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Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10% of all cell
surface protein (1). The tandem intracellular phosphatase
domains of CD45 are responsible for the regulation of the
src family kinase members Lck and Fyn through
dephosphorylation of the negative regulatory tyrosine residue found in
the carboxyl terminus of the kinase. This regulation of Lck and Fyn has
been demonstrated to be important for both T cell activation and
thymocyte development using both CD45-deficient cell lines as well as
gene-targeted mice (2, 3). The external domain of CD45 is
large, and heterogeneous with respect to both size and carbohydrate
content. There are three alternative splice exons, 4–6, in the
extracellular region whose usage is variable depending on the
developmental stage of the cell (4). Within the
alternatively spliced exons are numerous sites for O-linked
carbohydrate attachment. As well, there is an abundance of
N-linked carbohydrate sites mostly found outside of the
alternatively spliced exons (5). A biological role for the
external domain remains elusive; however, addition of mAbs specific for
the extracellular domain of CD45 in fetal thymic organ culture disrupts
normal thymic selection events (6). Therefore, it is
possible that the external domain of CD45 is involved in the process of
thymocyte development. Carbohydrate additions to proteins are evolutionarily conserved and extremely important for a number of different processes, such as protein folding, transport, and ligand binding. The processing of N-linked carbohydrate is quite complex, tightly controlled, and ultimately decides the fate of that particular glycoprotein, whether it is appropriate transport and function or degradation (7, 8). The machinery involved in carbohydrate processing lies within the endoplasmic reticulum (ER)3 and Golgi apparatus. The ER machinery is primarily responsible for ensuring proper folding through the actions of enzymes such as glucosidase I and II (9, 10, 11); calnexin and calreticulin (12, 13); and UDP-glucose glucosyltransferase (14). The carbohydrate-modifying enzymes within the Golgi stacks ultimately shape the final structure of the carbohydrate through various cleavages and additions by enzymes such as the mannosidases (15), and various glycosyltransferases.
Numerous examples exist in which carbohydrate is extremely important for biological function of a protein, as in the case of the selectins (16), the dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) and ICAM-2 (17), or ICAM-3 (18) interaction, and the recognition of bacterial Ags by the complement system (19). For the most part, these examples feature a lectin binding to its ligand via the carbohydrate displayed by the ligand. Therefore, to achieve proper biological outcomes, the carbohydrate processing by the cell expressing the ligand is extremely important. We have previously demonstrated that CD45 and glucosidase II (GII) associate by way of a mannose-dependent lectin interaction (20). In this study, we show that the carbohydrate structure found on CD45 changes as immature thymocytes mature into T cells. This change in carbohydrate structure probably involves an intrinsic change in the carbohydrate-processing machinery of the cell. An example of such a change in CD45 carbohydrate during development is the induction of the CT1 epitope after stimulation through the pre-TCR (21). The presence of CD45 that can be bound by mannose-binding protein (MBP) or related lectins on immature cells could be involved in the process of T cell maturation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse T lymphoma cells SAKRTLS.12.1 (SAKR), BW5147 (BW), and
their CD45-deficient derivatives (SAKR/T200- and
BW/T200-) were maintained as described
previously (22). Thymocytes and splenocytes were isolated
by gentle teasing of the thymus or spleen from C57BL/6 mice and used
immediately after isolation. Cloned CTL AB.1 was maintained as
previously described (23). mAb I3/2.3 that recognizes a
pan-specific determinant within the CD45 extracellular domain was
kindly provided by Dr. I. Trowbridge (Salk Institute, La Jolla, CA).
Fluorochrome-coupled anti-CD4 and anti-CD8 mAbs RM4-4 and
53-6.7, respectively, were purchased from BD PharMingen (Mississauga,
Ontario, Canada). The hybridoma secreting the class I MHC-specific mAb
M1/42.3.9.8 was purchased from American Type Culture Collection
(Manassas, VA). Rabbit antisera H2 and J37, specific to GII
and the
intracellular region of CD45, respectively, were previously described
(20, 24). Rabbit antiserum L177 was generated to a peptide
fragment corresponding to the alternatively spliced Box A1 region of
GII
coupled to keyhole limpet hemocyanin (25).
