From the Laboratory of Immunobiology, Department of
Cancer Immunology & AIDS, Dana-Farber Cancer Institute and
§ Department of Medicine, Harvard Medical School, Boston,
Massachusetts 02115,
Department of Biological Sciences, Imperial
College of Science, Technology & Medicine, London SW7 2AY, United
Kingdom, §§ M-SCAN Research and Training Center,
Silwood Park, Ascot SL5 7PZ, United Kingdom,
M-SCAN Inc., West Chester,
Pennsylvania19380, and ** Department of Cellular and
Molecular Medicine, Howard Hughes Medical Institute, University of
California, San Diego, California 92093
Received for publication, October 11, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Bidentate interaction of a T-cell receptor and
CD8 Within complex biological systems, glycans serve as key structural
and functional elements (1). Cell surface glycans are altered during
cell differentiation and activation in conjunction with changing
glycoprotein expression pattern (2). The precise chemistry of glycan
modification requires that the vertebrate genome encodes a variety of
enzymes that modify various classes of glycans. For example, more than
a dozen sialyltransferases with unique substrate specificities and
expression patterns operate at the level of the Golgi apparatus (3).
Glycosylation has particular relevance in the immune system where
cell surface proteins and lipids involved in immune recognition and
regulation are typically modified by various glycan structures during
cell differentiation and activation (4, 5). One particular example is
the T-cell surface glycoprotein, CD8.
The CD8 cell surface molecule is critical for the development and
activation of T-cells whose T-cell receptors
(TCR)1 recognize peptides
bound to major histocompatibility complex class I (MHCI) molecules (6,
7). This co-receptor is encoded by two distinct genes, CD8 Mass Spectrometry Analysis--
The tryptic peptide extracts
were cleaned by reverse-phase (C18) trapping and eluted by 1:1 MeOH:HOH
with 1% acetic acid or acetonitrile:0.01% trifluoroacetic acid or
acetonitrile:0.01% formic acid in various experiments. The eluted
volume (2-3 µl) was loaded in a nanospray tip and analyzed by ES
mass spectrometry (MS) and tandem MS/MS analysis on either ABI QStar
Pulsar or Micromass Q-TOF quadrupole time-of-flight mass spectrometers.
In some experiments, the same procedure was applied to gel pieces with
the addition of PNGase prior to tryptic digestion.
Nano-LCMS and MS/MS experiments on SP and DP in-gel digest extracts
were carried out on a Q-TOF instrument using a 75-µm C18 reverse-phase column eluted with a gradient of acetonitrile in 0.01%
formic acid at a flow rate of 200 nl/min. Data-dependent acquisition of MS/MS spectra was controlled by setting threshold ionization values for doubly, triply, and quadruply charged ions, and
collision energies were programmed in relation to the values seen to
produce good fragmentation in the earlier nanospray experiments.
The Peanut Agglutinin (PNA) Lectin Detects Glycosylation
Differences on Developing Thymocytes and Binds CD8 Mass Spectrometry Analysis of CD8
An early comparative study of CD8
Subtracting this peptide mass (1581.93 Da) from the experimentally
determined mass of the glycopeptide (3291.49 Da) then allowed the total
carbohydrate mass to be calculated as 1709.56 corresponding to
NeuGc2Hex3HexNAc3, in agreement
with the sugar fragment ions described earlier. The b ion peptide
fragment series (m/z 215, 314, 413, 528, 627, and
740) is visible at relatively low collision energies, which in our
experience would not cause total carbohydrate elimination from the
fragments. This strongly suggests that threonine 113 is not
glycosylated (Fig. 4). In additional variable collision energy
experiments, the proline y" ion fragments at m/z
843 and 473 were seen to carry glycosyl substituents via signals at
m/z 1046 (843 + HexNAc), 1208 (843 + HexHexNAc),
1249 (843 + HexNAc2), 676 (473 + HexNAc), and 838 (473 + HexHexNAc). A consideration of these data together with the sugar
fragment ions observed (both at low mass and as neutral losses from the
quasimolecular ion) suggested that threonines 120, 121, and 124 are
each O-linked to core 1 (HexHexNAc) structures, two of which
are capped with N-glycolylneuraminic acid. The virtual
absence of the m/z 823.88 signal from the
corresponding DP preparation provided the first molecular evidence of
differential sialylation of the CD8
Because of the complexity of the nanospray MS spectra of the total
CD8
A consideration of the mass differences among the 1040, 1117, and 1194 signals together with the MS/MS data, which showed that the peptide
portion of the differing molecules began with the same LTVVDV ...
