(Received for publication, November 7, 1995)
From the
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
CD4CD8
CD3
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.
The serum mannan-binding protein (S-MBP) ()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
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.
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, 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
(black/solid
area in panel a), 2 mM EDTA + no CaCl
(black/solid area in panel b), or 10 mM CaCl
+ 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).
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).
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).
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
-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. (
)
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
-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) .