Cell surface biotinylation, cell lysis, immunoprecipitation, MBP pull-down assays, reconstitution assay, and endoglycosidase treatment
Cell surface biotinylation was performed with 50 µl of 10 mM sulfo-normal human serum biotin (Pierce, Rockford, IL) per 5 x 107 cells/ml in PBS for 10 min at room temperature. The reaction was quenched by washing cells twice in PBS containing 5 mM glycine. All cells were lysed at a density of 5 x 107/ml in 0.5% Nonidet P-40 (Pierce) and TBS buffer, and incubated on ice for 20 min. Postnuclear supernatants were incubated for 1–2 h with I3/2-coupled Sepharose 4B at 4°C with rotation. Immunoprecipitates were washed three times with lysis buffer before the addition of reducing sample buffer and boiling. For MBP pull-down assays, cell surface biotinylated lysates were made to 1.25 M NaCl, 20 mM CaCl2 with or without 5 mM glucose or mannose before addition of immobilized MBP (Pierce), and then incubated for 4 h at 4°C with rotation. Pull-downs were washed three times with lysis buffer containing 1.25 M NaCl and 20 mM CaCl2. For elution of MBP-bound proteins, immobilized MBP was incubated with lysis buffer containing 1.25 M NaCl and 5 mM EDTA for 3 x 10 min. Eluted proteins were then either subjected to I3/2 immunoprecipitation or streptavidin pull-down for examining either bound CD45 or total bound cell surface proteins, respectively. Reconstitution assays were performed as previously described (20). Briefly, CD45 devoid of GII was incubated with a CD45-deficient, GII-containing lysate for 1 h at 4°C, followed by washing three times with lysis buffer. Immunoprecipitates were treated with endoglycosidase (Endo) H and F (Calbiochem, La Jolla, CA) in PBS containing 0.1% SDS, 1% 2-ME for 16 h at 33°C.
PAGE and immunoblotting
Proteins were resolved on 7.5% polyacrylamide gels and transferred to polyvinylidene difluoride-Immobilon (Millipore, Bedford, MA), as described previously (26). Western blot analysis was conducted with the indicated antiserum, followed by protein Ahrp, or with streptavidinhrp (Pierce), and visualized by ECL (PerkinElmer Life Sciences, Norwalk, CT).
Serum MBP purification
Rabbit serum MBP was purified as previously described (27). Briefly, whole rabbit serum proteins (Sigma, St. Louis, MO) were precipitated with polyethylene glycol 6000 and resuspended in TBS containing 1.25 M NaCl and 50 mM CaCl2 (MBP-binding buffer), and applied to an equilibrated 10-ml mannan-agarose (Sigma) column at 4°C. The column was washed with 20 vol of MBP-binding buffer before eluting bound proteins with TBS containing 1.25 M NaCl and 5 mM EDTA (MBP elution buffer). Fractions were collected and analyzed by SDS-PAGE and silver staining for presence of MBP. Fractions containing MBP were pooled, made to 50 mM CaCl2, and reapplied to a 2-ml mannan-agarose column. The column was washed with 20 vol of MBP-binding buffer before elution. Fractions were analyzed for MBP by silver staining and concentrated by centrifugal filtration. Concentrated MBP was biotinylated as per Pierce biotinylation kit (Pierce). Biotinylated MBP was detected by Western blotting using streptavidinhrp, as described above.
GII inhibitor treatment
AB.1 cells were treated with 2 mM N-methyl
deoxynorjirmycin (NMdNM; Oxford Glycosystems, Wakefield, MO) for
20 h. Cells were harvested, cell surface biotinylated, and lysed,
as described above. Cell lysates were examined for GII enzymatic
activity, as previously described (20), using the colorimetric
p-nitrophenyl -D glucopyranoside
(Sigma).
