From the aOxford Glycobiology Institute, Department of Biochemistry, the University of Oxford, South Parks Road, Oxford OX1 3QU, cDivision of Structural Biology, Henry Wellcome Building for Genomic Medicine, the University of Oxford, Roosevelt Drive, Oxford OX3 7BN, dOxford Centre for Molecular Sciences, Central Chemistry Laboratory, the University of Oxford, South Parks Road, Oxford OX1 3QU, fNuffield Department of Clinical Medicine, the University of Oxford, Oxford Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom, and the hDepartment of Pathology and Immunology, Monash Medical School, Commercial Road, Monash University, Prahran, Victoria 3181, Australia
Received for publication, December 20, 2002 , and in revised form, March 31, 2003.
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ABSTRACT |
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INTRODUCTION |
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CD8 is a glycoprotein consisting of disulfide-linked subunits, and
, which are encoded by two closely linked genes near the immunoglobulin
locus (3). Despite
sharing relatively low sequence similarity (
20%), the CD8 subunits are
structurally related and predicted to have identical topologies
(4). Each chain consists of an
extracellular immunoglobulin superfamily (IgSF) V-set domain attached to
hydrophobic transmembrane sequences and short cytoplasmic tails via extended,
disulfide-linked stalk-like peptides of 4851 (
-chain) or
3742 (
-chain) residues (reviewed by Gao et al.
(5)). The IgSF domains in CD8
exhibit highly variable N-linked glycosylation; in humans, only a
single site on the
-chain is glycosylated, whereas in mice the
-
and
-chains have three and one sites, respectively. The ratio seen in
the mouse is reversed for rat CD8. In all species, the stalk-like region of
each chain is rich in proline, serine, and threonine residues and is
O-glycosylated. Amino acid sequencing of rat CD8
has indicated
that the four threonine residues 122, 126, 132, and 134 clustered at the
membrane-distal end of the stalk are occupied with O-glycans
(6). The cytoplasmic domain of
CD8
is attached to the tyrosine kinase p56lck
(reviewed in Ref. 7). Most T
cells express CD8 as an
heterodimer, although a homodimeric form
(CD8
) is found on subsets of intraepithelial T lymphocytes of
the gut,
T cells, NK cells, and lymphoid-related dendritic
cells (reviewed by Zamoyska
(8)). CD8
and
CD8
T cells undergo different pathways of selection, perhaps
reflecting an underlying functional specialization within the CD8 T cell
population (9).
The interaction between CD8 and class I MHCp ligands was
initially investigated using cell adhesion assays
(10). More recently, the
affinities and kinetics of the interaction of soluble forms of human and mouse
CD8
with human and mouse class I MHC, respectively
(11,
12), and of human CD4 and
murine MHC class II (13), have
been determined using surface plasmon resonance-based methods. In both cases,
the interactions were shown to have extraordinarily low affinities and
extremely rapid dissociation kinetics. Crystallographic analyses of the
complexes of CD8
/class I MHCp from humans and mice
(14,
15) indicate that CD8 binds
the membrane-proximal
2 and
3 domains of MHCp and makes
additional contacts with
2-microglobulin. An analogous
interaction was revealed by the crystal structure of the complex of human CD4
domains 1 and 2, and murine MHC class II
(16), insofar as CD4 binds a
membrane-proximal cavity formed by residues from both the
2 and
2
domains of class II. It has been proposed that the weak interactions between
the TCR and MHCp may be enhanced by simultaneous interactions involving CD8
(17). However, CD8
interactions are generally at least 10-fold weaker than those involving the
TCR, and CD4 interactions are possibly even weaker. Therefore, although they
will contribute somewhat to the overall interaction, it seems more likely that
the key function of the co-receptors is to recruit sufficient
p56lck to pre-formed TCR·MHCp complexes to
consolidate the early signaling response
(11).