sequence, allowed the assignment of the glycopeptide structures of
these ions as CD8 Key Differences in CD8
A comparison of CD8 SP and DP glycopeptides revealed a key difference
in the O- but not the N-linked glycopeptides.
This difference is the almost complete absence of
non-sialylated Hex3HexNAc3 structures in CD8
SPs (Figs. 5 and 6). Conversely, double and triple Sia-capped Hex3HexNAc3 CD8 CD8 The current biochemical analysis favors reorientation of the globular
head domains of CD8. We find that on the noted stalk threonines, CD8 The structure of O-glycan adducts revealed herein also
limits the likelihood that galectins are operating to cross-link
galactose residues on neighboring CD8 Our findings show that the genetically programmed alteration of CD8 heterodimer with a peptide-MHCI complex is required
for the generation of cytotoxic T-lymphocytes. During thymic
development, the modification of CD8
glycans influences major
histocompatibility complex class I binding to T-cell precursors called
thymocytes. ES mass spectrometry (MS) and tandem MS/MS analysis were
used to identify the changes occurring in the CD8
-glycopeptides
during T-cell development. Several threonine residues proximal to the
CD8
Ig headpiece are glycosylated with core-type 1 O-glycans. Non-sialylated glycoforms are present in
immature thymocytes but are virtually absent in mature thymocytes.
These results suggest how sialylation in a discrete segment of the
CD8
stalk by ST3Gal-1 sialyltransferase creates a molecular
developmental switch that affects ligand binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
, whose polypeptide products are expressed in one of
two forms, CD8
homodimers or CD8
heterodimers (8, 9). Most
T-cells mature within the thymus and express cell surface CD8
receptors. Previously, we and others (10, 24) have shown that
immature thymocytes bind peptide-MHCI (pMHCI) tetramers more avidly
than mature thymocytes. The binding difference is the result of a
developmentally regulated glycosylation modification involving sialic
acid residues. Evidence of this is the increased CD8
-MHCI avidity
of mature thymocytes following treatment with neuraminidase, an enzyme
that removes sialic acid residues from cell surface molecules.
Moreover, the sialyltransferase ST3Gal-1, which specifically sialylates
core 1-O-glycans, is involved in controlling the
differential binding as evidenced by decreased CD8
-MHCI avidity
after induction of ST3Gal-1 (10). Given that CD8
glycans change
during thymic development (10, 11), we examined the physical nature of
CD8
O-glycosylation. Through the application of recent
advances in mass spectrometry (12), we have been able to identify a
developmental change in CD8
stalk glycosylation, which functions as
a molecular switch to critically affect ligand binding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sample Preparation--
Unfractionated thymocytes from
C57BL/6 mice (~2-4 × 109/experiment) were lysed in
buffer containing 1% Triton X-100. Resulting lysates were processed as
described previously (10) using anti-CD8
mAb YTS 156.77 for
immunoprecipitation. After separation on two-dimensional non-reducing/reducing SDS-PAGE, the proteins were stained with Gel-Code Blue reagent and the double positive (DP) and single positive
(SP) CD8
bands were excised. The gel slices were digested with
either trypsin or N-glycanase (PNGase) followed by
trypsin using conditions described previously (13).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
Within the
thymus, T-cell precursors move through a series of developmental stages
distinguishable by different CD4 and CD8 cell surface expression
patterns (14). CD4
CD8
double negative (DN)
cells progress to the CD4+CD8+ DP stage in the
thymic cortex, and upon successful selection, mature into either
CD4+ or CD8+ SP T-cells in the thymic medulla
(Fig. 1a). The DP to SP
transition is dependent on TCR ligation by pMHC molecules containing
self-peptides (15). Varied T-cell surface glycosylation patterns
detected by the plant lectin, PNA, also mark thymic developmental
progression. PNA binds to core 1-O-glycans bearing terminal
galactose residues (Gal
1-3GalNAc
Ser-Thr), staining immature
cortical thymocytes strongly (PNAhigh) and mature medullary
thymocytes weakly (PNAlow) (Fig. 1b) (16, 17).
