©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Unique CD45 Glycoform Recognized by the Serum Mannan-binding Protein in Immature Thymocytes (*)

(Received for publication, November 7, 1995)

Kazuhide Uemura Yasunori Yokota Yasunori Kozutsumi Toshisuke Kawasaki (§)

From the Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The serum-mannan binding protein (S-MBP) is a calcium-dependent C-type lectin specific for mannose and N-acetylglucosamine. S-MBP is known as a host defense factor involved in innate immunity, where the target ligands for S-MBP should be on the surface of exogenous microorganisms. In this study, we tried to find endogenous ligands for this endogenous lectin. Among the cells tested, only the lymphocytes from thymus of BALB/c mice expressed ligands for S-MBP on their surface, those from bone marrow, spleen, mesenteric lymph nodes and peripheral blood all being negative. Interestingly, among the thymocytes, only the immature thymocytes with the CD4CD8CD3 phenotype expressed ligands for S-MBP, and ligands for S-MBP decreased on their maturation. A major cell surface glycoprotein bearing S-MBP ligands was isolated and identified as CD45RO, which is a transmembrane protein with tyrosine phosphatase activity. Deglycosylation experiments with N-glycanase and endoglycosidase H indicated that the S-MBP ligands on thymic CD45 are high mannose type or hybrid type N-linked oligosaccharides. This unique presentation of S-MBP ligands on this special CD45 isoform suggested the possibility that the oligosaccharide portion of CD45 on immature thymocytes is associated with the maturation, development or selection events of thymocytes.


INTRODUCTION

The serum mannan-binding protein (S-MBP) (^1)has been isolated from various mammalian sera and characterized as a calcium-dependent C-type lectin, which recognizes mannose and N-acetylglucosamine(1, 2, 3, 4) . S-MBP is synthesized by the liver as a mixture of oligomers consisting of 9-15 identical subunits of about 31 kDa(5) . Each subunit has a collagen-like domain at its NH(2) terminus and a carbohydrate-recognition domain at its COOH terminus. We previously demonstrated that S-MBP activates the complement pathway, which is antibody- and C1q-independent(6, 7) , and is called the ``lectin pathway''(8) . S-MBP exhibits complement-dependent bactericidal activity (9) and also acts directly as an opsonin(10) . A variety of microorganisms have manno-oligosaccharide structures on their surface, while mammalian cells generally do not. For this reason, S-MBP specifically recognizes exogenous microorganisms. However, we also demonstrated that S-MBP exhibits complement-dependent cytotoxic activity toward mammalian cells that express high mannose type oligosaccharides(11) , suggesting the possibility that S-MBP eliminates abnormal mammalian cells that express high mannose type oligosaccharides on their surface.

The present study was undertaken to determine whether or not mammalian cells have cell surface oligosaccharides recognized by S-MBP. We found that only the lymphocytes from the thymus of BALB/c mice expressed S-MBP ligands. Then, the major glycoprotein which carried S-MBP ligands was identified as CD45 (T-200, leukocyte common antigen). This thymic CD45 carried characteristic oligosaccharides that were not carried by the CD45 molecules from other tissues. The biological significance of this finding is discussed with regard to the function of this transmembrane protein with tyrosine phosphatase activity.


EXPERIMENTAL PROCEDURES

Preparation of FITC-labeled Rabbit S-MBP, Sepharose 4B Rabbit S-MBP, and Anti-S-MBP Polyclonal Antibodies

S-MBP was purified from normal rabbits (Japan Bio-supply) using an affinity column of Sepharose 4B-mannan, as described previously(1) . For fluorescein isothiocyanate (FITC) labeling, 1 mg of rabbit S-MBP was incubated with 100 µg of FITC (Sigma) in 50 mM borate buffer (pH 9.2) containing 200 mM NaCl, 20 mM CaCl(2), and 100 mM mannose for 18 h at 4 °C. For the preparation of Sepharose 4B-rabbit S-MBP, rabbit S-MBP was coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) according to the manufacturer's instructions. Guinea pig anti-rabbit S-MBP IgG was prepared as described previously(1) .