An additional level of complexity regarding these interactions arises from
the recent suggestion that ligand binding by CD8 is regulated in vivo
by changes in its O-glycosylation. Specifically, Moody et
al. (18) and Daniels
et al. (19) each
report that the binding of tetrameric forms of MHCp in a CD8-dependent,
non-cognate manner to double-positive thymocytes diminishes as
O-glycan sialylation increases and the thymocytes progress through
positive selection. To account for this effect, sialylation-induced changes in
the structural properties of the stalk, either involving electrostatic
repulsion between the two chains
(18) or chain extension
effects (19), were proposed to
affect the ligand-binding site even though this is located 30 Å
from the stalk. As has already been noted
(20), thermodynamic
considerations make an electrostatic repulsion-based mechanism for these
effects extremely unlikely. In the present study, we test the second proposed
mechanism, i.e. that O-glycan sialylation modulates the
extension of the stalk-like region of CD8. Our findings suggest that
sialylation has little, if any, effect on the overall structure of CD8.
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EXPERIMENTAL PROCEDURES |
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EnzymesSequencing-grade exoglycosidase and peptide
N-glycosidase F (PNGaseF, EC 3.5.1.52
[EC]
) enzymes were obtained from
Glyko Ltd. (Upper Heyford, UK) unless specified. Incubation conditions for
single enzyme digests were as described by Rudd et al.
(21), using the buffers
provided by the enzyme manufacturers. When enzyme arrays were used, the buffer
was 50 mM sodium acetate, pH 5.5. Exoglycosidases were used at the
following concentrations. Arthrobacter ureafaciens sialidase (EC
3.2.1.18
[EC]
), 12 units/ml; almond meal -fucosidase (EC 3.2.1.111
[EC]
),
3 milliunits/ml; bovine testes
-galactosidase (EC 3.2.1.23
[EC]
), 12
units/ml; Streptococcus pneumoniae
-hexosaminidase (EC
3.2.1.30
[EC]
), 120 units/ml; and jack bean
-mannosidase (EC 3.2.1.24
[EC]
), 100
milliunits/ml.
Preparation of sCD8 and sCD8abFour
recombinant soluble forms of CD8 were prepared as described previously
(22). Briefly,
sCD8
was produced by co-expressing sCD8
with sCD8
constructs (Fig. 1) in both
Chinese hamster ovary (CHO) K1 cells (referred to as sCD8
K1 and
sCD8
K1) and the mutant CHO cell line Lec3.2.8.1 (referred to as
sCD8
Lec and sCD8
Lec). The sCD8
was
purified to homogeneity by OX-8 affinity and/or ion-exchange chromatography
followed by gel filtration and the sCD8
by immunoaffinity
chromatography on an antibody (OX-8) column. For ultracentrifugation-based
structural comparisons with these mammalian cell-expressed forms of murine
CD8, Escherichia coli-expressed unglycosylated human
sCD8
consisting of residues 1120 of the mature protein,
referred to here as sCD8
E, was prepared as described in detail
elsewhere (23).
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Hydrazine Release, Re-N-acetylation, and Labeling of N- and
O-GlycansApproximately 50 µg of each protein was dialyzed
against 0.1% trifluoroacetic acid and then lyophilized. N-Glycans
were released by hydrazine at 95 °C (N* mode) and
purified using a GlycoPrep 1000 (Oxford GlycoSciences Ltd., Abingdon, UK).
These hydrazinolysis conditions represent a compromise between achieving
non-selective release, maximizing yield, and minimizing released sugar
degradation (24,
25). Manual hydrazinolysis for
selective release of O-glycans was performed as described previously
(24,
26). For in-gel release of the
glycans, the - and
-chains of CHO K1 sCD8 were separated on a
one-dimensional SDS-PAGE gel that was stained with Coomassie Blue. The
N-linked glycans were released from gel slices by incubation with
PNGaseF as described previously
(27).
Labeling and High Performance Liquid Chromatography (HPLC) Fluorescent labeling with 2-aminobenzamide (2-AB) was performed as described by Bigge et al. (28) using the kit provided by Glyko Ltd. HPLC was performed essentially as described by Guile et al. (29). The procedures for analyzing N-glycans were as described by Rudd et al. (21) and for O-glycans as described by Royle et al. (30). Glucose units (29) were calculated from retention time by reference to a standard consisting of a partially hydrolyzed dextran ladder using a prototype version of PeakTime, a program developed by Dr. E. Hart at the Oxford Glycobiology Institute.
Simultaneous Exoglycosidase Sequencing of the Released Glycan PoolExogylcosidase sequencing was performed as described previously (31, 32). Normal phase HPLC separations (NP-HPLC) were performed on a GlycoSep®-N chromatography column (Glyko Ltd.) using low salt conditions, and structures were assigned as described previously (29).
