Interaction of Human Macrophage C-type Lectin withO-Linked N-Acetylgalactosamine Residues on Mucin Glycopeptides

A fluorescein-labeled synthetic peptide, PTTTPITTTTK, was converted into O-glycosylated glycopeptides with various numbers of attachedN-acetyl-d-galactosamines (GalNAcs) by in vitro glycosylation with UDP-GalNAc and a microsomal fraction of LS174T human colon carcinoma cells. Glycopeptides with 1, 3, 5, and 6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Their sequences were determined by a peptide sequencer to be PTTTGalNAcPITTTTK for G1, PTGalNAcTTPITGalNAcTGalNAcTTK for G3, PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G6. A calcium-type human macrophage lectin (HML) was prepared in a recombinant form, and its interaction with these glycopeptides was investigated by surface plasmon resonance (SPR) spectroscopy and fluorescence polarization. The affinity of recombinant HML (rHML) for immobilized glycopeptides increased, as revealed by SPR, in parallel with the number of GalNAc. The highest affinity was obtained when the G6-peptide was immobilized at high density. Fluorescence polarization equilibrium-binding assays also revealed that the affinity of rHML for soluble gly-copeptides increased, depending on the number of attached GalNAcs. Carbohydrate recognition domain (CRD) fragments of HML were prepared, and their affinity for these four glycopeptides was also determined, this affinity was apparently lower than that of rHML. Affinity constants of rHML for the G3- and G5-peptides were 11- and 38-fold higher, respectively, than for the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylatedO-linked glycopeptides should be due to the trimer formation of rHML. A fluorescein-labeled synthetic peptide, PTTTPITTTTK, was converted into O-glycosylated glycopeptides with various numbers of attachedN-acetyl-d-galactosamines (GalNAcs) by in vitro glycosylation with UDP-GalNAc and a microsomal fraction of LS174T human colon carcinoma cells. Glycopeptides with 1, 3, 5, and 6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Their sequences were determined by a peptide sequencer to be PTTTGalNAcPITTTTK for G1, PTGalNAcTTPITGalNAcTGalNAcTTK for G3, PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G6. A calcium-type human macrophage lectin (HML) was prepared in a recombinant form, and its interaction with these glycopeptides was investigated by surface plasmon resonance (SPR) spectroscopy and fluorescence polarization. The affinity of recombinant HML (rHML) for immobilized glycopeptides increased, as revealed by SPR, in parallel with the number of GalNAc. The highest affinity was obtained when the G6-peptide was immobilized at high density. Fluorescence polarization equilibrium-binding assays also revealed that the affinity of rHML for soluble gly-copeptides increased, depending on the number of attached GalNAcs. Carbohydrate recognition domain (CRD) fragments of HML were prepared, and their affinity for these four glycopeptides was also determined, this affinity was apparently lower than that of rHML. Affinity constants of rHML for the G3- and G5-peptides were 11- and 38-fold higher, respectively, than for the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylatedO-linked glycopeptides should be due to the trimer formation of rHML. Calcium-dependent animal lectins (C-type lectins) 1The abbreviations used are: C-type lectin, calcium-dependent animal lectin; Ca-HBS, HEPES buffer containing CaCl2 and NaCl; CRD, carbohydrate recognition domain; DSS, disuccinimidyl suberate; EGS, ethylene glycolbis(succinimidylsuccinate); ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; FP, fluorescence polarization; HML, human macrophage lectin; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Manα-BPP, mannose α-biotinylated polyacrylamide polymer; rHML, recombinant human macrophage lectin; PTH, phenylthiohydantoin; saccharide-BPP, biotinylated saccharide-polyacrylamide polymers; PAGE, polyacrylamide gel electrophoresis; SPR, surface plasmon resonance.1The abbreviations used are: C-type lectin, calcium-dependent