Flow-cytometric Analysis

Lymphocytes were prepared from the thigh bone marrow, thymus, spleen, mesenteric lymph nodes, and peripheral blood of 5-week-old normal BALB/c mice (Nippon SLC). Single cell suspensions prepared from these lymphoid tissues and peripheral blood were centrifuged to collect the cells. The cells were washed with 20 mM Tris-buffered saline (pH 7.5) containing 2 mM EDTA to remove endogenous S-MBP. Debris and erythrocytes were removed by density gradient centrifugation using M-SMF (Nippon Kotai), a lymphocyte separation solution. Approximately 1 times 10^6 cells were stained with 7 µg of FITC-labeled rabbit S-MBP in 50 µl of 20 mM Tris-buffered saline (pH 7.5) containing 10 mM CaCl(2), 0.1% sodium azide and 2% heat-inactivated fetal calf serum (HyClone). For two-color analysis, the following mAbs were used: Phycoerythrin(PE)-labeled anti-mouse CD4 (Coulter Immunology), FITC-labeled anti-mouse CD8 (Life Technologies, Inc.), PE-labeled anti-CD8 (Coulter Immunology), and PE-labeled anti-CD3 (145-2C11; Pharmingen). After washing, the cells were analyzed with a FACScan flow cytometer with LYSIS II software (Becton-Dickinson). Dead cells were excluded by staining with propidium iodide (Sigma) and selective gating, and lymphocyte populations were gated by a combination of forward light scatter and 90° side scatter.

Cell Surface Labeling and Preparation of Cell Lysates

Cells (1 times 10^8/ml) were incubated in 20 mM phosphate-buffered saline (pH 8.0) containing 3.2 mg/ml sulfo-NHS-biotin (Pierce) at 4 °C for 30 min with occasional swirling. The reaction was stopped by the addition of phosphate-buffered saline containing 20 mM glycine (pH 7.4). After washing, the cells were lysed at 1 times 10^8/ml for 30 min on ice in 50 mM Hepes-buffered saline (pH 7.4) containing 1% Nonidet P-40 (Nonidet P-40) and protease inhibitors (100 µg/ml phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 100 µg/ml benzamidine). Nuclei and insoluble substances were removed by centrifugation at 400 times g for 10 min and then at 100,000 times g for 30 min, respectively, and the supernatant was saved as the cell lysate.

Affinity Chromatography and SDS-PAGE

CaCl(2) was added to the cell lysate to 20 mM, and then the mixture was applied to an affinity column of Sepharose 4B-rabbit S-MBP. After washing the column with 50 mM Hepes-buffered saline (pH 7.4) containing 20 mM CaCl(2) and 0.1% Nonidet P-40, the substances bound to the column were eluted with 50 mM Hepes-buffered saline (pH 7.4) containing 2 mM EDTA and 0.1% Nonidet P-40 or 50 mM Hepes-buffered saline (pH 7.4) containing 20 mM CaCl(2), 100 mM mannose and 0.1% Nonidet P-40. The eluted proteins were resolved by SDS-PAGE on a 5-20% gradient gel (Atto), and then transferred to a nitrocellulose membrane. The membrane was treated with 20 mM Tris-buffered saline (pH 7.5) containing 3% bovine serum albumin. After incubation of the membrane with horseradish peroxidase-conjugated streptavidin (1:1000 dilution, Vector) for 60 min at room temperature, surface-biotinylated proteins were visualized with 4-chloro-1-naphthol (Wako).

Immunoprecipitation of CD45

The cell lysates and eluate from the Sepharose 4B-rabbit S-MBP column were incubated with anti-CD45 mAb (30F11.1, Pharmingen) in 50 mM Hepes-buffered saline (pH 7.4) containing 5 mM EDTA, 0.5% Nonidet P-40, and 0.25% gelatin for 2 h at 4 °C. Protein G-Sepharose beads (Sigma) were added to the mixture, and then the incubation was continued for another 18 h at 4 °C. The resultant immune complexes were washed, resolved by SDS-PAGE on a 7.5% gel(12) , and then transferred to a nitrocellulose membrane. Biotinylated proteins were detected on the membrane as described above.