FACS analysis
A total of 1 x 106 cells was incubated with either 10 µg/ml biotinylated MBP, or 5 µg/ml fluorochrome-conjugated anti-CD4 or anti-CD8 for 20 min on ice in PBS containing 1 mM CaCl2. For MBP detection, the cells were incubated with streptavidinFITC for an additional 20 min on ice in PBS containing 1 mM CaCl2. All cells were then fixed in 1% paraformaldehyde before analysis.
Generation of Con A thymocyte blasts
Freshly isolated C57BL/6 thymocytes were incubated with 2 µg/ml Con A (Amersham Pharmacia Biotech, Baie d’Urfé, Quebec, Canada) and IL-2 for 3 days. The cultures were then split and allowed to proliferate for 4 days in the presence of IL-2. Thymocyte blasts were then harvested and either lysed or used for FACS analysis, as above.
Purification of splenic T cells
Freshly isolated C57BL/6 splenocytes were passed over a mouse T
cell immunocolumn (Cedarlane Laboratories, Hornsby, Ontario, Canada),
which purifies T cells based on negative selection. Splenic T cell
preparations were >80% T cells, as determined by FACS analysis for
TCR.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To gain additional insight into the possible biological role that
the association between CD45 and GII plays, we examined whether the
association occurred in all cell types or in a subset of cells. CD45
immunoprecipitates from lysates of cells of different developmental
stages were performed and analyzed by Western blot for the presence of
GII. GII was only coimmunoprecipitated with CD45 in lysates from BW5147
and thymocytes, while virtually no GII was detected in CD45
immunoprecipitates from a CTL clone, AB.1, or splenic T cells (Fig. 1
). A small amount of GII was seen in
CD45 immunoprecipitates from the purified splenic T cells, but only
after overexposure of the Western blot (data not shown). These data
suggest that the association between CD45 and GII is developmentally
regulated and only occurs in immature cells.
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Because the association between CD45 and GII is based on a lectin
interaction, requires the active site of GII, and can be inhibited
by mannose (20), we wished to examine the mannose content
of the carbohydrate on CD45 from various cell types using another
lectin. We chose to use MBP, as it is specific for mannose, and has
been previously shown to bind CD45 (28). Lysates from cell
surface-biotinylated SAKR, a T lymphoma, thymocytes, and AB.1 were
incubated with immobilized MBP in the presence of
Ca2+ to recover all proteins capable of binding
MBP. The MBP beads were washed, and bound proteins were eluted with 5
mM EDTA and then captured with anti-CD45-coated beads. The captured
CD45 was resolved by SDS-PAGE and detected by Western blotting. CD45
immunoprecipitates from each of the cell types were also performed.
Using streptavidin to specifically detect cell surface CD45 illustrated
that MBP can recognize cell surface CD45 in immature cells such as SAKR
and thymocytes, but not from mature cells such as AB.1 (Fig. 2A). Examining total MBP-bound
CD45 reveals that there are two different forms of CD45 recognized by
MBP, only one of which is readily cell surface biotinylated (Fig. 2A). These two different forms are likely to be different
glycoforms of CD45RO, as Western blotting for CD45RB showed only minor
amounts of CD45RB brought down by MBP, and the CD45RB has a higher
relative m.w. than did the uppermost band in the MBP-bound CD45
immunoblot (data not shown).
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B).