animal lectin; Ca-HBS, HEPES buffer containing CaCl2 and NaCl; CRD, carbohydrate recognition domain; DSS, disuccinimidyl suberate; EGS, ethylene glycolbis(succinimidylsuccinate); ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; FP, fluorescence polarization; HML, human macrophage lectin; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Manα-BPP, mannose α-biotinylated polyacrylamide polymer; rHML, recombinant human macrophage lectin; PTH, phenylthiohydantoin; saccharide-BPP, biotinylated saccharide-polyacrylamide polymers; PAGE, polyacrylamide gel electrophoresis; SPR, surface plasmon resonance. (1Drickamer K. J. Biol. Chem. 1988; 263: 9557-9560Abstract Full Text PDF PubMed Google Scholar) comprise a large family of recognition molecules in the immune system. Their specificity and potential regulatory functions demand a perspective far beyond the traditional concept of lectins (1Drickamer K. J. Biol. Chem. 1988; 263: 9557-9560Abstract Full Text PDF PubMed Google Scholar, 2Gabius H.J. Eur. J. Biochem. 1997; 243: 543-576Crossref PubMed Scopus (480) Google Scholar). A C-type lectin specific for galactose (Gal) andN-acetyl-d-galactosamine (GalNAc) as a monosaccharide has been found in histiocytic macrophages in mice (3Mizuochi S. Akimoto Y. Imai Y. Hirano H. Irimura T. Glycobiology. 1997; 7: 137-146Crossref PubMed Scopus (43) Google Scholar). A very similar lectin is also expressed in human macrophages as preferentially bound glycopeptides containing consecutive serine (Ser) and threonine (Thr) residues with attached GalNAc, which is well known as a carcinoma-associated Tn antigen (4Suzuki N. Yamamoto K. Toyoshima S. Osawa T. Irimura T. J. Immunol. 1996; 156: 128-135PubMed Google Scholar). While these C-type lectins are assumed to play roles in the recognition of malignant cells, their immunological roles are actually more diverse (5Kawakami K. Yamamoto K. Toyoshima S. Osawa T. Irimura T. Jpn. J. Cancer Res. 1994; 85: 744-749Crossref PubMed Scopus (34) Google Scholar, 6Imai Y. Akimoto Y. Mizuochi S. Kimura T. Hirano H. Irimura T. Immunology. 1995; 86: 591-598PubMed Google Scholar, 7Sakamaki T. Imai Y. Irimura T. J. Leukocyte Biol. 1995; 57: 407-414Crossref PubMed Scopus (26) Google Scholar). In rats, a lectin homologous to these molecules was shown to be up-regulated in the area of chronic rejection of heart transplants in rats (8Russell M.E. Utans U. Wallace A.F. Liang P. Arceci R.J. Karnovsky M.J. Wyner L.R. Yamashita Y. Tarn C. J. Clin. Invest. 1994; 94: 722-730Crossref PubMed Scopus (49) Google Scholar). It has long been known that other C-type lectins produced by hepatocytes are involved in asialoglycoprotein uptake from the circulation; macrophage C-type lectins from rodents seem to function in the same fashion (5Kawakami K. Yamamoto K. Toyoshima S. Osawa T. Irimura T. Jpn. J. Cancer Res. 1994; 85: 744-749Crossref PubMed Scopus (34) Google Scholar, 9Ozaki K. Ii M. Itoh N. Kawasaki T. J. Biol. Chem. 1992; 267: 9229-9235Abstract Full Text PDF PubMed Google Scholar).Because the interactions of some lectins with simple monosaccharides are sometimes weak, the direct binding of these lectins with a monosaccharide may be difficult to determine. If the cooperative effects of polyvalent carbohydrate ligands are present, however, and the lectin has multivalency, a dramatic increase in the affinity could prevail (2Gabius H.J. Eur. J. Biochem. 1997; 243: 543-576Crossref PubMed Scopus (480) Google Scholar, 10Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (995) Google Scholar, 11Rini J.M. Annu. Rev. Biophys. Biomed. 1995; 24: 551-577Crossref PubMed Scopus (424) Google Scholar). Members of the C-type lectin family often form oligomeric structures through the stalk regions containing the α-helical domain; this provides an advantage in polyvalent binding (12Beavil A.J. Edmeades R.L. Gould H.J. Sutton B.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 753-757Crossref PubMed Scopus (100) Google Scholar, 13Dierks S.E. Bartlett W.C. Edmeades R.L. Gould H.J. Rao M. Conrad D.H. J. Immunol. 