S-MBP Blotting of CD45 and Its Deglycosylated Form on Nitrocellulose Membranes

Cell lysates were applied to an affinity column of biotinylated anti-CD45 mAb (30F11.1, Pharmingen) coupled to immobilized avidin (Pierce). After washing the column with 20 mM phosphate buffer (pH 6.4) containing 500 mM NaCl and 2 mM CHAPS, the substances bound to the column were eluted with 50 mM glycine-HCl (pH 2.5) containing 150 mM NaCl and 2 mM CHAPS. The eluate was immediately adjusted to pH 7.5 with Tris base. For deglycosylation experiments, affinity-purified CD45 was denatured by boiling in the presence of 0.1% SDS and 50 mM beta-mercaptoethanol, and then treated with N-glycanase (Genzyme) or endoglycosidase H (Seikagaku Kogyo) according to the manufacturer's instructions. Deglycosylated and control samples were resolved by SDS-PAGE on a 7.5% gel (12) and then transferred to a nitrocellulose membrane. After blocking the membrane with the buffer containing 3% bovine serum albumin as described above, the membrane was first incubated with 100 µg/ml rabbit S-MBP for 2 h, second with 20 µg/ml guinea pig anti-rabbit S-MBP IgG for 2 h, and finally with 20 µg/ml horseradish peroxidase-conjugated anti-guinea pig IgG for 90 min. Each incubation was performed at room temperature in 20 mM Tris-buffered saline (pH 7.5) containing 20 mM CaCl(2), 1% bovine serum albumin and 0.1% sodium azide, followed by three washes with 20 mM Tris-buffered saline (pH 7.5) containing 20 mM CaCl(2) and 0.05% Tween 20, except for the last incubation, for which Tween 20 was omitted. The protein bands were visualized with a chromogenic substrate kit (Konica).


RESULTS

Expression of S-MBP Ligands on Mouse Lymphocytes

The expression of S-MBP ligands on the surface of lymphocytes was investigated with FITC-labeled rabbit S-MBP by flow cytometry. Lymphocytes obtained from bone marrow, thymus, spleen, mesenteric lymph nodes, and peripheral blood of 5-week-old male BALB/c mice were incubated with FITC-labeled rabbit S-MBP in the presence of Ca and then analyzed with a flow cytometer. As shown in Fig. 1A, thymocytes were stained heavily with FITC-labeled rabbit S-MBP, the level of which was about 100-fold higher than the control value, whereas other lymphocytes did not show significant staining. These results indicated that thymocytes can be differentiated into two groups with regard to reactivity to S-MBP. The binding of rabbit S-MBP to the surface of thymocytes was sugar-specific and calcium-dependent, since when thymocytes were stained in a buffer containing 2 mM EDTA but no Ca, or 100 mM mannose and 10 mM Ca, the strong staining with FITC-labeled rabbit S-MBP disappeared (Fig. 1B). Interestingly, a minor population of thymocytes was stained only weakly (Fig. 1, panels A-b and B-a), suggesting the possibility that the level of S-MBP ligands on the cell surface varies among the subpopulations of thymocytes. In order to examine this, double staining of thymocytes with FITC-labeled rabbit S-MBP and PE-labeled mAbs specific for CD4, CD8, or CD3 was carried out. The thymocytes obtained from a 5-week-old male BALB/c mouse consisted of double-positive (CD4CD8) cells, single-positive (CD4CD8 or CD4CD8) cells, and double-negative (CD4CD8) cells, in the proportions of 83%, 15%, and 2%, respectively (Fig. 2, panel a). As shown in Fig. 2(panels b and c), approximately 99% and 96% of the cells expressing high levels of S-MBP ligands were CD4- and CD8-positive, respectively. Furthermore, CD4- or CD8-negative cells expressed much lower levels of S-MBP ligands than CD4- or CD8-positive cells, indicating that the cells lacking either CD4 or CD8 (CD4CD8, CD4CD8, CD4CD8 cells) did not express high levels of MBP ligands. On the other hand, a reciprocal correlation was found between the level of S-MBP ligands and that of CD3: all the cells expressing low levels of CD3 expressed high levels of S-MBP ligands, while most of the cells expressing high levels of CD3 expressed low levels of S-MBP ligands (Fig. 2, panel d). These results of flow cytometry indicated that immature thymocytes with the CD4CD8CD3 phenotype express high levels of S-MBP ligands on their surface, and that the S-MBP ligands disappear on maturation of the thymocytes. It is well known that immature CD4CD8CD3 thmocytes are localized in the cortex. This was confirmed by histochemical staining of the thymus with FITC-labeled rabbit S-MBP, bright staining being seen in the cortex but not in the medulla (data not shown).