This MBP binding is Ca2+ dependent and sugar
specific, as the binding is inhibited by the inclusion of EDTA or the
addition of mannose (data not shown). Interestingly, CD45-deficient
variants of SAKR (Fig. 2C) and BW (data not shown) both bind
MBP, but at 50% the level seen in wild-type cells. Consistent with
these data is the observation that in MBP pull-downs of cell
surface-biotinylated lysates, we see that CD45 is the predominant
MBP-bound protein; however, it is certainly not the only protein (data
not shown), as previously reported (28). These data
suggest that cell surface CD45 from immature cells is the major
glycoprotein recognized by MBP, but there appear to be other minor cell
surface glycoproteins that can be recognized by MBP. Developmental differences in the amount of MBP binding and CD45-GII association
We have demonstrated that the ability of MBP to bind CD45 and the
amount of GII associated with CD45 correlates with the maturation state
of the cell. Next, we wanted to examine these changes more closely
using ex vivo thymocytes. Ex vivo thymocyte populations contain a
mixture of immature double-negative and double-positive cells, as well
as mature single-positive cells. By using three-color flow cytometric
analysis, one can separate the four different developmental populations
of bulk thymocytes and compare the level of MBP binding to each
population. Three-color analysis of bulk thymocytes demonstrated that
there is a 2- to 3-fold higher level of MBP binding on immature
double-positive thymocytes compared with either immature
double-negative or mature single-positive thymocytes (Fig. 3A). As an alternative
approach to compare developmental differences with respect to MBP
binding and CD45-GII association, Con A stimulation of thymocytes was
performed. Stimulation of thymocytes with Con A leads to an increase in
the percentage of single-positive thymocytes and a corresponding
decrease in the percentage of double-positive cells (Fig. 3B). Comparing the bulk thymocytes and Con A thymocyte
blasts for MBP binding in FACS analysis, there is approximately a
2-fold decrease in MBP binding by the Con A thymocyte blasts (Fig. 3C). Coordinate with the decrease in MBP binding, we see an
approximate 2-fold decrease in the amount of GII bound to CD45 in the
Con A thymocyte blasts (Fig. 3D). These data suggest that
there is an overall change in the mannose content of CD45 as
double-positive thymocytes mature into single-positive thymocytes.
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To this point, a correlation existed between the cell types in
which the CD45-GII association occurred and the cell types in which MBP
could recognize CD45. We then sought to determine whether perhaps both
MBP and GII could recognize the same pool of CD45. To this end, we
performed a reconstitution assay in which MBP-purified CD45 from BW5147
was incubated with a lysate of BW/T200- cells,
the CD45-deficient variant of BW5147. The bound proteins were separated
by SDS-PAGE and visualized by Western blotting with GII-specific Abs.
GII from the BW/T200- was able to bind
MBP-purified CD45, which suggests that the same pool of CD45 is capable
of associating with both MBP and GII (Fig. 4).
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Because of the ability of both GII and MBP to recognize CD45 on
immature cells, and our previous data demonstrating that GII only
associates with Endo H-sensitive carbohydrate on CD45 (24), we
postulated that immature cells express higher levels of Endo
H-sensitive carbohydrate on the cell surface than mature cells. To test
this hypothesis, we performed a CD45 immunoprecipitate from cell
surface-biotinylated lysates from various cells. The bound proteins
were treated with either Endo H or Endo F under reducing and denaturing
conditions, resolved by SDS-PAGE, and analyzed by blotting with
streptavidin. Upon treatment of cell surface CD45 from either BW,
thymocytes, AB.1, or splenic T cells with Endo H, there is a shift in
the mobility of CD45 owing to the presence of immature high mannose or
hybrid type carbohydrate (Fig. 5). This
shift is not as dramatic as treatment with Endo F, which suggests there
is a mixture of both Endo H-sensitive and resistant carbohydrate. In
examining control glycoproteins such as class I MHC, CD44, and LFA-1,
we see virtually no Endo H-sensitive carbohydrate (Fig. 5 and data not
shown). Therefore, cell surface CD45 from all cells examined contains a
mixture of both immature and mature carbohydrate, while other
glycoproteins examined contain only mature carbohydrate. Although the
high level of Endo H-sensitive carbohydrate on CD45 expressed on the
cell surface is surprising, it does not appear to be the basis for the
preferential GII and MBP binding to CD45 from the immature cells.