1993; 150: 2372-2382PubMed Google Scholar). A similar observation has been made in the binding of carbohydrate-specific antibodies (14MacKenzie C.R. Hirama T. Deng S. Bundle D.R. Narang S.A. Young N.M. J. Biol. Chem. 1996; 271: 1527-1533Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). For example, synthetic neoglycoproteins and saccharide-derivatized polyacrylamide polymers have been used to represent a multivalent ligand that has a high affinity for lectins (15Bovin N.T. Korchagina E.Y. Zemlyanukhina T.V. Byramova N.E. Galanina O.E. Zemlyakov A.E. Ivanov A.E. Zubov V.P. Mochalova L.V. Glycoconjugate J. 1993; 10: 142-151Crossref PubMed Scopus (206) Google Scholar, 16Adler P. Wood S.J. Lee Y.C. Lee R.T. Petri Jr., W.A. Schnaar R.L. J. Biol. Chem. 1995; 270: 5164-5171Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In other studies, the clustering effects of carbohydrate chains were evaluated in detail through the use of trivalent glycosides (17Lee R.T. Lee Y.C. Glycoconjugate J. 1987; 4: 317-328Crossref Scopus (99) Google Scholar, 18Lee Y.C. FASEB J. 1992; 6: 3193-3200Crossref PubMed Scopus (210) Google Scholar, 19Lodish H.F. Trends Biochem. Sci. 1991; 16: 374-377Abstract Full Text PDF PubMed Scopus (102) Google Scholar). Although the multivalency of carbohydrate ligands is seen in a variety of native glycoproteins, particularly O-linked glycoproteins, its functions in carbohydrate-recognition pathogenic processes are not well known. Furthermore, differences in the kinetics of protein-carbohydrate interactions between monosaccharide and multivalent ligands were unknown until recently. The first step toward determining the significance of HML in the pathogenesis of a variety of diseases was the cDNA cloning and sequencing of human macrophage lectin (HML), described in a previous report (4Suzuki N. Yamamoto K. Toyoshima S. Osawa T. Irimura T. J. Immunol. 1996; 156: 128-135PubMed Google Scholar). The next step should be to elucidate the nature of natural ligand for this lectin. In the previous study, HML was demonstrated to preferentially bind clusters ofN-acetylgalactosamine linked to Ser or Thr in a carcinoma-associated mucin-like configuration (4Suzuki N. Yamamoto K. Toyoshima S. Osawa T. Irimura T. J. Immunol. 1996; 156: 128-135PubMed Google Scholar).Epithelial mucins are characterized by their tandem repeat domains, which contain many Ser and Thr residues (20Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 57: 607-634Crossref PubMed Scopus (858) Google Scholar, 21Kim Y.S. Gum J. Brockhausen I. Glycoconjugate J. 1996; 13: 693-707Crossref PubMed Scopus (268) Google Scholar). The tandem repeat portion of the MUC2 mucin consists of 23 amino acids, including 14 Thr residues; these Thr residues are reported to be glycosylated up to 78% in the LS174T colon carcinoma cell line (22Byrd J.C. Nardelli J. Siddiqui B. Kim Y.S. Cancer Res. 1988; 48: 6678-6685PubMed Google Scholar). Although the tandem repeat domain of MUC2 mucin in other cells is also thought to be highlyO-glycosylated, whether the glycosylation patterns in different cells and tissues are unique is not yet understood. Mucin architecture represents an ideal framework for a variety of multiple ligand arrangements that should serve as a recognition unit. In the present study, we applied the surface plasmon resonance (SPR) biosensor and fluorescence polarization (FP) spectroscopy to analyze ligand-density dependence in carbohydrate-protein interactions.DISCUSSIONTo examine the interaction of HML with GalNAc residues arranged on peptides, we prepared short synthetic glycopeptides corresponding to a portion of the tandem repeat domain of MUC2 mucin with different numbers of attached GalNAc residues. Molecular sizes were determined by MALDI-TOF MS, and the locations of the incorporated residues were determined by peptide sequencing. Kinetics of the binding were examined in both the fluid-solid phase with SPR and the fluid-fluid phase with FP; both equilibrium-binding assays of the interaction of rHML with immobilized glycopeptides revealed that the number of GalNAc residues does affect K D. Affinity