Figure 1: Expression of S-MBP ligands on the surface of murine lymphocytes. A, cells obtained from bone marrow (panel a), thymus (panel b), spleen (panel c), lymph nodes (panel d), and peripheral blood (panel e) of a 5-week-old BALB/c mouse were stained with FITC-labeled rabbit S-MBP in the presence of 10 mM CaCl(2), and then analyzed by flow cytometry (black/solid area). For negative controls, autofluorescence of the cells was measured in each experiment (white/blank area). Lymphocyte populations were gated by forward light scatter and 90° side scatter in each measurement. B, Ca dependence and sugar specificity of the binding of S-MBP to thymocytes. Thymocytes were stained with FITC-conjugated rabbit S-MBP in the presence of 10 mM CaCl(2) (black/solid area in panel a), 2 mM EDTA + no CaCl(2) (black/solid area in panel b), or 10 mM CaCl(2) + 100 mM mannose (black/solid area in panel c). Autofluorescence of the cells was shown in each panel with white/blank area.




Figure 2: Expression of S-MBP ligands on thymocyte subpopulations. Thymocytes obtained from a 5-week-old BALB/c mouse were analyzed by two-color flow cytometry, using FITC-labeled anti-CD8 and PE-labeled anti-CD4 (panel a), FITC-labeled rabbit S-MBP and PE-labeled anti-CD4 (panel b), FITC-labeled rabbit S-MBP and PE-labeled anti-CD8 (panel c), or FITC-labeled rabbit S-MBP and PE-labeled anti-CD3 (panel d).



Isolation of Cell Surface Glycoproteins Carrying S-MBP Ligands from Thymocytes

In order to identify the components on thymocytes that bear S-MBP ligands, thymocytes were surface-labeled with sulfo-NHS-biotin, and then the labeled cells were lysed with Nonidet P-40 and the lysate was applied to an affinity column of Sepharose 4B-rabbit S-MBP in the presence of 20 mM Ca. Proteins bound to the column were eluted with 2 mM EDTA or 100 mM mannose, resolved by SDS-PAGE, and then visualized with horseradish peroxidase-conjugated streptavidin. To our surprise, only a single major protein was detected in the eluate from the column, there being a large number of cell surface-labeled proteins in the lysate (Fig. 3A, lanes 2 and 3). The apparent molecular weight of this cell surface protein was estimated to be 175 kDa by SDS-PAGE on a 7.5% gel (data not shown). This indicates that the 175-kDa protein is a glycoprotein carrying oligosaccharide ligands for S-MBP.


Figure 3: A, isolation of cell surface glycoproteins carrying S-MBP ligands from thymocytes. Thymocytes were surface-labeled with sulfo-NHS-biotin and then lysed in 1% Nonidet P-40-containing buffer, and then the lysate was applied to a Sepharose 4B-rabbit serum S-MBP column as described under ``Experimental Procedures.'' Aliquots of the lysate (lane 1), the eluate with EDTA-containing buffer (lane 2), and the eluate with mannose-containing buffer (lane 3) were resolved by SDS-PAGE on a 5-20% gradient gel, and the labeled proteins were transferred to a nitrocellulose membrane and detected with horseradish peroxidase-conjugated streptavidin. The positions of molecular size markers are shown in the left margin. B, identification of the 175-kDa protein on the surface of thymocytes as CD45 by immunoprecipitation. The lysate of surface-labeled thymocytes and the eluate with EDTA-containing buffer from the Sepharose 4B-rabbit serum S-MBP column were immunoprecipitated with anti-mouse CD45 (lanes 2 and 4, respectively), washed, and then resolved by SDS-PAGE, and the labeled proteins were detected with horseradish peroxidase-conjugated streptavidin. As negative controls, the lysate and the eluate with EDTA-containing buffer from the Sepharose 4B-rabbit serum S-MBP column were immunoprecipitated with normal rat IgG (lanes 1 and 3, respectively).