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In a recent report by Hansen et al. (29), it was
shown that MBP from a number of different species comes in two
different forms, denoted MBP-A and MBP-C. The difference between the
two forms lies in the amino terminus, in which MBP-A has three
cysteines, whereas MBP-C has only two. This difference leads to MBP-A
being capable of forming hexamers, while MBP-C is found predominantly
in dimers or trimers (29). As well, it was demonstrated
through sugar competition, that MBP-A can be inhibited from binding
mannan with a significantly lower concentration of glucose that can
MBP-C, while the concentration of mannose required to compete off
either form of MBP from mannan is comparable (29). In
examining the binding of CD45 by MBP with either glucose or mannose as
competitors, the presence of two different glycoforms of CD45 bound by
MBP was revealed (Fig. 6). Cell
surface-biotinylated lysates from SAKR, BW5147, and thymocytes were
subjected to an MBP pull-down assay without any competitor, or in the
presence of either glucose or mannose. We then compared the cell
surface vs the total CD45 bound to MBP in each case. In the SAKR and
thymocyte lysates, only the higher m.w. glycoform of CD45 is readily
surface biotinylated, and that form disappears with the inclusion of
glucose or mannose (Fig. 6). The lower m.w. glycoform is only slightly
sensitive to glucose, but is entirely competed from binding by mannose
(Fig. 6). A similar pattern is seen with the BW5147 cells, except the
upper form is more resistant to competition with glucose (Fig. 6).
These data suggest that there are two glycoforms of CD45 bound by MBP,
each with differential sensitivity to glucose competition.
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A strong correlation exists between cell types in which CD45 and
GII associate and in which cell surface CD45 is bound by MBP. To
determine whether or not GII activity directly influences the amount of
cell surface CD45 bound by MBP, we treated AB.1 cells, which do not
normally have endogenous MBP ligands on their cell surface, with the
GII inhibitor N-methyl deoxynorjirmycin (NMdNM) overnight
and examined them for the presence of MBP ligands. The overnight
treatment of AB.1 with 2 mM NMdNM resulted in a 50% inhibition of GII
activity, as measured by a colorimetric substrate (data not shown).
This inhibition of GII activity resulted in a 56% decrease in cell
surface class I MHC expression, a 44% decrease in surface CD45
expression, and a 321% increase in MBP binding, as measured by FACS
analysis (Fig. 7A). Cell
surface expression of class I MHC has been previously shown to be
dependent on GII activity (30); however, it appears that
CD45 cell surface expression is also somewhat dependent on GII
activity. The increase in MBP binding after GII inhibition directly
links GII activity with the production of MBP ligands. Furthermore, in
examining whether or not CD45 specifically acquires the appropriate
carbohydrate for MBP recognition, an MBP pull-down from cell
surface-biotinylated AB.1 lysates either untreated or NMdNM treated
showed that there is a significant increase in the amount of cell
surface CD45 recognized by MBP in the GII-inhibited AB.1 cells (Fig. 7B). Taken together, these results demonstrate that GII has
the ability to regulate the presence of cell surface MBP ligands.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The association between CD45 and GII is seen in immature cells, but not
mature cells (Fig. 1). This change in the ability of GII to bind CD45
may be due to a number of different factors, including changes in GII
or changes in CD45. In examining the isoform expression of GII in
immature vs mature cells, we find comparable amounts of GII that
contain the first alternatively spliced sequence (Box A1), which are
the only isoforms capable of associating with CD45 (20),
as well as other GII isoforms (data not shown). Therefore, GII
isoform expression does not appear to be significantly different
between mature and immature cells. Previous data using a transfection
system indicated that all isoforms of CD45 are capable of associating
with GII (data not shown), and examination of two different isoforms of
CD45 in BW cells, CD45RO, and CD45RB revealed that both isoforms are
capable of associating with GII (data not shown). These data suggest
that all isoforms of CD45 are capable of associating with GII within
the same cell type. These data support the notion that the ability of
CD45 to associate with GII is intrinsic to the cell, and not dependent
on the isoform of CD45 found within that cell.