of the G3-peptide (PTGalNAcTTPITGalNAcTGalNAcTT[K]) for HML was 11-fold greater than that of the G1-peptide (PTTTGalNAcPITTTTK) and that of the G5-peptide was 38-fold greater; when K D values were normalized according to the number of GalNAc residues, the increases were 3.5- and 7.6-fold, respectively.Differences in affinity for CRD fragments of G3 and G5 relative to G1, based on the molar amount of glycopeptide, were 2- and 6-fold, respectively; differences based on the number of GalNAc residues were 0.7- and 1.2-fold, respectively. Therefore, rHML fragments but not CRD fragments revealed a drastic increase in affinity that depended on the number of GalNAc residues present. The differences among G1-, G3-, and G5-peptides should be due to the increased avidity of polyvalent ligands. This effect was more prominent with rHML than with CRD fragments due to the oligomeric nature of rHML, whereas CRD fragments apparently remained in monomeric in aqueous solutions (Fig. 1).The G5- and G6-peptides were shown to contain three consecutive Thr-linked GalNAc residues, i.e. Tn antigen. Anti-trimeric Tn mAb (26Reddish M.A. Jackson L. Koganty R.R. Qiu D. Hong W. Longenecker B.M. Glycoconjugate J. 1997; 14: 549-560Crossref PubMed Scopus (72) Google Scholar) was shown to bind these glycopeptides. It has already been shown that rat hepatic lectin, a type of trimeric C-type lectin, also bound preferentially to clustered GalNAc residues, as shown by using synthetic cluster glycosides based on Tris (17Lee R.T. Lee Y.C. Glycoconjugate J. 1987; 4: 317-328Crossref Scopus (99) Google Scholar, 18Lee Y.C. FASEB J. 1992; 6: 3193-3200Crossref PubMed Scopus (210) Google Scholar). Therefore, in addition to the impact of increased number of GalNAc residues, three consecutive Thr attached with GalNAc residues might also contribute to the high binding affinity for HML.Dense immobilization of all of the synthetic glycopeptides leads to high affinity association. The difference between densely immobilized and sparsely immobilized glycopeptides seemed to be due to the dramatic decrease in the dissociation rates (Table I). Although the organization of the densely immobilized glycopeptides on the membranes was unknown, the binding of anti-trimetric Tn mAb strongly suggested that some GalNAc residues from separate glycopeptides were located very close to each other; thus, the clustering of GalNAc residues may well be one of the more important factors in the high affinity of HML (Fig. 6). Because the immobilization was done via biotin-streptavidin, it may be unconceivable that the distance of GalNAc would be as close as to mimic a single amino acid distance between them. Thus, the spacing among the carbohydrate chains may contribute to macromolecular ligand interactions with HML, as observed in amebic lectin binding (16Adler P. Wood S.J. Lee Y.C. Lee R.T. Petri Jr., W.A. Schnaar R.L. J. Biol. Chem. 1995; 270: 5164-5171Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). There may be another level at which clusters in macromolecular carbohydrate ligands contribute to high affinity (18Lee Y.C. FASEB J. 1992; 6: 3193-3200Crossref PubMed Scopus (210) Google Scholar). Simulation by x-ray crystallography (29Sheriff S. Chang C.Y. Ezekowitz A.B. Nat. Struct. Biol. 1994; 1: 789-793Crossref PubMed Scopus (212) Google Scholar) has revealed three binding sites of trimeric mannose binding protein spaced approximately 4.5 nm apart. If HML also accommodates such a structure, GalNAc residues spaced more sparsely than trimeric Tn might be required for viable ligand-carbohydrate interactions.The results of FP equilibrium-binding assays, with one exception, were consistent with the findings from the experiments using the SPR biosensor, K D for G1-peptides from SPR (1.0 μm for rHML) was higher than K D based on FP binding data (6.8 μm for rHML and 8.3 μm for CRD). The difference might be due to an increase in conformational stability on solid surfaces. It is also probable that the solid phase configuration was more appropriate for the formation of the multiple attachment sites