Trypsin Digestion50 µg of each sample
(sCD8K1 and sCD8
Lec) was dissolved in 200 µl of
6 M guanidine HCl, 0.5 M Tris, pH 8.0, with 3 µl of
0.5 M dithiothreitol and reduced by incubation at 37 °C for 2
h. The sample was then alkylated by addition of 3 µl of 4-vinylpyridine and
incubated for a further 1.5 h in the dark at 37 °C and then dialyzed
against 50 mM NH4HCO3. Samples were dried,
then resuspended in 40 µl of trypsin solution (1.25 µg/ml in 25
mM NH4HCO3 10% acetonitrile), and incubated
overnight at 37 °C. Peptides were extracted with a C18 ZipTip (Millipore
Corp., Bedford, MA) and analyzed directly by MALDI mass spectrometry. A fifth
of each sample was desialylated with A. ureafaciens sialidase, and
the glycopeptides were analyzed by MALDI after extraction with a C18
ZipTip.
MALDI Mass Spectrometry of GlycopeptidesSamples were
purified using C18 ZipTips (Millipore, Bedford, MA) prior to analysis by
MALDI-TOF mass spectrometry
(33). Positive ion MALDI-TOF
mass spectra were recorded with a Micromass TOFSpec 2E reflectron-TOF mass
spectrometer (Micromass Ltd., Atlas Park, Simonsway, Manchester, UK) fitted
with delayed extraction and a nitrogen laser (337 nm). The extraction voltage
was 20 kV; the pulse voltage was 3200 V, and the delay was 500 ns. Samples
were prepared by mixing 0.3 µl of the solution of tryptic peptides and
glycopeptides on the MALDI target together with the matrix solution (0.3 µl
from a solution of -cyano-4-hydroxycinnamic acid (10 mg/ml) in 7:3
(v/v) acetonitrile, 0.1% trifluoroacetic acid) and allowing it to dry at room
temperature. The resulting molecular weights were used to search the sequence
data base maintained at the European Molecular Biology Laboratory (EMBL) in
Heidelberg, Germany.
MALDI Mass Spectrometry of Released GlycansUnderivatized, released glycans were purified using a Nafion 117 membrane (Aldrich) and examined by MALDI mass spectrometry with the TOFSpec instrument in positive ion reflectron mode. Samples (0.3 µl in water) were mixed with a saturated solution of 2,5-dihydroxybenzoic acid on the MALDI target and allowed to dry at room temperature. The sample was then recrystallized from ethanol. Operating conditions for the mass spectrometer are as follows: extraction voltage, 20 kV; pulse voltage, 3200 V. The instrument was calibrated with dextran oligomers. Monoisotopic masses of the [M + Na]+ ions are listed in Tables I and II; all masses were within 0.1 mass unit of the calculated values.
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Q-TOF Mass Spectrometry of GlycopeptidesElectrospray MS and MS/MS spectra were recorded with a Micromass Q-TOF mass spectrometer fitted with a Z-spray ion source and a nanoflow injection system. The ion source temperature was 150 °C; the needle voltage was 1668 V, and the cone voltage was 80 V. Samples were infused in 1:1 double-distilled water/acetonitrile containing 0.1% formic acid at about 20 nl/min from borosilicate capillary needles. For MS/MS, the collision energy was 3540 V for the doubly charged [M + 2H]2+ ions from the C-terminal glycopeptide ions and 100110 V for the singly charged ions. The mass window for parent ion selection was about 4 Da. Argon at 20 pounds/square inch was used as the collision gas. Sample acquisition and processing were performed with the Micromass MassLynx data system.
