View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Mass spectrum of the NGLs derived from the sulfated N-glycan, pLH-F3-2, isolated from lutropin. The molecular ions and fragment ions correspond to those of the monoantennary N-glycan sequence illustrated, with and without a core fucose residue, which was described previously as one of the major oligosaccharide species in human lutropin (reference 10).

Monosaccharides and Blood Group Oligosaccharides.
The monosaccharides, 4-sulfated GalNAc, 6-sulfated GalNAc, and 4,6-disulfated GalNAc were from Sigma Chemical Co. Lacto-N-neotetraose (LNNT) was from Dextra. 3'-sialyl-Lewis^a pentasaccharide, 3S-Le^a, was from the Institute of Food Substances (Moscow). The following oligosaccharides were synthesized chemically: 3'-sialyl-Lewis^x pentasaccharide, 3S-Le^x 24; 3'-sulfated-Le^x pentasaccharide, 3Su-Le^x 25; 3'-sulfated-Le^a pentasaccharide, 3Su-Le^a 26; and 6'-sulfated-Le^x pentasaccharide, 6Su-Le^x 25. The 3'-sulfated-lacto-N-tetraose, 3Su-LNT, and 3'-sulfated-LNNT, 3Su-LNNT, were prepared by mild acid hydrolysis 27 of 3Su-Le^a and 3Su-Le^x, respectively.

NGLs.
NGLs, unless otherwise stated, were prepared by the conjugation of oligosaccharides to the aminophospholipid L-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE; Fluka), followed by isolation from the reaction mixtures by TLC as described previously 28. A modified procedure was used for the preparation of NGLs from CS oligosaccharides. In brief, lyophilized CS oligosaccharides (100 nmol) were dissolved in 5 µl H₂O, and 100 µl of DHPE solution (5 mg/ml in CHCl₃/MeOH 1:3, vol/vol) was added, followed by 20 µl of freshly prepared tetrabutylammonium cyanoborohydride (20 µg/µl in MeOH). The reaction mixtures were heated at 60°C for 70 h, conjugation was monitored by high performance TLC, and NGLs were purified using silica minicolumns (500 mg silica, Sep-Pak^® Vac Cartridge; Waters Corp.) essentially as described previously 29.

Biotinylated Oligosaccharides.
Oligosaccharides were conjugated to 6-(biotinyl)-aminocaproyl-hydrazide (BACH; Sigma Chemical Co.) and characterized as described previously 30 31. In brief, the oligosaccharides were reacted with BACH for 16 h in methanol/water 95:5, vol/vol, purified by HPLC, and its chemical structures were corroborated by mass spectrometry.

Liquid Secondary Ion Mass Spectrometry.
Negative-ion liquid secondary ion mass spectrometry (LSIMS) was carried out on a VG Analytical ZAB-2E mass spectrometer (VG Analytical) equipped with a cesium ion gun operated at 25 keV with an emission current of 0.5 µA. For the native and biotinylated oligosaccharides, 1 µg of sample was used for analysis with thioglycerol as the liquid matrix. NGLs were analyzed by in situ TLC/LSIMS with a mixture of diethanolamine/tetramethylurea/m-nitrobenzyl alcohol (2:2:1, by volume) as the matrix as described previously 32.

Binding Experiments.
Binding of Cys-MR-Fc or of Cys-DEC-Fc, 0.5 µg/well (preincubated with anti–human IgG, at a chimera to anti-IgG ratio of 1:3 [wt/wt]) to glycoproteins or to NGLs (reaction volumes 50 µl), was assayed essentially as described previously for L-selectin 33. For inhibition of Cys-MR binding to hLH (0.5 µg hLH added/well), the Cys-MR-Fc was used at 0.25 µg/well, which gave an absorbance in the range of 0.75–0.79 corresponding to 50% of maximum absorbance. Cys-MR binding was also examined to serglycin immobilized on microwells (1.5 µg added/well) and treated at 30°C for 4 h with either chondroitinase ABC (50 mU in 50 µl of 10 mM phosphate buffer, 150 mM NaCl, pH 7.5 [PBS]), with the heat-denatured enzyme, with heparinase I (2.5 U), or with PBS alone. After washing four times with PBS, binding of Cys-MR-Fc, 0.5 µg added/well, was assayed as above.