preferred by oligomeric lectin than are glycopeptides in solutions. Collectively, slightly lower constants from FP assays in fluid-fluid phases than those from SPR measurements seemed to be reasonable. From FP assays conducted under ideally diluted conditions, K D values between monovalent lectin and GalNAc monosaccharide can be obtained by the Scatchard plot (Fig. 6). All K D values reported here were based on the monovalent ligand-monovalent receptor model. Such analysis was used because the binding of multivalent lectins to multivalent glycopeptides is inherently more complex than are antigen-antibody interactions, and thus it is difficult to simulate the factors involved in a simple equation (14MacKenzie C.R. Hirama T. Deng S. Bundle D.R. Narang S.A. Young N.M. J. Biol. Chem. 1996; 271: 1527-1533Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The results, however, fit well with the single-site binding model. It is possible, then, that, regardless of the valency, binding of rHML might be an "all-or-nothing" event with no intermediate forms, as discussed by Adler and co-workers (16Adler P. Wood S.J. Lee Y.C. Lee R.T. Petri Jr., W.A. Schnaar R.L. J. Biol. Chem. 1995; 270: 5164-5171Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar).In microtiter assays, rHML bound only GalNAc α- or β-monosaccharide-BPPs linked directly to the spacer arm on the polymer backbone. A GalNAc residue located away from the polymer backbone, such as those in the Forrsman disaccharide and the blood group A-type trisaccharide (Fig. 2), did not have strong affinity with HML. On the contrary, a low albeit significant affinity for HML by these oligosaccharides was detected through measurement on the SPR biosensor (data not shown). Such a discrepancy might be due to different steric hindrances in the access of this lectin to the binding sites using these two methods. The preferred ligand structure for HML binding was multiple GalNAc residues attached to linear backbones, regardless of the specific synthetic polymers or oligopeptides. As noted above, the Galβ residue attached to the same polymer neither bound directly to the lectin nor inhibited the binding of GalNAc to rHML. These results were consistent with our previous data, which showed that rHML bound neither N-linked carbohydrate chains with terminal Gal residues nor the N-terminal octapeptide of human glycophorin A with three consecutive desializedO-linked carbohydrate chains (4Suzuki N. Yamamoto K. Toyoshima S. Osawa T. Irimura T. J. Immunol. 1996; 156: 128-135PubMed Google Scholar). Greater specificity for GalNAc than Gal was also observed with a human hepatic lectin (30Iobst S. Drickamer K. J. Biol. Chem. 1996; 271: 6686-6693Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), in contrast to a mouse macrophage lectin that preferentially bound Gal instead of GalNAc residues (31Imai Y. Irimura T. J. Immunol. Methods. 1994; 171: 23-31Crossref PubMed Scopus (16) Google Scholar, 32Hosoi T. Imai Y. Irimura T. Glycobiology. 1998; 8: 791-798Crossref PubMed Scopus (14) Google Scholar). Using chimeric and mutagenized CRD fragments of hepatic and macrophage lectin, amino acid residues likely to be involved in the selective binding of GalNAc in the human hepatic lectin were predicted (30Iobst S. Drickamer K. J. Biol. Chem. 1996; 271: 6686-6693Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) (His-256 and Asn-208). These amino acids apparently correspond to His-260 and Tyr-212 in HML, which may be the most important residue in the interaction between HML and GalNAc. HML could serve as an useful model to further investigate the nature of GalNAc-protein interactions.When solutions of either rHML or CRD fragments were incubated in the presence of cross-linking agents, a homotrimer of rHML was the only major oligomeric lectin protein (Fig. 1). The CRD fragments lacking the HML stalk region did not appear to form oligomers. Other members of the C-type lectin family, including CD23, CD72, rat asialoglycoprotein receptor, chicken hepatic receptor, Kupffer cell receptor, and mannose-binding protein, are also known to form oligomers (12Beavil A.J. Edmeades R.L. Gould H.J. Sutton B.