The change in PNA reactivity is attributable to the induction of the
ST3Gal-1 sialyltransferase within the hematopoietic compartment that
catalyzes the addition of sialic acid (Sia) residues in a
2-3
linkage to terminal galactose (Sia
2-3Gal
1-3GalNAc
Ser-Thr), capping the PNA binding site in medullary thymocytes (Fig. 1, b and c) (18). Genetic disruption of ST3Gal-1
causes PNAhigh reactivity to persist into the medullary
thymocyte compartment (Fig. 1b) (2). Five likely
possibilities for O-glycan structures that could bind PNA in
the cortex are presented in Fig. 1c. Among the small group
of thymocyte cell surface molecules identified as being PNA-reactive
are CD45, CD43, and CD8 (19). In particular, the CD8
chain is a
major component of the differential PNA binding observed on immature
thymocytes (Fig. 1d).
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Fig. 1.
Alterations in PNA binding during thymic
maturation. a, stages of thymocyte maturation defined
by changes in CD4 and CD8 cell surface expression and PNA reactivity.
b, thymic tissue sections (×50) showing the cortical
(C) and medullary (M) regions as defined by
hematoxylin and eosin (H & E) and PNA reactivity
(PNA) in wild-type (WT), and
ST3Gal-1 /
mice. c, cortical
O-glycan structures that may exist and can bind PNA are
depicted. In the medulla, 1-3 represent sialylated core 1 structures resulting from the action of ST3Gal-1 and subsequent
modifications. d, PNA-stained thymic tissue sections (×200
DIC images) from WT and CD8
/
mice showing that
CD8
is a major binder of PNA in the cortex.
Glycans--
To identify
N-and O-linked glycan sites on CD8
and define
glycosylation changes associated with the DP to CD8 SP thymocyte transition, mass spectrometry was used to analyze tryptic peptides of
CD8
prepared from immunoprecipitates. CD8
proteins were immunoprecipitated from lysates of cell surface-labeled DP and CD8 SP
thymocytes sorted by MoFlo, using Sepharose-coupled anti-CD8
mAb and
separated on two-dimensional non-reducing/reducing SDS-PAGE gels as
described previously (10). Whereas three distinct pairs of CD8
heterodimers (
38Kd
30Kd,
38Kd
29Kd, and
'33Kd
29Kd) are evident on DP thymocytes, CD8
heterogeneity is reduced upon DP
to SP maturation (Fig. 2a). By
the CD8 SP stage, a single 38-kDa CD8
subunit is paired with a major
30-kDa CD8
glycoform. Note that aside from the 33-kDa CD8
'
cytoplasmic RNA splice variant found in DP thymocytes, CD8
is not
detectably altered during thymic maturation as assessed by the
two-dimensional gel analysis. A composite pattern is obtained from
silver staining of proteins immunoprecipitated from unfractionated
thymocytes and run in the two-dimensional gel system (see
"Experimental Procedures"). Since DP thymocytes comprise 80% of
thymocytes while the CD8 SP fraction accounts for merely 3-5%, the
total thymocyte CD8
immunoprecipitation pattern is most similar
to that of the isolated DP thymocytes. The two-dimensional gel pattern
of CD8
proteins precipitated from sorted DP and CD8 SP thymocytes
provided a ready basis to obtain "DP" and "CD8 SP"
thymocyte-derived gel slices as indicated in the Fig. 2b
(inset). The excised gel slices were then digested with
either trypsin or PNGase followed by trypsin using the conditions described previously (13). Following the in-gel digestion and extraction, the purified peptide mixture was analyzed using ES on Q-TOF
geometry tandem hybrid instrumentation (20) in both MS and MS/MS modes
(see "Experimental Procedures").