Analytical UltracentrifugationAnalytical
ultracentrifugation experiments were performed using a Beckman Optima XL(I)
analytical ultracentrifuge, which is equipped with absorbance and interference
optics. Samples of sCD8E, sCD8
K1, and
sCD8
Lec were spun at 40,000 rpm at 20 °C. Sample
distributions were recorded, using the interference or absorbance systems, at
2-min intervals. The data were analyzed using the Sedfit software
(34,
35). The partial specific
volumes and masses of sCD8
K1, sCD8
Lec, and
sCD8
E were calculated using the known amino acid composition of
CD8 and the glycosylation analysis of sCD8
K1 and
sCD8
Lec reported herein. The calculation was accomplished using
a derivative program of AtoB
(36). The sedimentation
coefficients of sCD8 calculated over a concentration range were extrapolated
to infinite dilution and corrected for buffer viscosity and density compared
with water as a standard solvent. The program Sedfit was used to calculate the
frictional ratio of each sample f/f0, this being
the ratio between the experimental frictional coefficient and the frictional
coefficient of a spherical species of the same mass and partial specific
volume. The frictional and sedimentation coefficients obtained were corrected
for hydration effects for a series of different levels of hydration as
previously described (37),
yielding "dry" Perrin functions Pexp and
sedimentation coefficients
(0) (S)
(38):
![]() | (Eq. 1) |
![]() | (Eq. 2) |
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RESULTS |
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CD8 ConstructsFor the present study, four recombinant
soluble forms of glycosylated CD8 were prepared as described previously
(22). Briefly,
sCD8 consists of the mouse CD8
chain (residues
1130 of the mature polypeptide) expressed as a soluble, homodimeric
fusion protein with 17 residues of the rat CD8
stalk-like peptide
(residues 122138) containing the O-linked sugars and the OX-8
anti-rat CD8 monoclonal antibody epitope
(Fig. 1a). Overall,
the stalk-like region of this protein, measured from the first residue beyond
-strand G of the murine
-chain V-set IgSF domain, consists of 26
residues. sCD8
was generated by co-transfecting the sCD8
construct (Fig. 1a)
together with a construct consisting of residues 1116 of the mouse
-chain sequence attached to the 23 residues forming the C terminus of
the sCD8
construct (Fig.
1b). The sCD8 isoforms were expressed in CHO K1 and
Lec3.2.8.1 cells and are referred to as the K1 and Lec derivatives,
respectively. For structural comparison with the glycosylated murine proteins,
unglycosylated human sCD8
consisting of residues 1120 of
the mature polypeptide, i.e. completely lacking the stalk-like region
of CD8, was expressed in E. coli as described in detail elsewhere
(23). This protein, for which
a crystal structure exists
(14), is referred to as
sCD8
E.
Glycosylation Analysis, N-Linked GlycansThe
N-glycans were released by hydrazinolysis from purified
sCD8K1, sCD8
K1, and sCD8
K1 expressed in
CHO K1 cells, and sCD8
Lec and sCD8
Lec expressed in
Lec3.2.8.1 cells. The glycan pools were analyzed directly by MALDI-TOF mass
spectrometry to obtain the composition of the constituent isobaric
monosaccharides (Tables I,
II,
III) or labeled with 2-AB for
analysis by NP-HPLC (Fig. 2 and
Tables I,
II,
III). The HPLC procedure
separates glycans on the basis of their hydrophilicity which, in practice,
corresponds very closely to molecular weight
(29). HPLC chromatograms were
calibrated against a standard dextran hydrolysate, enabling sample retention
times to be expressed as glucose units. Preliminary structural assignments
were made by comparison with glucose unit values in a data base of standard
N-glycans (21). The
glycosylation profiles of the
and
constructs, and
of a non-physiological form of sCD8 consisting of the
homodimer,
were all very similar (Fig.
2a, top 3 panels), indicating that there is
little difference in the glycan processing of the
- and
-chains
and that the N-glycosylation sites are equally accessible to the
processing enzymes.
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The identity of the N-glycans was confirmed by exoglycosidase
digestion of the pool of labeled glycans and re-analysis by NP-HPLC
(representative data for sCD8 is shown in
Fig. 2b). The relative
proportions of each glycan, derived from integration of the fluorescence
intensities of the peaks (Fig.
2a), are shown in
Table I. About a third of the
glycans (34%) were of the oligomannose types
Man5,6,7,8GlcNAc2. The remaining glycans were all
bi-antennary complex-type glycans. Of these, 57% were sialylated, mostly as
the mono-sialylated form. Thirty nine percent of the bi-antennary glycans were
core-fucosylated (Table I).
In contrast, sCD8Lec and sCD8
Lec contained only
oligomannose glycans (Fig.
2a, two lower profiles), which digested with
jack bean
-mannosidase to Man1GlcNAc2
(Fig. 2c). This glycan
profile was expected because Lec3.2.8.1 cells are mutated at the gene encoding
GlcNAc transferase T1 required for the formation of complex-type
N-glycans (Lec1 mutations; see Ref.