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 753-757Crossref PubMed Scopus (100) Google Scholar, 29Sheriff S. Chang C.Y. Ezekowitz A.B. Nat. Struct. Biol. 1994; 1: 789-793Crossref PubMed Scopus (212) Google Scholar). The primary amino acid sequence of the stalk region of rHML has a structure known to be involved in oligomer formation. When the amino acid sequence of the stalk region (from Ile-87 to Glu-153) deduced from the nucleotide sequence was arranged in order to accommodate two heptads (a, b, c, d, e, f, and g), hydrophobic residues were arranged at positions a and d in a way that should result in an α-helix coiled-coil. Thus, HML forms a trimeric configuration and would have high affinity for ligands with GalNAc residues such as in the case of CD23 with IgE (13Dierks S.E. Bartlett W.C. Edmeades R.L. Gould H.J. Rao M. Conrad D.H. J. Immunol. 1993; 150: 2372-2382PubMed Google Scholar).We have determined affinity constants between HML and peptides with a single GalNAc residue using FP equilibrium-binding assays. A carbohydrate density-dependent increase of affinity for the lectin was revealed, and the increase was more prominent with intact rHML than with CRD fragments. In the present study, variations in the distance between multiple GalNAc residues on a single peptide were not systematically investigated. More dispersed arrangements of GalNAc residues than those used in this study may generate more preferable configurations as functional ligands for HML, and therefore this will be an important subject for future studies. Calcium-dependent animal lectins (C-type lectins) 1The abbreviations used are: C-type lectin, calcium-dependent animal lectin; Ca-HBS, HEPES buffer containing CaCl2 and NaCl; CRD, carbohydrate recognition domain; DSS, disuccinimidyl suberate; EGS, ethylene glycolbis(succinimidylsuccinate); ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; FP, fluorescence polarization; HML, human macrophage lectin; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Manα-BPP, mannose α-biotinylated polyacrylamide polymer; rHML, recombinant human macrophage lectin; PTH, phenylthiohydantoin; saccharide-BPP, biotinylated saccharide-polyacrylamide polymers; PAGE, polyacrylamide gel electrophoresis; SPR, surface plasmon resonance.1The abbreviations used are: C-type lectin, calcium-dependent animal lectin; Ca-HBS, HEPES buffer containing CaCl2 and NaCl; CRD, carbohydrate recognition domain; DSS, disuccinimidyl suberate; EGS, ethylene glycolbis(succinimidylsuccinate); ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; FP, fluorescence polarization; HML, human macrophage lectin; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Manα-BPP, mannose α-biotinylated polyacrylamide polymer; rHML, recombinant human macrophage lectin; PTH, phenylthiohydantoin; saccharide-BPP, biotinylated saccharide-polyacrylamide polymers; PAGE, polyacrylamide gel electrophoresis; SPR, surface plasmon resonance. (1Drickamer K. J. Biol. Chem. 1988; 263: 9557-9560Abstract Full Text PDF PubMed Google Scholar) comprise a large family of recognition molecules in the immune system. Their specificity and potential regulatory functions demand a perspective far beyond the traditional concept of lectins (1Drickamer K. J. Biol. Chem. 1988; 263: 9557-9560Abstract Full Text PDF PubMed Google Scholar, 2Gabius H.J. Eur. J. Biochem. 1997; 243: 543-576Crossref PubMed Scopus (480) Google Scholar). A C-type lectin specific for galactose (Gal) andN-acetyl-d-galactosamine (GalNAc) as a monosaccharide has been found in histiocytic macrophages in mice (3Mizuochi S. Akimoto Y. Imai Y. Hirano H. Irimura T. Glycobiology. 1997; 7: 137-146Crossref PubMed Scopus (43) Google Scholar). A very similar lectin is also expressed in human macrophages as preferentially bound glycopeptides containing consecutive serine (Ser) and threonine (Thr) residues with attached GalNAc, which is well known as a carcinoma-associated Tn antigen (4Suzuki N. Yamamoto K. Toyoshima S. Osawa T. Irimura T. J. Immunol. 