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Fig. 2.
Two-dimensional nonreducing/reducing 10%
SDS-PAGE separation of thymic CD8
molecules. a, surface biotin-labeled CD8 proteins
immunoprecipitated with CD8
mAb (YTS 156.77) from lysates of sorted
DP and CD8 SP C57BL/6 thymocytes. b, CD8 proteins
precipitated from total thymocytes (~2.5 × 109)
stained with silver. The DP and CD8 SP gel slice regions used for MS
analysis of CD8
are marked in the inset to the
right.
SP and DP preparations run by
nanospray ES-MS from a formic acid/acetonitrile solution showed a clear
quadruply charged signal at m/z 823.88 (corresponding to M = 3,291.49) in the SP sample, which was
virtually absent in the corresponding DP analysis. Fig.
3 shows the MS/MS spectrum of this ion at
moderate collision energies of 30-50 eV. The spectrum is the sum of
data obtained at 30, 40, and 50 eV. The spectrum shows definitive
evidence of glycosylation via major signals at m/z 204 (HexNAc), 366 (HexHexNAc), 290 (NeuGc
minus H2O), 308 (NeuGc), and 673 (NeuGcHexHexNAc). Hex
denotes any six-carbon neutral sugar, including glucose, galactose, and
mannose, whereas HexNAc is a six-carbon sugar with an
N-acetylated amino group at position 2. NeuGc is formed by
an enzyme that catalyzes the hydroxylation of the N-acetyl
group attached to C5 of the nine-carbon sialic acid
backbone. The mouse CD8
sialic acids identified were of the glycolyl
variety as expected rather than the N-acetyl (NeuAc)-type that predominates in mammalian brain tissue (1). The collision energies
were chosen to provide both carbohydrate and peptide backbone
fragmentation (21) in an effort to identify the CD8 peptide sequence
carrying the glycosylation. Signals observed at
m/z 215, 314, 413, 528, 627, and 740 were
interpreted as N-terminal peptide fragments, b ions (20), assignable to
a sequence ... VVDV(L/I) ... , which is present in CD8
in tryptic peptide-(112-125), LTVVDVLPTTAPTKK (Figs. 3 and
4). The threonines at positions 113, 120, 121, and 124 represented possible sugar attachment sites via
O-glycosylation. A free (non-glycosylated) peptide of this
sequence would be expected to show intense C-terminal ammonium ion (y")
fragmentation (20, 21) corresponding to fragmentation at the labile
L-P and A-P bonds, giving calculated nominal masses of 715 and 345, respectively. Neither of these signals is present in Fig. 3. However,
with increasing collision energy, which causes preferential cleavage of
sugar residues, prominent signals at m/z 843 and
473 begin to appear, which are 128 Da higher in mass. These
signals were assigned to C-terminal proline cleavage fragments PTTAPTKK
and PTKK, respectively, thus proving that the peptide backbone for the
823.884+-peptide is in fact the CD8
-(112-126) sequence
LTVVDVLPTTAPTKK. The fact that the 473 ion was only created at higher
collision energies showed that at least threonine 124 is
glycosylated.
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Fig. 3.
Collisional activation MS/MS spectrum of
m/z 823.884+
corresponding to CD8 stalk region
glycopeptide-(112-126). A tryptic digest of CD8
SP was
subjected to ES-MS analysis, and m/z
823.884+ was selected for MS/MS experiments. Moderate
collision energies (30-50 eV) were utilized in order to provide both
carbohydrate and peptide backbone information. The charge states of the
ions are colored to simplify the spectrum. Singly charged
species are red, doubly charged are green, and
triply charged are blue.