45). The N-glycans of
sCD8 expressed in Lec3.2.8.1 cells are, therefore, not only significantly
smaller but also uncharged compared with those of CHO K1 cell-derived sCD8,
consistent with previous studies
(46,
47).
sCD8K1 and sCD8
K1 were separated into their
constituent chains by SDS-PAGE (Fig.
3a), and the bands corresponding to the
-chains
(bands
1 and
2) and
-chain (band
) were excised.
sCD8
chains from both the
and
constructs
ran as doublets on SDS-PAGE (Fig.
3a). Protein sequencing analysis of
1 and
2
by mass spectrometry confirmed that both bands were derived from the
-chain (data not shown). The doublet was not due to the existence of
distinct sCD8
glycoforms because it remained after digestion with
PNGaseF (data not shown). Importantly, identical heterogeneity was also
apparent in Lec3.2.8.1-derived protein (data not shown), but its source
remains unresolved. The profiles of the glycans determined by NP-HPLC revealed
differences in the proportions of glycans present in the
- and
-chains; the most abundant glycan on the
-chain was the
mono-sialylated bi-antennary glycan, N4, whereas for the
-chain, the
fucosylated non-sialyated glycan, N3, was most abundant and another structure,
N5, was absent (Fig.
3b; Table
III).
Glycosylation Analysis, O-Linked GlycansO-Glycans were
selectively released by a procedure optimized to minimize
"peeling" (24,
26) from the purified
proteins. The profiles of the sCD8K1, sCD8
K1, and
sCD8
K1 O-glycans are almost identical
(Fig. 4a, top
three panels). Exoglycosidase sequencing
(Fig. 4b;
Table IV) indicated that these
glycans consisted of the type 1 core Gal-
1,3GalNAc disaccharide (O1) and
its sialylated tri- (O2) and tetrasaccharide derivatives (O3). This pattern is
consistent with the known absence of the core 2-branching
N-acetylglucosaminyltransferase in CHO K1 cells
(48), which is required for
the synthesis of larger O-glycan structures branching at C6 of the
core GalNAc.
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The NP-HPLC profile of sCD8Lec O-glycans
(Fig. 4a) was
characterized by the complete absence of the di-sialylated O3-glycan structure
and a substantial reduction in the mono-sialylated structure
(Fig. 4c and
Table IV). Therefore, the key
difference between the two forms of sCD8
is that although 86% of
the core 1 O-glycans are mono- and di-sialylated in CHO K1 cells, 82%
of the Lec3.2.8.1-derived core 1 O-glycans are non-sialylated. This
glycan composition is consistent with the known defects in Lec3.2.8.1 cells
(4244),
which are predicted to result in the production of single GalNAc residues.
However, although hydrazinolysis cleaves single GalNAc residues from proteins,
some monosaccharides are removed along with peptides at a subsequent paper
chromatography step. Therefore, in principle, the NP-HPLC analysis could
overestimate the degree of sialylation of sCD8
Lec. In order to
determine whether or not single GalNAc residues occupy any of the sites in
sCD8
Lec, mass spectrometric analysis of the tryptic fragments of
sCD8
was undertaken.
Electrospray mass spectrometry and subsequent MS/MS fragmentation of the [M
+ H]+ ions of tryptic peptides derived from sCD8Lec
and sCD8
K1 produced a cleavage profile consistent with that of a
15-amino acid C-terminal
-chain glycopeptide with the sequence
APTPVPPPTGTPRPL (examples are given in Fig.
5). This peptide contains three of the four threonine residues in
the sCD8 stalk likely to be sites of O-glycan attachment
(6). The fragmentation pattern
indicated that, when present, the hexoses are attached to the HexNAc
residues.
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MALDI mass spectrometry (Fig.
