 1 and 2/3 (RHL 1, RHL 2/3). Despite numerous studies, the composition of a functional ASGP-R has remained unclear. We examined this question in rat hepatoma tissue culture (HTC) cells (which lack endogenous ASGP-R) that were co-transfected with cDNAs for both RHL 1 and RHL 2/3. The original population was cloned, but derivatives were unstable. We therefore used fluorescence-activated cell sorting to separate a subpopulation of cells (positive) that specifically endocytosed fluoresceinated asialoorosomucoid (ASOR) from one that did not (negative). We then used indirect immunofluorescence with polypeptide-specific ASGP-R antibodies, immunoanalysis, and binding and uptake studies with two Gal ligands (ASOR and NAc-galactosylated poly-L-lysine (Gal-Lys] to further define the ASGP-R status in these two populations. As reported by others, we found that expression of both RHL 1 and RHL 2/3 in the positive cells resulted in binding, uptake and degradation of ASOR, the most commonly used ASGP-R ligand. The negative cells expressed only RHL 1 and neither bound nor processed ASOR. However, the presence of RHL 1 was sufficient for specific high affinity binding and processing of the synthetic ligand, Gal-Lys, by negative cells. These results show that RHL 1 can function as an ASGP-R, given a highly galactosylated ligand, and that RHL 2/3 must play an important role in the organization of native ASGP-R in the membrane.

Asialoglycoprotein receptor genes are linked on chromosome 11 in the mouse.

The asialoglycoprotein receptor on the hepatocyte plasma membrane recognizes galactose-terminated glycoproteins and internalizes them for subsequent degradation in lysosomes. The rat receptor, also known as rat hepatic lectin (RHL), is comprised of three protein subunits called RHL-1, RHL-2, and RHL-3; two genes code for RHL-1 and RHL-2/3, respectively. We have cloned and sequenced the gene for RHL-2/3, and demonstrated that homologous asialoglycoprotein receptor genes exist in the mouse genome. Biochemical studies have demonstrated that receptor subunits exhibit the same temporal expression during development and function in a coordinate manner. This study asks if mouse receptor genes are linked and thus could possibly respond to shared cis-acting regulatory elements. Using restriction fragment length polymorphisms (RFLPs) and recombinant inbred lines, we mapped two closely linked mouse hepatic lectin (MHL) genes to chromosome 11. Asgr is designated to name asialoglycoprotein gene loci. Coordinate regulation of this linked gene family is discussed.

Partial structure of the gene for chicken cartilage proteoglycan core protein.

A genomic DNA fragment (gCORE-1), encoding a portion of the cartilage proteoglycan core protein, has been isolated from a phage library using cDNA as a probe. The genomic insert is about 17 kilobase pairs; two BamHI fragments of the insert (1.3 and 4.8 kilobase pairs) contain most of the hybridizable sequences found in the cDNA. Sequence analysis of these fragments shows that they contain a total of five exons that encompass 216 amino acid residues, all of which are identical to those of the corresponding cDNA sequence. Three of the exons, which are adjacent to one another, are very similar to the corresponding exons in the gene of a rat hepatic lectin as well as to an exon in the gene of human pulmonary surfactant-associated protein. There is a strong degree of conservation of amino acid sequences encoded in the three genes, although there is no similarity between their introns. The sizes of the five exons in gCORE-1, except for one (which is indeterminate because only a partial cDNA sequence is available), are less than 184 base pairs, whereas the sizes of the introns range from 218 to greater than 2629 base pairs. Four of the introns interrupt an exon codon at either their donor or acceptor sites, between the first and second nucleotides. Only one intron does not split a codon. Intron and exon boundary sites are in agreement with known consensus sequences for introns. The dispersed distribution and relatively small size of the exons, if representative of the entire gene, suggest that the complete gene which codes for the core protein may be quite sizable.

Rabbit and rat hepatic lectins have two sugar-combining sites per monomeric unit

Binding characteristics of the galactose/N-acetylgalactosamine-specific, hepatic lectins of rabbit and rat were studied using small, high-affinity ligands containing two and three N-acetylgalactosamine residues per molecule [Lee, R. T. and Lee, Y. C. (1987) Glycoconjugate J. 4, 317-328]. These N-acetylgalactosamine cluster ligands have the receptor-ligand dissociation constants in nanomolar range, so that the lectin-ligand interaction can easily studied by an equilibrium (gel chromatography) or non-equilibrium (fast filtration assay) method. The results suggest that there exist on the average two N-acetylgalactosamine-combining sites per monomeric unit of both the rabbit and rat lectins.

Structural similarity between the macrophage lectin specific for galactose/N-acetylgalactosamine and the hepatic asialoglycoprotein binding protein

The rat peritoneal macrophage lectin specific for galactose/N-acetylgalactosamine was shown to be a homologue of the hepatic asialoglycoprotein binding protein (rat hepatic lectin, RHL). The macrophage lectin was immunochemically crossreactive with the major form of RHL (RHL-1) but not with the minor forms (RHL-2 and -3). The overall homology between the macrophage lectin and RHL-1 was confirmed by peptide maps of their lysyl endopeptidase digests on reverse-phase HPLC. Despite these similarities, however, the macrophage lectin was distinct from HRL-1 as revealed by the differences in the NH2-terminal 20 amino acid sequences of these two lectins.