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Fig. 4.
CD8 glycosylation
sites. a, CD8
heterodimer schematic. Amino
acid numbers show the start of the mature CD8
protein, stalk region,
transmembrane segment, cytoplasmic tail, and the C terminus. The single
N-linked glycosylation site and five O-linked
glycosylation sites are designated. b, CD8
amino acid
sequence. Recovered tryptic peptides are indicated by boxes
with shaded boxes denoting the glycopeptides. The
longer form of the stalk glycopeptide includes the four residues
indicated by the overhead bracket. The five sites of
O-glycosylation, all threonine residues, are
highlighted. The single CD8
N-linked
glycosylation site is marked with an asterisk. The two
cysteine residues forming an intrachain disulfide bond within the
Ig-like domain are indicated by closed circles, whereas
those forming interchain bonds with CD8
are marked by open
circles.
SP/DP glycoproteins.
digests, a further preparation of SP and DP CD8
was then
examined by nanoLC-MS and nanoLC -MS/MS (see "Experimental Procedures"). These data were important in allowing the unambiguous confirmation of the glycosylation state of the remainder of the CD8
stalk region. This was achieved by locating
N-glycolylneuraminic acid-containing signals at
m/z 10404+, 11174+, and
11944+. These glycopeptides were found eluting at 24.8 min,
26.7/27.4 min (doublet), and 28.1 min respectively, compared with the
CD8
-(112-126) disialyl glycopeptide described earlier in the
nanospray experiment, which eluted at 30.2/31.1 min as evidenced by
signals at 8234+ and 10983+.
-(112-130)-LTVVDVLPTTAPTKKTTLK carrying
NeuGc1Hex5HexNAc5,
NeuGc2Hex5HexNAc5, and
NeuGc3Hex5HexNAc5 glycosylation,
respectively. The equivalent non-sialyl species was also
observed at m/z
9634+/12843+ eluting at 23.7 min. The
carbohydrate-related signals in the MS/MS spectra of this extended
glycopeptide suggest two additional core 1-type HexHexNAc glycans at
threonines 127 and 128, leading to five substitutions in all (Fig. 4).
Interestingly, comparing the signal strengths of the short (112-126)-
and long (112-130)-peptides with the degrees of sialylation observed
suggests that there is minimal additional sialylation on the Thr-127
and Thr-128 glycans relative to that observed on the peptide containing
Thr-120, Thr-121, and Thr-124. Also, comparing the degree of
sialylation between the SP and DP preparations in this nanoLC-MS and
-MS/MS experiment indicates heavier sialylation of the SP-derived
structures, although in both sets of data, the mono-sialyl structure is
predominant. Relatively higher levels of non-sialylated structures were
observed in the DP preparation. The predicted N-linked
glycopeptide CD8
-(1-17), also seen in the earlier nanospray
experiment, is seen principally as standard core-fucosylated
biantennary structures carrying two sialylated antennae
(m/z 14113+), one sialylated
antenna together with one
-galactose-capped antenna
(m/z 13633+) or two
-galactose-capped antennae (m/z
13153+).
O-linked Glycopeptides Linked to T-cell
Maturation--
Since the comparison of the nanoLC-MS and nanospray
experiments then showed little or no suppression in the nanospray data and because the "residence time" for MS/MS analysis is much longer in nanospray leading to better quality data in complex studies such as
these, subsequent comparative analyses were conducted using nanospray
ESMS. Fig. 5 gives the comparative data
for a CD8
SP/DP preparation. The data show an almost complete
absence of non-sialylated HexHexNAc structures in the SP sample
compared with the DP via signals at
m/z 893, 963, and 1284, whereas the principal
sialylation state in both SP and DP is mono-sialyl
(m/z 995 and 1040) as seen in the other studies.