Binding of Cys-MR-Fc to biotinylated oligosaccharides was assayed essentially as described previously for plant lectins 30 and selectins 31. In brief, biotinylated oligosaccharides were immobilized in high capacity streptavidin-coated microtiter wells (Boehringer Mannheim) in TBS (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl). Cys-MR-Fc chimera was at 5 µg/well (preincubated for 1 h with rabbit anti–human IgG, at a chimera to anti-IgG ratio of 1:3 [wt/wt]). The diluent used was TBS containing 0.5% (wt/vol) BSA, and reaction volumes were 200 µl. Binding was detected with peroxidase-labeled protein A (10 µg/ml) using ABTS (2,2'-azinobis[3-ethylbenzthiazoline-sulfonic acid]) as substrate, and absorbance was read at 405 nm.

Histochemical Experiments.
Histochemical staining of spleens from nonimmunized C57 mice and mice 9–10 d after intraperitoneal immunization with 100 µg of chicken gammaglobulin precipitated in alum (Jackson ImmunoResearch Laboratories) was performed essentially as described 13. In brief, 8-µm cryostat sections were fixed for 12 min in acetone at 4°C or in 2% formaldehyde in PBS at 18°C. Fixed sections were washed in PBS, and Fc receptors were blocked for 10 min at 18°C using rat anti–mouse CD16/CD32 (Becton Dickinson) at 10 µg/ml in 5% (vol/vol) donkey serum-PBS. Sections were incubated for 30 min at 18°C with the Cys-MR-Fc chimera at 10 µg/ml in donkey serum-PBS. Binding was detected using fluorescein-labeled donkey anti–human IgG (Jackson ImmunoResearch Laboratories) for 30 min at 18°C, and sections were viewed with a Zeiss ultraviolet epifluorescence microscope. Specificity of staining was corroborated by the lack of staining of sections similarly incubated with a recombinant human Fc dimer (gift of Dr. E. Hofer, University of Vienna, Vienna, Austria). B cell areas in the splenic white pulp were identified by staining with rat anti–mouse complement receptor type 1 (CD35; Becton Dickinson) at 10 µg/ml. Antibody binding was detected using rhodamine-labeled donkey anti–rat IgG (Jackson ImmunoResearch Laboratories).

In certain experiments, the fixed spleen sections were treated for 30 min at 37°C with chondroitinase ABC (500 mU/ml) or with heparinase I (100 mU/ml) diluted in PBS containing 0.05% (vol/vol) Tween 20 and 1 mM PMSF (Sigma Chemical Co.). Enzyme-treated sections were stained in both single and dual immunolabeling experiments with a rabbit antiserum (1:100 dilution) specific for the core regions of cleaved CS chains (Biogenesis Ltd.) and with the Cys-MR-Fc. Binding of the CS antibody was detected using rhodamine-labeled donkey anti–rabbit IgG (Jackson ImmunoResearch Laboratories). The staining patterns for the Cys-MR-Fc and the CS antibody were viewed simultaneously with a Zeiss LSM 510 confocal microscope, and apparent colocalization in selected areas was confirmed by Z sectioning.

For inhibition studies, Cys-MR-Fc was incubated for 1 h at 18°C with the monosaccharides GalNAc, 4-sulfated GalNAc, or 6-sulfated GalNAc (Sigma Chemical Co.) at 5 mg/ml before application onto spleen sections.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In accord with previous data, the Cys-MR-Fc gave a binding signal with lutropin. In contrast, no binding was detected with Cys-DEC ( Fig. 2 A, inset). The NGL derived from the sulfated N-glycan, F3-2, isolated from lutropin was also strongly bound by Cys-MR-Fc ( Fig. 2 A). As the Cys-MR binding was inhibitable with 4-sulfated N-acetylgalactosamine (concentration giving 50% inhibition of binding [IC₅₀] 0.18 mM), and less strongly with 6-sulfated and 4,6-disulfated N-acetylgalactosamine (IC₅₀ values 1.34 and 0.85 µM, respectively; Fig. 3 A), we examined the protein for recognition of glycans with various sulfation patterns as described below, and see Table .

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Binding experiments with Cys-MR and Cys-DEC. NGLs (A–C), lutropin (A, inset), and biotinylated oligosaccharides (D) were immobilized on microwells, and the binding of Cys-MR-Fc or Cys-DEC-Fc, 5 µg/well, was assayed as described in Materials and Methods.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Inhibition of the binding of Cys-MR to lutropin by sulfated monosaccharides and glycosaminoglycans. hLH, 0.5 µg/well, was coated onto microwells, and the binding of Cys-MR-Fc, 0.25 µg/well, in the presence of the sulfated monosaccharides (A) or glycosaminoglycans (B), was assayed as described in Materials and Methods.