6a) showed that in excess of 92% of the tryptic
glycopeptides of sCD8Lec had all three sites occupied
(m/z = 2107.1, 2269.1, 2431.3, and 2593.3); 7% had two sites
occupied (m/z 1903.2, 2066.1, and 2227.2), and 1% had one
site occupied (m/z 1701.6 and 1863.6). The relative amounts
of the fully occupied, sCD8
Lec glycopeptide glycoforms are also
apparent in the MALDI-MS profile of the whole tryptic digest. Relative to the
amount of the glycoform consisting of single GalNAc residues at each of the
three glycosylation sites (m/z = 2107.1), glycoforms with
one (m/z = 2269.1), two (m/z = 2431.3),
and three (m/z = 2593.3) additional hexose sugars were
present at 107, 62, and 13%, respectively. By taking these ratios into
account, and the results of the NP-HPLC analysis
(Fig. 4, a and
c; Table
IV),
70% of the O-glycans present on
Lec3.2.8.1-derived CD8 consisted of single GalNAc residues; the remainder were
extended with
1,3-galactose to form type 1 core Gal-
1,3GalNAc, and
only 18% of these (
6% of the O-glycans overall) were
sialylated.
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Similar MALDI-MS analysis was performed on sCD8K1
(Fig. 6b). This showed
that 100% of the available sites were occupied with GalNAc residues and that
96% of the glycopeptides (m/z 2593.2) contained an
additional hexose (galactose) residue at each site. From the HPLC analysis of
the glycans (Table IV), 55% of
the O-glycans containing galactose were monosialylated and 30% were
disialylated. Thus, at least 82% of all the O-glycans present in the
CHO K1-derived CD8
were sialylated (i.e. 85% of the 96%
core 1 O-glycans detected by MALDI-MS).
Analytical UltracentrifugationsCD8K1,
sCD8
Lec, and sCD8
E have apparent sedimentation
coefficients at 293 K (
) that vary
inversely with concentration, presumably due to the limiting effects of
macromolecular crowding on diffusion (Fig.
7) (49).
Extrapolation to zero protein concentration yields values for
unaffected by crowding effects,
(0). These values are listed in
Table V together with the
molecular weight, partial specific volume (
), dependence of s on
concentration (s(c)), frictional ratio
(f/f0), solvent-corrected s
(
), and the Perrin function
(Pexp). Where necessary, the calculations are based on
hydration weights of 0.10.5 g of H2O/g protein. The
calculated mass values are sufficiently close to the mass of
sCD8
K1 obtained by MALDI-TOF (56,871 Da, data not shown) to be
used in the calculations. As expected, sCD8
K1 and
sCD8
Lec have much larger s and P values than
sCD8
E, reflecting the presence of the stalk-like region and
N- and O-glycosylation of the CHO K1- and Lec3.2.8.1-derived
proteins. Critically, the P values, which provide shape information,
are very similar for sCD8
K1 and sCD8
Lec (1.61
versus 1.54) even though the two proteins have substantially
different sedimentation coefficients (3.73 versus 3.13), due to the
different sizes of the N- and O-glycans attached to each protein.
|
|
To aid in the interpretation of these effects, we calculated the
hydrodynamic properties of low resolution molecular models of each protein.
The calculated s and P values for sCD8E,
i.e. 2.61 and 1.14, respectively, modeled explicitly on the crystal
structure of this protein (14)
for
= 0.3 g/g, are very similar to the experimental values
(i.e. 2.65 and 1.12) and indicate that in solution, as in crystals,
this form of sCD8 lacking the stalk-like region is very compact
(Table V;
Fig. 8a). The
sCD8
Lec and sCD8
K1 models were based on the
crystal structure of the ligand-binding domain of murine CD8
(15), the sequence of the
stalk-like regions, and the foregoing glycosylation analysis. The hydrodynamic
properties of a model of sCD8
K1 with a highly extended parallel
stalk (i.e.
2.6Å per residue, the likely upper limit for
mucin-like polypeptides) are also in good agreement with the experimental
values (i.e. s and P values of 4.05 and 1.53 at
=
0.3 g/g, versus 3.73 and 1.61, respectively;
Fig. 8c). Crucially, a
model of sCD8
Lec with the same highly extended,
parallel stalk also gives calculated s and P values that
closely match the experimental values (3.23 and 1.53 at
= 0.3 g/g,
versus 3.13 and 1.54, respectively;
Fig. 8b).
|
To confirm that hydrodynamic modeling is sensitive to the overall structure
of CD8, the predicted hydrodynamic properties of models of
sCD8Lec, in which the stalk is absent to different degrees or is
not fully extended, were determined (Fig.
8d). In combination, models 46 in
Fig. 8d approximate
the hydrodynamic properties of an essentially unconstrained, flexible stalk.