Fig. 6 is an expansion of the
m/z 893 region of Fig. 5 showing the comparative
relative abundance of the key signal at m/z 893 for SP and DP and its absence in the SP sample. Note that the absence
of non-sialylated signals in SP is not attributable to the low relative
abundance of this sample, because the signals at
m/z 995 and 1040 in the same approximate mass
range of Fig. 5 are clearly visible. Significantly, the relative degree
of sialylation of the N-linked structures attached to
peptide-(1-17) is the same for both SP and DP preparations as
exemplified by the signals at m/z 1022 and 1059 or by m/z 1363 and 1411.
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Fig. 5.
Nanospray ES-MS spectra of a
CD8 -derived from SP and DP thymocytes.
A and B, the m/z 850-1125
region for SP and DP, respectively. C and D, the
m/z 1125-1420 region for SP and DP,
respectively. For the ease of interpretation, N-linked
structures are labeled in green, and O-linked
structures are labeled in red. The annotations show the
peptide sequence numbers and the total sugar composition for each
glycopeptide-derived signal.
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Fig. 6.
Expansion of the
m/z 893 region of Fig. 5
showing the abundance between SP and DP and the relative absence of the
key signal relating to non-sialylated HexHexNAc structures in SP
compared with its presence in DP thymocytes.
-(112-126)-peptides occur in
greater abundance in SP rather than DP, although these differences are
smaller by comparison. Unexpectedly, our structural studies have
revealed that both SP and DP are mainly mono-sialylated in the stalk
region despite the presence of five core-type 1 O-glycan
substitutions (Thr-120, Thr-121, Thr-124, Thr-127, and Thr-128) (Fig.
4). Sialylation occurs principally within the 120-124 sequence, and
there appears to be little or no additional sialylation of residues 127 and 128. Since SP thymocytes are PNAlow, this finding
suggests that the majority of CD8
stalk O-glycans are
inaccessible to this lectin. Thus, the key change between DP and SP is
core-type 1 sialylation at a single site in the 121-124 stalk segment.
This site is presumably that recognized by the peanut lectin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
rather than CD8
was previously shown to be the
critical co-receptor on thymocytes for MHCI binding (10, 22). In CD8
knock-out mice, for example, CD8
homodimers are expressed on the
surface of thymocytes but fail to support significant MHCI binding
activity as assessed by pMHCI tetramers using flow cytometry. Moreover,
the varied glycosylation pattern of CD8
at different thymic
developmental stages correlates with the noted change in the CD8
ligand binding activity whereby DP thymocytes interact with MHCI more
avidly than CD8 SP thymocytes. Several hypotheses have been offered to
account for this developmentally programmed alteration in CD8
co-receptor MHCI ligand-binding function (10, 23-25). First, the
sialylation of the CD8
stalk may affect the orientation of the
co-receptor globular head domains relative to the T-cell membrane
and/or CD8
domain-domain association strength, modulating the
ability of the distal binding surface of the CD8
Ig-like domain
to clamp MHCI (10). Second, sialic acid residues might reduce the
clustering of CD8
molecules on thymocyte surfaces because of
repulsion of the negatively charged sugars, preventing MHCI binding as
detected by pMHCI tetramers. Third, multivalent
mammalian lectins such as galectins (26), known to control clustering
of certain T-cell surface glycoproteins (27), might "pre-cluster"
non-sialylated CD8 co-receptors in DP thymocytes but not sialylated
glycan-bearing counterparts on CD8 SP thymocytes. Fourth, the effects
might be attributed to sialylation of molecules other than CD8
given the previous lack of direct evidence for developmentally
controlled sialic acid addition to the CD8 co-receptor itself.
harbors a sialic acid linked to a core 1 disaccharide that lacks a
N-acetyllactosamine, indicating an absence of both core 2 O-glycans and elongated core 1 glycans, which might alter lateral mobility of CD8
in the plasma membrane by virtue of larger hydrodynamic radii. Consistent with this view, we observed no
differences in the distribution of CD8
co-receptors on the surface of DP and CD8 SP thymocytes (10). Furthermore, CD8
remains
constitutively concentrated in cholesterol-sphingolipid-rich plasma
membrane microdomains due, at least in part, to CD8
cytoplasmic tail
palmitoylation (28).