1996; 156: 128-135PubMed Google Scholar). While these C-type lectins are assumed to play roles in the recognition of malignant cells, their immunological roles are actually more diverse (5Kawakami K. Yamamoto K. Toyoshima S. Osawa T. Irimura T. Jpn. J. Cancer Res. 1994; 85: 744-749Crossref PubMed Scopus (34) Google Scholar, 6Imai Y. Akimoto Y. Mizuochi S. Kimura T. Hirano H. Irimura T. Immunology. 1995; 86: 591-598PubMed Google Scholar, 7Sakamaki T. Imai Y. Irimura T. J. Leukocyte Biol. 1995; 57: 407-414Crossref PubMed Scopus (26) Google Scholar). In rats, a lectin homologous to these molecules was shown to be up-regulated in the area of chronic rejection of heart transplants in rats (8Russell M.E. Utans U. Wallace A.F. Liang P. Arceci R.J. Karnovsky M.J. Wyner L.R. Yamashita Y. Tarn C. J. Clin. Invest. 1994; 94: 722-730Crossref PubMed Scopus (49) Google Scholar). It has long been known that other C-type lectins produced by hepatocytes are involved in asialoglycoprotein uptake from the circulation; macrophage C-type lectins from rodents seem to function in the same fashion (5Kawakami K. Yamamoto K. Toyoshima S. Osawa T. Irimura T. Jpn. J. Cancer Res. 1994; 85: 744-749Crossref PubMed Scopus (34) Google Scholar, 9Ozaki K. Ii M. Itoh N. Kawasaki T. J. Biol. Chem. 1992; 267: 9229-9235Abstract Full Text PDF PubMed Google Scholar). Because the interactions of some lectins with simple monosaccharides are sometimes weak, the direct binding of these lectins with a monosaccharide may be difficult to determine. If the cooperative effects of polyvalent carbohydrate ligands are present, however, and the lectin has multivalency, a dramatic increase in the affinity could prevail (2Gabius H.J. Eur. J. Biochem. 1997; 243: 543-576Crossref PubMed Scopus (480) Google Scholar, 10Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (995) Google Scholar, 11Rini J.M. Annu. Rev. Biophys. Biomed. 1995; 24: 551-577Crossref PubMed Scopus (424) Google Scholar). Members of the C-type lectin family often form oligomeric structures through the stalk regions containing the α-helical domain; this provides an advantage in polyvalent binding (12Beavil A.J. Edmeades R.L. Gould H.J. Sutton B.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 753-757Crossref PubMed Scopus (100) Google Scholar, 13Dierks S.E. Bartlett W.C. Edmeades R.L. Gould H.J. Rao M. Conrad D.H. J. Immunol. 1993; 150: 2372-2382PubMed Google Scholar). A similar observation has been made in the binding of carbohydrate-specific antibodies (14MacKenzie C.R. Hirama T. Deng S. Bundle D.R. Narang S.A. Young N.M. J. Biol. Chem. 1996; 271: 1527-1533Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). For example, synthetic neoglycoproteins and saccharide-derivatized polyacrylamide polymers have been used to represent a multivalent ligand that has a high affinity for lectins (15Bovin N.T. Korchagina E.Y. Zemlyanukhina T.V. Byramova N.E. Galanina O.E. Zemlyakov A.E. Ivanov A.E. Zubov V.P. Mochalova L.V. Glycoconjugate J. 1993; 10: 142-151Crossref PubMed Scopus (206) Google Scholar, 16Adler P. Wood S.J. Lee Y.C. Lee R.T. Petri Jr., W.A. Schnaar R.L. J. Biol. Chem. 1995; 270: 5164-5171Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In other studies, the clustering effects of carbohydrate chains were evaluated in detail through the use of trivalent glycosides (17Lee R.T. Lee Y.C. Glycoconjugate J. 1987; 4: 317-328Crossref Scopus (99) Google Scholar, 18Lee Y.C. FASEB J. 1992; 6: 3193-3200Crossref PubMed Scopus (210) Google Scholar, 19Lodish H.F. Trends Biochem. Sci. 1991; 16: 374-377Abstract Full Text PDF PubMed Scopus (102) Google Scholar). Although the multivalency of carbohydrate ligands is seen in a variety of native glycoproteins, particularly O-linked glycoproteins, its functions in carbohydrate-recognition pathogenic processes are not well known. Furthermore, differences in the kinetics of protein-carbohydrate interactions between monosaccharide and multivalent ligands were unknown until recently. The first step toward determining the significance of HML in the pathogenesis of a variet