Along with the comparisons between the models of the unglycosylated and
glycosylated forms of sCD8
(Fig. 8, ac)
this shows that, in combination, s and P are capable of
discriminating between models of CD8 whose stalk-like regions have distinct
conformational properties.
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DISCUSSION |
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The presence of the -chain in sCD8
, or its absence in
, might have been expected to give rise to different
N-glycosylation patterns, particularly given the very low level of
protein sequence conservation. Although differing slightly in proportion, the
N-glycans of CHO K1 cell-derived sCD8
and
were essentially of the same type, suggesting that the
glycosylation sites are equally well exposed on both chains and accessible to
the relevant processing enzymes. The N-glycan structures are
restricted and consist of oligomannose and biantennary complex-type glycans.
This glycan profile is presumably a consequence of the three-dimensional
structure of the protein, as CHO K1 cells are known to be capable of
processing tri- and tetra-antennary sialylated glycans on other glycoproteins,
such as recombinant tissue plasminogen activator
(50) and erythropoietin
(51). Overall, however, our
results argue strongly against the possibility that structure-based
N- glycosylation differences are responsible for any functional
differentiation of the
- and
-isoforms of CD8.
The O-glycosylation in CHO cells is also very restricted and probably much simpler than for CD8 expressed on lymphocytes. In CHO K1 cells, the O-glycans consist of mono- and di-sialylated core 1 structures. Differences in O-glycosylation in the Lec3.2.8.1 cell line are likely due to the Lec2 and Lec8 mutations, which result in defective CMP-sialic acid and UDP-galactose translocation into the Golgi, respectively (43, 44). Lectin-binding studies of the O-glycosylation of recombinant glycophorin A expressed in Lec8 cells (52) indicated that the glycans are truncated and non-sialylated, and this is confirmed here for sCD8 expressed in Lec3.2.8.1 cells. Most of the O-linked glycans were predicted to be restricted to single GalNAc residues, although leakiness of the Lec2 and Lec8 phenotypes may generate O-linked structures with galactose and/or sialic acid residues added to the GalNAc.2 A 15-amino acid fragment, identified as the glycopeptide from the C terminus (position 133147), was present as three distinct glycoforms, with the dominant species bearing a single GalNAc sugar at each of the three threonine residues. However, this glycopeptide is also present as glycoforms that contain an additional hexose residue at one or more of these sites, and mono-sialylated core 1 disaccharide was detected by sialidase digestion and HPLC analysis (Fig. 4c), confirming that the Lec3.2.8.1 mutant is indeed leaky.
Numerous studies have shown that, in leukocytes, O-glycosylation is very complex and varies in an activation-dependent and tissue-specific manner. For example, marked changes in core 2 branching and 6-GlcNAc transferase activities have been associated with T cell maturation, and a thymus-specific core 26GlcNAc-transferase has now been identified (53, 54). Of most relevance to the present study, the sialylation of core 1 O-linked glycans is known to be up-regulated during thymocyte maturation (55, 56). Changes in O-glycan processing of this nature could have at least two functional outcomes. First, novel ligands for lectin-like receptors might be generated, such as the sialyl Lex determinant and the associated structures serving as ligands for selectin- and galectin-mediated cell-cell adhesion molecules (e.g. Ref. 57). A second possibility is that modifications of O-glycans alter the structure of O-glycosylated glycopeptides, indirectly modulating the presentation or conformation of the ligand-binding sites of cell surface molecules. Two groups have independently proposed that the binding function of CD8 is regulated in this way. Moody et al. (18) have suggested that the developmentally programmed sialylation of core 1 O-glycan structures by the galactose-sialyltransferase, ST3, alters the quaternary structure of the globular head domain of CD8, reducing its capacity to "clamp" MHC class I. Similarly, Daniels et al. (19) showed that CD8 binds MHC class I tetramers less avidly and that it becomes less effective as an adhesion molecule as sialylation increases during thymocyte maturation. They argue that changes in the flexibility or extension of the stalk-like region of CD8 may be critical for optimal ligand binding.
This proposal raises the question of whether or not the structural
properties of CD8 can in fact be altered by sialylation. Previously, the
structural effects of O-glycans had been thought to depend only on
steric interactions between the peptide-linked GalNAc and the adjacent amino
acids of the polypeptide
(5860).
This conclusion was based on the analysis of mucins but was not confirmed for
cell surface proteins with much shorter stalk-like polypeptides, such as CD8.