The NGLs of the CS-B and CS-A pentasaccharides, CS-B5 and CS-A5, were bound by Cys-MR-Fc ( Fig. 2A and Fig. B). These pentasaccharides have a common terminal GalNAc(4S) (Table ). In contrast to the pentasaccharide, the CS-A hexasaccharide, CS-A6, which terminates with the unsaturated uronic acid, UAβ1-3, did not give a detectable binding signal with Cys-MR-Fc despite the presence of three GalNAc(4S) residues along the oligosaccharide chain (Table ). First, these results show that Cys-MR recognizes GalNAc(4S) in a terminal (nonreducing end), rather than an internal position. Therefore, the lack of inhibitory activity in the original sample of CS-A polysaccharide suggests that the amount of terminal GalNAc(4S) is likely to be lower than in the CS-B polymer. Second, the carbohydrate-binding site of Cys-MR can accommodate oligosaccharides terminating with GalNAc(4S) joined by β1-4 linkage to glucuronic acid or iduronic acid, as well as to N-acetylglucosamine, as found on the N-glycans of lutropin. The CS-C pentasaccharide, CS-C5, containing predominantly the terminal GalNAc(6S)β1-4GlcAβ1-3 sequence (Table ), gave a negligible binding signal with Cys-MR-Fc, in accordance with the weak inhibitory activity of the monosaccharide GalNAc(6S) relative to GalNAc(4S).

Cys-MR Recognition of Sulfated Blood Group Chains.
We evaluated Cys-MR binding to the NGLs and biotinylated forms of sulfated oligosaccharides of the blood group series, based on the type 1 and type 2 backbone sequences with galactose joined by β1-3 and β1-4 linkage to N-acetylglucosamine: Galβ1-3GlcNAc and Galβ1-4GlcNAc, respectively ( Fig. 2A, Fig. C, and Fig. D). Clearly, there is recognition of sulfated forms of both types of blood group chains when there is sulfate at position 3 of the terminal galactose. Moreover, there is binding to the corresponding sulfated Le^a and Le^x sequences, which contain fucose joined by 1-4 and 1-3 linkage to the N-acetylglucosamine of the type 1 and type 2 backbones, respectively. Overall, there is a preference for the type 2 analogues over the type 1, and for the nonfucosylated chains over the fucosylated forms. There was no detectable binding to the 6-sulfated-Le^x sequence, or to the 3S-Le^a and 3S-Le^x sequences.

Inhibition by Sulfated N-Acetylgalactosamine of the Cys-MR Binding to Cells in the Spleen.
Cys-MR-Fc showed strong staining of large cells with granular cytoplasm in the marginal zone of spleen from naive and immunized mice, as described previously by Martínez-Pomares et al. ( 13; Fig. 4 A). There were weaker punctate staining of groups of cells in the splenic white pulp within B cell areas ( Fig. 5 B), which were identified by their strong staining with anti-CD35. Staining by Cys-MR-Fc of cells in this region was greater in immunized mice. There was no staining of spleen sections from naive or immunized mice when similarly incubated with the recombinant human Fc dimer (results not shown).

View larger version (1300K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Immunofluorescence micrographs of sections of naive mouse spleen showing inhibition of Cys-MR binding in the presence of sulfated monosaccharides. Sections were overlaid with Cys-MR-Fc, 10 µg/ml, in the presence of PBS (A), 4-sulfated N-acetylgalactosamine (B), 6-sulfated N-acetylgalactosamine (C), or N-acetylgalactosamine (D), 5 mg/ml, and binding was detected with fluorescein-labeled donkey anti–human IgG as described in Materials and Methods. A'–D' are the corresponding phase–contrast micrographs. Similar results were obtained using spleen sections from immunized mice (not shown). Original magnifications: x650.

View larger version (108K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Confocal images of a dual immunolabeling experiment showing the similar distribution and limited colocalization of the binding of Cys-MR and an antibody to the core regions of CS chains within the B cell areas of the splenic white pulp. Before immunolabeling, spleen sections from immunized mice were treated with chondroitinase ABC as described in Materials and Methods. The sections were thereafter overlaid with a rabbit antiserum, directed at the core region of cleaved CS chains, and Cys-MR-Fc, followed by rhodamine-labeled anti–rabbit IgG (anti-CS binding, A) and fluorescein-labeled anti–human IgG (Cys-MR binding, B). The two staining patterns are shown superimposed in C. The area within the white square in C is shown enlarged in D. Original magnifications: (A–C) x2,000; (D) x10,000.