We and others have also shown that there is a higher amount of MBP
binding to cell surface CD45 (Fig. 2A) in immature cells
(28). Because the CD45-deficient variants of the BW and
SAKR cells also bind MBP (Fig. 2C), and MBP appears to pull
down a limited set of cell surface proteins other than CD45 (data not
shown), we suspect that there is an overall change in the
carbohydrate-processing machinery within immature cells, giving rise to
carbohydrate structures capable of being bound by MBP. The regulation
of which glycoproteins acquire carbohydrate capable of being bound by
MBP may involve GII, or other ER proteins; therefore, we are interested
in determining whether there are other proteins that associate with
GII, specifically Box A1 containing GII. In support of differential
carbohydrate processing in thymocytes, detection of calnexin-associated
CD3
on the cell surface with immature carbohydrate has been reported
(31). In this case, it appears that calnexin may regulate
the carbohydrate on CD3, whereas GII may regulate the CD45
carbohydrate.
The most striking data in support of developmentally regulated changes
in the carbohydrate structure on CD45 come from the analysis of ex vivo
thymocytes. Double-positive thymoctyes are recognized by MBP to a
significantly higher degree than either the double-negative or
single-positive subsets (Fig. 3A). As well, there is more
GII-bound CD45 in bulk thymocytes vs single-positive Con A thymocyte
blasts (Fig. 3D). Interestingly, during thymocyte
development, the activity of CD45 is most crucial at the
double-positive stage during positive selection, as illustrated by
CD45-deficient mice (32). Therefore, lectins expressed on
thymic stromal cells with similar specificity to MBP could bind to the
abundant CD45 glycoprotein, where their interaction could affect
adhesion, signaling, or plasma membrane localization, leading to
changes in thymocyte selection. The binding of CD45 to lectins on the
surface of thymic stromal cells may be of a low affinity and transient
in nature, but may allow for some high avidity interactions to take
place. This appears to be the case for the interaction between DC-SIGN
and ICAM-3, in which the interaction between DC-SIGN on the dendritic
cell and ICAM-3 on the T cell seems to be the initial adhesion event
allowing for the interaction between LFA-1 and ICAM-3
(18). The adhesion mediated by LFA-1 and ICAM-3 keeps the
dendritic cell and T cell in contact long enough for TCR engagement by
MHC-peptide (18).
Since a soluble lectin, MBP, has been demonstrated to bind CD45, it is also possible that a soluble, mannose-specific lectin found within the thymus could bind CD45. The binding of CD45 by a soluble lectin may constrain CD45 in a particular spatial organization that may impact signaling thresholds. This was recently demonstrated to occur for the TCR, in which binding of the TCR by galectin-1 decreased TCR clustering (33). In light of the work published by Johnson et al. (34), describing the movement of a small fraction of CD45 into the immunological synapse, it is possible that a soluble mannose-specific lectin-binding CD45 will perform a similar function to galectin-1 and spatially restrict the movement of CD45. By restricting the movement of CD45 into and out of the immunological synapse, signaling thresholds may be altered. The altering of signaling thresholds may be particularly important for thymic selection events.
The use of monosaccharides to inhibit the MBP binding of CD45
demonstrated the existence of two glycoforms of CD45. Both glycoforms
of CD45 could be inhibited from binding MBP by mannose, while only the
higher m.w. glycoform was competed by glucose (Fig. 6). These data
suggest that MBP may be binding CD45 via terminal mannose or terminal
glucose. In analyzing the total carbohydrate on CD45 from immature SAKR
cells for the presence of terminal glucose using Endo H digestion
followed by mannosidase treatment, which is less active on carbohydrate
containing terminal glucose, we found that there were two distinct
pools of CD45 carbohydrate. One pool was susceptible to mannosidase
treatment, while the other was not, which suggests the presence of
terminal glucose on a portion of CD45 carbohydrate (data not shown). It
is not clear which pool of carbohydrate was from cell surface CD45;
however, since the ratios of mannosidase-sensitive to insensitive
carbohydrate were 1:1, we believe at least some the
mannosidase-insensitive carbohydrate was cell surface derived. In fact,
the calnexin-associated, cell surface CD3 was found to contain
terminal glucose on its Endo H-sensitive carbohydrate by a similar
method (31). In order for the cell surface CD45
carbohydrate to contain terminal glucose or mannose, there must be a
mechanism in place to protect those sugars. Those residues may be
protected from processing by a number of different mechanisms,
including physical protection by interacting proteins, such as GII, or
sequestration within the ER and Golgi, thereby preventing access to the
carbohydrate.