stalk adducts. Galectin-1 has
been implicated in thymocyte apoptosis through the recognition of core 2 O-glycans on CD43 and CD45 (27, 29). Galectin-3 binding to
1-6-branched lactosamine chains produced by the Mgat5
gene on TCR N-glycans is reported to inhibit T-cell
activation, perhaps by altering TCR clustering (30). Most mammalian
galectins bind preferentially to galactose on polylactosamine, although
some may bind to other galactose linkages (31, 32). Detailed
site-specific assignment of Sia adducts on the CD8
stalk confirms
that the addition of sialic acid per se modulates CD8
co-receptor function.
glycosylation during thymocyte differentiation from immature DP to
mature SP stages is restricted to the O-glycans without concurrent changes in the N-linked structures. These
O-linked sites (Thr-120, Thr-121, Thr-124, Thr-127, and
Thr-128) localize to a segment of the CD8 stalk immediately abutting
the CD8
Ig-like domain. O-Linked glycans are attached to
none of the 14 other serine or threonine residues in the examined
tryptic fragments. Three of the five threonines (Thr-120, Thr-124, and
Thr-128) are conserved in all of the CD8
homologues sequenced to
date, residing within or adjacent to the lysine-rich segment that is
unique to the CD8
stalk (10). In view of both the weak association
between CD8
and CD8
head regions (10), an uncharacteristic
feature for Ig-like domain heterodimers and the almost certain
requirement for participation of CD8
and CD8
CDR-like loops in
the binding to the MHCI
3 domain (by extension from
crystallographic analysis of two CD8
·pMHCI complexes) (33, 34),
sialylation in this specific region may impact significantly on
CD8
binding to MHCI. The addition of sialic acid to the CD8
stalk could facilitate neutralization of positive charges on the
adjacent stalk lysine residues (Lys-125, Lys-126, Lys-130, and Lys-132
to Lys-135), probably permitting the stalk to assume a retracted rather
than fully extended configuration or resulting in other conformational changes. By altering CD8
domain-domain association and/or
disposition of the CD8 globular headpiece relative to the cell surface,
CD8
stalk O-glycans create a molecular switch regulating
MHCI binding. That sialic acid addition to core 1 O-glycans
during thymic ontogeny is a conserved feature of vertebrate development
(2) with CD8
representing a major thymic PNA-binding protein (Fig.
1d) underscores the essential nature of this molecular
switch. Additional chemical details regarding the dynamic glycobiology
of CD8 will be important, not only for understanding the co-receptor
function of thymocytes but that of naive, memory and effector
CD8 peripheral T-cells.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI45022 (to E. L. R.) and HL57345 (to J. D. M.) and grants from the Wellcome Trust and Biotechnology and Biological Sciences Research Council (to H. R. M. and A. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Both authors contributed equally to this work.
¶¶ Supported as an investigator of the Howard Hughes Medical Institute.
To whom correspondence should be addressed: Laboratory
of Immunobiology, Dana-Farber Cancer Institute and Department of
Medicine, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3412; Fax:
617-632-3351; E-mail: Ellis_reinherz@dfci.Harvard.edu.
Published, JBC Papers in Press, November 28, 2002, DOI 10.1074/jbc.M210468200
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ABBREVIATIONS |
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The abbreviations used are: TCR, T-cell receptor; MHCI, major histocompatibility complex class I; pMHCI, peptide-MHCI; mAb, monoclonal antibody; DP, double positive; SP, single positive; PNGase, N-glycanase; ES, electrospray; MS, mass spectrometry; LC, liquid chromatography; DN, double negative; PNA, peanut agglutinin; Sia, sialic acid; NeuGc, N-glycolylneuraminic acid; NeuAc, N-acetylneuraminic acid; HexNAc, N-acetylhexosamine; GalNAc, N-acetylgalactosamine.
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