Distribution of CS Immunostaining and Cys-MR Binding in the White Pulp of Spleen, and Binding of Cys-MR to Proteoglycans Secreted by Monocytes.
As cells of the immune system are known to produce cell-associated and secreted forms of CS proteoglycans, the amounts and proportions of which may change after various stimuli 34 35 36, we compared the immunostaining pattern for CS chains with that of Cys-MR staining. In both naive and immunized mice, there was similar distribution of the staining for CS chains and Cys-MR binding within the B cell area of the white pulp ( Fig. 5A and Fig. B), but they differed in the marginal zone (not shown). There was no fluorescein staining observed after incubation of sections with the recombinant human Fc dimer (not shown); in addition, there was no rhodamine staining observed in the absence of the antibody to CS (not shown). Although similar, the two staining patterns for Cys-MR-Fc and anti-CS in the B cell area overlapped only in limited areas (yellow areas in Fig. 5C and Fig. D). Moreover, there was no perceptible difference in the Cys-MR staining in these areas before and after treatment with chondroitinase ABC (results not shown). Thus, although substantial amounts of CS chains are present, it is likely that the Cys-MR binding forms are relatively minor components.

To directly evaluate the ability of Cys-MR to interact with chondroitin 4-sulfate proteoglycans produced by cells of the hematopoietic and immune systems 34 35 36 37, we examined its binding to a preparation of proteoglycans secreted by a monocytic cell line and known to be rich in serglycin. There was specific binding to the proteoglycan immobilized on microwells ( Fig. 6), and treatment with chondroitinase ABC, but not heparinase, abolished the binding. We conclude that Cys-MR interacts with these soluble proteoglycans via CS chains.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Binding of Cys-MR to serglycin and abolition of binding after treatment of the proteoglycan with chondroitinase ABC but not heparinase. Serglycin was coated onto microwells, 1.5 µg added/well, and incubated with chondroitinase ABC (50 mU), the heat-denatured chondroitinase ABC, heparinase I (2.5 U), or PBS. The binding of Cys-MR-Fc, 0.5 µg/well, was assayed as described in Materials and Methods.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The mechanism whereby the Cys-MR can accommodate the three classes of oligosaccharide ligand is elucidated by the structural study of the Cys-MR complexed with 4-sulfated N-acetylgalactosamine 38. The crystal structure shows that the sulfate group is involved in no less than six hydrogen bonds with the protein; the N-acetyl group has no contact, however, with the binding site of Cys-MR. This explains our finding that a sulfated galactose can be bound as well as a sulfated N-acetylgalactosamine. A structural explanation is also provided for the weak inhibitory activity we have observed in the present investigation for the 6-sulfated N-acetylgalactosamine (IC₅₀ 1.3 mM) compared with that for the 4-sulfated form (IC₅₀ 0.18 mM). Molecular modeling by Liu et al. 38 based on the crystal structure shows that for optimum binding, the sulfate group must be at C-3 or C-4 of the terminal monosaccharide. When GalNAc is sulfated at C-6, it does not make favorable van der Waals contacts with Trp117 of the Cys-MR binding site. The modeling studies also show that the addition of a monosaccharide at C-3 of the 4-sulfated N-acetylgalactosamine would hinder the binding of the sulfate. This accounts for our observation that the hexasaccharide CS-A6 with a terminal uronic acid linked to C-3 of N-acetylgalactosamine was not bound by Cys-MR. We can now also explain the preferential binding of Cys-MR to the 3Su-Le^x over the 3Su-Le^a analogue. The modeling, based on the crystal structure, shows that the sulfated galactose and the fucose of Le^x can be stacked readily against Trp117, whereas in the Le^a analogue, there is steric hindrance between the N-acetyl group and Asn102 in the Cys-MR binding site.

In contrast to Cys-MR, the cysteine-rich domain of DEC-205, another member of the MR family, did not give a binding signal with lutropin in the present investigation. The structural basis for this finding is apparent from the x-ray crystallographic study of the Cys-MR–ligand complex by Liu et al. 38 which defines the amino acid residues used to bind the sulfated carbohydrates. The amino acids in the analogous region are lacking in the Cys-DEC sequence.