Identification of the 175-kDa Protein on the Surface of Thymocytes as an Isoform of CD45

Mouse CD45, which is also called the leukocyte common antigen or T200, comprises a family of transmembrane glycoproteins abundantly expressed on the surface of nucleated hematopoietic cells(13) . CD45 exists as isoforms ranging from 175 to 235 kDa, and the lowest molecular weight isoform is expressed mainly on thymocytes(14) . In order to study whether the 175-kDa glycoprotein is a CD45 isoform or not, immunoprecipitation of the 175-kDa glycoprotein with 30F11.1, an mAb recognizing an epitope common to all mouse CD45 isoforms, was carried out. As shown in Fig. 3B, an isoform of CD45 was immunoprecipitated with 30F11.1 not only from the lysate of thymocytes but also from the eluate from the S-MBP column, while no labeled protein was immunoprecipitated with normal rat IgG. Furthermore, the 175-kDa glycoprotein isolated from the S-MBP column was shown to be reactive with mAb 30F11.1 upon Western blot analysis (data not shown). These data indicated that the 175-kDa glycoprotein that carries S-MBP ligands is the lowest molecular weight isoform of CD45, which is referred to as CD45RO.

Deglycosylation Analysis of Thymic CD45

S-MBP binds to various types of glycoproteins with terminal mannose and N-acetylglucosamine residues. In order to characterize the S-MBP ligands carried by thymic CD45, oligosaccharides were removed enzymatically from thymic CD45, and then the reactivity of the deglycosylated thymic CD45 toward rabbit S-MBP was examined. As shown in Fig. 4(lanes 1 and 3), the 175-kDa band contains S-MBP ligands and was stained well on S-MBP blotting. These ligands were removed completely by treatment with N-glycanase, which releases all kinds of N-linked oligosaccharides (Fig. 4, lane 2), and also with endoglycosidase H, which releases high mannose type and hybrid type oligosaccharides from glycoproteins (Fig. 4, lane 4). These results indicated that the S-MBP ligands carried by the thymic CD45 are high mannose or hybrid type oligosaccharides with terminal mannose and/or N-acetyl glucosamine residues. It should be noted that upon silver staining after N-glycanase and endoglycosidase H treatment, respectively, two major bands corresponding to apparent M(r) of 150 and 140 kDa (Fig. 4, lane 6), and one major band corresponding to an apparent M(r) of 160 kDa (Fig. 4, lane 8) were detected. These results were consistent with the view that approximately one-third or one-half of the oligosaccharide chains attached to the CD45 molecule are high mannose type or hybrid type oligosaccharides, the rest being complex type oligosaccharides. The second S-MBP reactive band exhibiting higher mobility on SDS-PAGE (Fig. 4, lanes 1 and 3, etc.) may be due to proteolytic degradation occurring during incubation with these enzymes.


Figure 4: Sensitivity of S-MBP ligands on thymic CD45 to N-glycanase and endoglycosidase H. CD45 was purified from the 1% Nonidet P-40 lysate of mouse thymocytes on an immunoaffinity column and then digested with N-glycanase (lanes 2 and 6) or endoglycosidase H (lanes 4 and 8). As negative controls, thymic CD45 was incubated without the enzyme under the same conditions as for N-glycanase (lanes 1 and 5) or endoglycosidase H (lanes 3 and 7) treatment. The reactivity of S-MBP toward undigested and digested thymic CD45 was examined by S-MBP blotting (lanes 1-4) as described under ``Experimental Procedures.'' Protein bands were visualized by silver staining (lanes 5-8).




DISCUSSION

The expression of S-MBP ligands on the surface of various lymphocytes from normal mice was examined. Immature thymocytes abundantly expressed S-MBP ligands, while lymphocytes from other lymphoid tissues and peripheral blood carried only trace amounts of S-MBP ligands. Moreover, S-MBP ligands on the surface of thymocytes decreased upon maturation of the thymocytes. It is known that cell surface carbohydrates change during thymocyte development. A plant lectin, peanut agglutinin (PNA), specific for galactose, was shown to bind to immature, but not mature thymocytes(15) . The cortex distribution of PNA ligands in the thymus resembles the results obtained for S-MBP ligands, but the nature of the proteins carrying PNA ligands has not been determined yet. The S-MBP ligands exposed on thymocytes should be different from PNA ligands, because S-MBP does not bind to galactose residues(1, 4) . However, it is possible that these two different types of ligand can be carried by the same polypeptide backbone.