In support of a model whereby GII directly regulates MBP binding,
treatment of mature cells with a GII-specific inhibitor results in an
increase in MBP binding to cell surface CD45 (Fig. 7). Since GII is
required for the removal of the terminal glucose residues from the
immature carbohydrate, inhibition of its activity most likely results
in the maintenance of a fraction of those glucose moieties. Therefore,
MBP may be binding to cell surface CD45 from GII-inhibited AB.1 cells
by way of terminal glucose residues. Since GII stably associates with
CD45 in immature cells, it is possible that the enzymatic activity of
GII is inhibited while bound to CD45, thus preserving the glucose
residue on the immature carbohydrate expressed on cell surface CD45.
This protection hypothesis could explain our detection of cell surface
CD45, which is capable of being bound by MBP, and is sensitive to
glucose competition (Fig. 6). MBP or other lectins with similar
specificity could bind to this pool of CD45.
In summary, it appears that the regulation of CD45 glycosylation changes during T cell development; however, future studies must be done to determine whether these modifications are important for T cell maturation. Given the recent interesting example of how carbohydrate modification and lectin interaction regulate T cell responsiveness (33), it is tempting to speculate that carbohydrate interactions with CD45 and other proteins could be important for regulating signaling thresholds during thymocyte development.
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Acknowledgments |
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antiserum. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Hanne L. Ostergaard, Department of Medical Microbiology and Immunology, 6-70 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, Canada, T6G 2S2. E-mail address: hanne.ostergaard{at}ualberta.ca
3 Abbreviations used in this paper: ER, endoplasmic reticulum; DC-SIGN, dendritic cell-specific ICAM-3 grabbing nonintegrin; Endo H/F, endoglycosidase H/F; GII, glucosidase II; MBP, mannose-binding protein; NMdNM, N-methyl deoxynorjirmycin.
Received for publication March 2, 2001. Accepted for publication July 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TCR proteins with calnexin and results specifically in accelerated degradation of nascent TCR proteins within the endoplasmic reticulum. EMBO J. 13:3678.[Medline]
-subunit of the nicotinic acetylcholine receptor. J. Biol. Chem. 273:17064.- and -subunits of -glucosidase II. J. Biol. Chem. 272:13117.- and -subunits of mouse glucosidase II in T lymphocytes. Glycobiology 9:277.This article has been cited by other articles:
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  R. Dawes, S. Petrova, Z. Liu, D. Wraith, P. C. L. Beverley, and E. Z. Tchilian Combinations of CD45 Isoforms Are Crucial for Immune Function and Disease J. Immunol., March 15, 2006; 176(6): 3417 - 3425. [Abstract] [Full Text] [PDF]
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 E. Z. Tchilian, R. Dawes, L. Hyland, M. Montoya, A. Le Bon, P. Borrow, S. Hou, D. Tough, and P. C. L. Beverley Altered CD45 isoform expression affects lymphocyte function in CD45 Tg mice Int. Immunol., September 1, 2004; 16(9): 1323 - 1332. [Abstract] [Full Text] [PDF]
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 T. Stanton, S. Boxall, K. Hirai, R. Dawes, S. Tonks, T. Yasui, Y. Kanaoka, N. Yuldasheva, O. Ishiko, W. Bodmer, et al. A high-frequency polymorphism in exon 6 of the CD45 tyrosine phosphatase gene (PTPRC) resulting in altered isoform expression PNAS, May 13, 2003; 100(10): 5997 - 6002. [Abstract] [Full Text] [PDF]
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 T. A. Baldwin and H. L. Ostergaard The Protein-tyrosine Phosphatase CD45 Reaches the Cell Surface via Golgi-dependent and -independent Pathways J. Biol. Chem., December 20, 2002; 277(52): 50333 - 50340. [Abstract] [Full Text] [PDF]
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