The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells.

The monoclonal antibody HNK-1 originally raised to an antigenic marker of natural killer cells also binds to selected regions in nervous tissue. The antigen is a carbohydrate that has attracted much interest as its expression is developmentally regulated in nervous tissue, and it is found, and proposed to be a ligand, on several of the adhesive glycoproteins of the nervous system. It is also expressed on glycolipids and proteoglycans, and is the target of monoclonal auto-antibodies that give rise to a demyelinating disease. The epitope, as characterized on glycolipids isolated from the nervous system, is expressed on 3-sulfated glucuronic acid joined by ␤1-3-linkage to a neolacto backbone. Here we exploit the neoglycolipid technology, in conjunction with immunodetection and in situ liquid secondary ion mass spectrometry, to characterize HNK-1-positive oligosaccharide chains derived by reductive alkaline release from total brain glycopeptides. The immunoreactive oligosaccharides detected are tetra-to octasaccharides that are very minor components among a heterogeneous population, each representing less than 0.1% of the starting material. Their peripheral and backbone sequences resemble those of the HNK-1-positive glycolipids. An unexpected finding is that they terminate not with N-acetylgalactosaminitol but with hexitol (2-substituted and 2,6-disubstituted). In a tetrasaccharide investigated in the greatest detail, the hexitol is identified as 2-substituted mannitol.Monoclonal antibodies raised with the aim of identifying developmentally regulated antigens and differentiation antigens that have biological functions are in many cases directed to oligosaccharides of glycoproteins or glycolipids (1). Prominent among such antigens are those based on backbone sequences of the poly-N-acetyllactosamine type that are O-or N-glycosidically linked to proteins or to a glucose core of glycolipids (1, 2), several of which are now established ligands for carbohydrate-binding proteins of animals (3-6); and there is much interest in the biological events elicited by the carbohydrate-protein interactions. HNK-1 antigen, recognized by a hybridoma antibody, is a carbohydrate antigen first identified as a marker of the natural killer cell population among lymphocytes (7), and later found to be expressed on glycolipids (8 -10) as well as several glycoproteins (11) and proteoglycans (12) of the nervous system, with a changing distribution at different stages of development (13-15). HNK-1 antigen, as characterized biochemically on glycolipids, is expressed on 3-sulfated glucuronic acid joined by ␤1-3-linkage to a (poly)Nacetyllactosamine backbone (8 -10). This carbohydrate sequence has been implicated as a ligand for the leukocyteendothelium adhesion molecules, L-and P-selectins (16,17) and for a number of cell-interaction systems in nervous tissue (11, 18), notably as a ligand for the major glycoprotein of myelin, P o , in the peripheral nervous system (19), the extracellular matrix protein, laminin (20), and an adhesive protein, amph...

Carbohydrate recognition by amyloid P component from human serum has been investigated by binding experiments using several glycosaminoglycans, polysaccharides and a series of structurally defined neoglycolipids and natural glycolipids. Two novel classes of carbohydrate ligands have been identified. The first is 6‐phosphorylated mannose as found on lysosomal hydrolases, and the second is the 3‐sulphated saccharides galactose, N‐acetyl‐galactosamine and glucuronic acid as found on sulphatide and other acidic glycolipids that occur in neural or kidney tissues or on subpopulations of lymphocytes. Binding to mannose‐6‐phosphate containing molecules and inhibition of binding by free mannose‐6‐phosphate and fructose‐1‐phosphate are features shared with mannose‐6‐phosphate receptors involved in trafficking of lysosomal enzymes. However, only amyloid P binding is inhibited by galactose‐6‐phosphate, mannose‐1‐phosphate and glucose‐6‐phosphate. These findings strengthen the possibility that amyloid P protein has a central role in amyloidogenic processes: first in formation of focal concentrations of lysosomal enzymes including proteases that generate fibril‐forming peptides from amyloidogenic proteins, and second in formation of multicomponent complexes that include sulphoglycolipids as well as glycosaminoglycans. The evidence that binding to all of the acidic ligands involves the same polypeptide domain on amyloid P protein, and inhibition data using diffusible, phosphorylated monosaccharides, is potentially important leads to novel drug designs aimed at preventing or even reversing amyloid deposition processes without interference with essential lysosomal trafficking pathways.

Mycoplasma pneumoniae infection in the human is often followed by a transient autoimmune hemolytic disorder characterized by high titer autoantibodies to a carbohydrate antigen, the I antigen. Because the major host cell receptor for the Mycoplasma is the sialylated form of this antigen, it is likely that the immunologic disorder is initiated by the microbe-saccharide interaction. Here we review briefly knowledge on the autoantibodies and the structures and distribution of the saccharide antigens and receptors. We discuss possible mechanisms for the triggering of autoantibody production and consider ways in which perturbation of various glycoprotein carriers of the carbohydrate ligands may elicit a variety of pathobiologic responses. We conclude by highlighting ideas on further molecular dissections of the elements of the microbe-host interaction.

The IgG 1 hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewis a (Le a ) and Le x types, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri-and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3′-sulfated Le a tetrasaccharide sequence, which is a potent ligand for the E-and L-selectins. The antibody gives a relatively low signal with the 3′-sulfated non-fucosylated backbone, and has no detectable cross-reaction with the 3′-sulfated Le x isomer, nor with sialyl-Le a and -Le x analogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.