A new finding in the present investigation is that the binding described for Cys-MR with cells both in the marginal zones and in the B cell areas of the naive and the immune spleen 13 is mediated by the binding site for sulfated carbohydrates. We further provide evidence that Cys-MR binds secreted proteoglycans of cells of the immune system, the major component of which is serglycin 36. Binding to serglycin is via the CS chains, which are known to be predominantly of chondroitin 4-sulfate type 37.

Serglycin accumulates in intracellular vesicles of cells of the immune system, and is secreted by the activated cells 36. The amounts of such proteoglycans that are retained in cells, or are secreted, differ after various stimuli 39. Thus, in lymphoid tissues in vivo, the serglycin has the potential to form a complex with Cys-MR and modulate the availability of the saccharide-binding site of both the transmembrane form and the secreted form of MR. It has been shown recently 17 that serglycin in the serum is rapidly taken up by hepatic endothelial cells (also referred to as sinusoidal scavenger endothelial cells), and that a hyaluronan receptor plays a major role in the uptake. We propose that interactions of Cys-MR with serglycin in lymphatic tissue fluid, or with serglycin in homogenates of hepatic cells, may underlie the reported lack of demonstrable binding activity of certain tissue isolates of MR toward sulfated oligosaccharides 8.

Chondroitin 4-sulfate chains occur also on cell membrane–associated proteoglycans of lymphocytes, monocytes, macrophages, and other cells of the hematopoietic system 34. Changes in their carbohydrate chains have been documented during cellular activation 36. Notable among chondroitin 4-sulfate–containing proteoglycans at the cell surface are several variants of the signal-transducing molecule CD44 40. It will be interesting to investigate if such glycosylation variants of CD44 are among counterreceptors for the Cys-MR.

An intriguing candidate counterreceptor for Cys-MR is the CS-modified form of the class II invariant chain (Ii) referred to as Ii-CS. While Ii-CS amounts to only a small proportion (2–5%) of Ii that associates with newly synthesized class II, it appears nevertheless to have an important role in antigen presentation functions 41. Evidence has been presented that expression of Ii-CS in antigen-presenting cells enhances antigen presentation and thereby enhances the triggering of primary antigen responses by T cells 41. Knowing that an abundant amount of MR is codistributed with class II molecules in the specialized antigen-loading MHC II compartment 42, there is the potential for Ii-CS interactions with Cys-MR, and we raise the possibility that such interactions may occur, and thus facilitate, the class II–Ii dissociation, simultaneously facilitating class II–peptide antigen binding.

Chondroitin sulfate chains occur, par excellence, on proteoglycans in extracellular matrices 34 43. The presence and the accessibility of Cys-MR ligands in extracellular matrices deserve investigation. Here may lie determinants of the trafficking of macrophages and their localization in pathological tissues, and also clues to the interactions of MR-bearing kidney mesangial, tracheal smooth muscle, and retinal pigment cells.

Carbohydrate sequences of the blood group family occur widely at the surface of cells. They occur on glycoproteins and glycolipids, and constitute a class of differentiation antigen, a reflection of the pronounced differentiation-associated changes they undergo in the branching patterns of their backbone regions and in the capping residues that they bear in their peripheral regions 44. The sialyl-Le^x and -Le^a sequences occur variously as markers of granulocytes, monocytes, or subsets of lymphocytes. The sulfo-Le^x and -Le^a sequences are differentially expressed on various normal and malignant epithelial cells 45, and they may constitute ligands that mediate interactions of cancer cells with the selectins. In its ability to bind to the sulfo-Le^a and -Le^x sequences, Cys-MR overlaps with the selectins 46, but it differs in its preference for the nonfucosylated analogues. The distribution of sulfated blood group chains on cells of the immune system needs to be determined. It will be interesting to investigate whether they occur on the acidic glycoforms of glycoproteins, such as sialoadhesin or CD45 of lymphoid tissue and the spleen, that are bound by Cys-MR 47 48.

So far, we have considered endogenous ligands for Cys-MR. The repertoire of sequences recognized by this domain raises the possibility that certain acidic microbial polysaccharides may be bound. Thus, it will be interesting to determine whether, in an analogous way to the mannose-binding C-type lectin domains on MR, the Cys-MR contributes to trapping of ligand-positive microbial pathogens, and to the delivery of ligand-positive antigens for processing and presentation.