The S-MBP ligands on thymocytes were carried specifically by a 175-kDa glycoprotein, which was identified as an isoform of CD45 (CD45RO) on immunoprecipitation and Western blot analysis. CD45 is a glycosylated transmembrane protein with tyrosine phosphatase activity and is required for T cell receptor-mediated signaling(15, 16) . CD45 is expressed as multiple isoforms (mass = 175-235 kDa), which are generated through alternative splicing of extracellular exons 4, 5, 6, and 7, and the profiles of the expression of CD45 isoforms are cell type- and differentiation stage-specific(13) . Correlation between thymocyte maturation and the expression of high molecular weight CD45 isoforms has been reported(14) . There are potential N-glycosylation sites and a cluster of O-glycosylation sites in the extracellular region of the high molecular weight CD45 isoform. The majority of the O-glycosylation sites occur on protein sequences encoded by variable exons 4, 5, and 6, which are not present in CD45RO. In contrast, many N-glycosylation sites are present in CD45RO (17) . This localization of potential N-glycosylation sites has led to the argument that oligosaccharides at these sites may be associated with some specific functions of the CD45RO isoform. For example, N-linked oligosaccharides containing alpha-2,6-linked sialic acids carried by CD45RO expressed on mature T cells have been shown to be ligands for CD22, a sialic acid-binding lectin expressed on B cells(18) . The interaction between CD22 on B cells and CD45 on T cells modulates T cell receptor-mediated signal transduction(19) . CD45 on immature thymocytes also plays an important role in maturation and clonal selection events(20, 21) . The unique expression of S-MBP ligands on thymic CD45RO suggests that the oligosaccharide ligands for S-MBP may be associated with the regulation of signal transduction or cell adhesion, which is required for thymocyte maturation and selection events. In fact, our preliminary experiments showed that S-MBP had some inhibitory effect on the anti-CD3 induced-apoptosis of thymocytes, suggesting that the binding of S-MBP to CD45 may regulate T cell receptor-mediated signal transduction. (^2)

The results of the deglycosylation experiment with N-glycanase indicate that the S-MBP ligands on thymic CD45 are N-linked oligosaccharides. S-MBP has been shown to preferentially bind to N-linked bi-antennary complex type oligosaccharides containing two terminal GlcNAc residues as well as to high mannose type oligosaccharides(22) . That the S-MBP ligands on thymic CD45 were sensitive to endoglycosidase H, which cleaves high mannose type and hybrid type oligosaccharides but not complex type oligosaccharides(23) , ruled out the possibility that N-linked bi-antennary complex type oligosaccharides are the major components of the S-MBP ligands on thymic CD45. The expression of unsialylated complex type oligosaccharides on thymic CD45 has been demonstrated by specific labeling with alpha-2,6-sialyltransferase(24) . This is consistent with the results of the deglycosylation experiments in this study. More than one-half of the oligosaccharide chains appear to be complex type oligosaccharides, although they are not involved in the binding of S-MBP. It is of interest that most of the oligosaccharide chains of CD45 from human peripheral lymphocytes are complex type chains(25) . A study is in progress to determine the structures of the oligosaccharide ligands for S-MBP on thymic CD45.

It is not yet known whether or not S-MBP binds to its ligands on thymic CD45 in vivo. Our preliminary data suggest that S-MBP mRNA is not expressed in the thymus (data not shown). In addition, a blood-thymus barrier exists in the thymus cortex(26) , which inhibits the entrance of macromolecules into the thymus cortex from the circulation. However, the transcapsular route, a by-pass of the blood-thymus barrier through which serum proteins reach the thymus, has been reported(27) . In addition, it is possible that some other endogenous lectin specific for mannose is present in the thymus and is associated with the regulation of the CD45RO molecules. In this regard, it should be noted that DEC-205, a membrane-bound protein that is homologous to the macrophage mannose receptor, is expressed on thymic dendrite cells(28) .


FOOTNOTES

*
This work was supported in part by the Special Coordination Funds of the Japanese Science and Technology Agency for Promoting Science and Technology, and also by a grant-in-aid for scientific research on priority areas from the Japanese Ministry of Education, Science and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-75-753-4572; Fax: 81-75-761-8949.

(^1)
The abbreviations used are: S-MBP; serum-mannan binding protein; MBP, mannan-binding protein; mAb, monoclonal antibody; CD, cluster of differentiation; PNA, peanut agglutinin; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; PE, phycoerythrin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

(^2)
K. Uemura, Y. Yokota, Y. Kozutsumi, and T. Kawasaki, unpublished observation.


ACKNOWLEDGEMENTS

We thank Hiroko Yamaguchi for secretarial assistance.


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