Comparison of the hydrodynamic data for sCD8K1 and
sCD8
Lec clearly indicates that O-glycans consisting of
a single GalNAc are as effective as sialylated core 1 glycans in extending the
stalk region of CD8. Therefore, our data generalize the concept that steric
interactions between the peptide-linked GalNAc and the adjacent amino acids of
the polypeptide account for the major structural effects of
O-glycans, regardless of the length of the polypeptide.
The hydrodynamic modeling suggests that the degree of extension may be as
great as 2.6 Å per residue. This is greater than for leukosialin (2
Å per residue (61)),
comparable with that for bovine and porcine submaxillary gland mucins (2.5
Å per residue (59,
62)) but much less than the
theoretical maximum (3.4 Å per residue). Remarkably, this apparent
degree of extension of the CD8 stalk is achieved at half the O-glycan
density of leukosialin (63),
i.e. approximately one glycan for every six amino acids. However, it
is necessary to acknowledge the weaknesses of hydrodynamic modeling, wherein a
static model is substituted for a dynamic molecule and the imperfectly
understood hydration of proteins has to be arbitrarily fixed. Whereas our data
indicate that the degree of extension is essentially indistinguishable for
sCD8K1 and sCD8
Lec, systematic errors could
confound a more quantitative analysis.
How can the results of Moody et al.
(18) and Daniels et
al. (19) be explained?
The monomeric, non-cognate affinity of CD8 for MHC class I molecules is
extremely low (11,
15) and much weaker than for
the TCR·MHCp. Interactions that are this weak are very likely to be
sensitive to avidity effects (i.e. density-dependent binding
effects), which can occur in the absence of structural changes at the level of
individual molecules. These effects are distinct from affinity
changes, which are structure-dependent. Given that neither group directly
tested the effect of CD8 sialylation on the monovalent binding affinity, in
the light of our data, we suggest that the binding changes observed by Moody
et al. (18) and
Daniels et al. (19)
are more likely the result of avidity changes. The simplest explanation is
that de- or unsialylated CD8 tends to aggregate, increasing the likelihood of
observable tetramer binding, for example. The differential sialylation of CD8
during thymopoiesis may simply occur coincidentally along with that of other
glycoproteins for which sialylated O-glycans are of greater
functional significance. The dominant thymocyte sialoglycoprotein is not CD8
but CD43 (64) after all. It
could be argued that our results are not representative of the behavior of CD8
in vivo, where the -isoform predominates, given that we
have characterized the solution properties of the
-isoform.
However, in all species, the analogous region of the
-chain of CD8 is
also rich in proline and threonine residues and is likely to have a very
similar structure to that of the
-chain. Therefore, although we cannot
rule out the possibility that, in contrast to the
-chain, the structure
of the
-chain is sensitive to sialylation, there is at present no
obvious structural basis for suspecting that this is the case. More generally,
as has been noted (65),
because many proteins are affected by manipulations of the type employed by
Moody et al. (18) and
Daniels et al. (19)
and glycosylation can affect proteins in several ways, it will often be
difficult to establish that the glycosylation of a particular protein is
important.
Finally, we note that the O-glycosylated region of the
-chain is significantly shorter than that of the
-chain (by nine
residues in mouse CD8, measured to the inter-chain disulfide). Given the same
degree of extension implied by the present hydrodynamic data, the
-chain
is likely to cause the
-chain to arch, favoring a docking interaction
with class I MHCp parallel with the cell surface, comparable with that seen in
the crystal structures (14,
15). It is now accepted that
the co-receptors and the TCR each have to bind the same MHCp molecule
(2). If this is initiated by
TCR binding to MHCp, as seems likely given the higher affinity of the TCR
interaction (66), both
co-receptors will be required to dock precisely to a binding site fixed
150 Å from the surface of the T cell. The question remains as to
why CD4 and CD8 have evolved such different solutions to this docking problem.
It seems relevant that cytotoxic CD8+ T cells are required to
recognize their targets largely in the absence of interactions involving other
similarly sized adhesive and co-stimulatory molecules, such as CD2 and CD28,
that facilitate CD4+ T cell contacts with their targets. The
orientating effects of the stalk, coupled with its inherent flexibility, may
overcome this limitation and facilitate CD8-class I MHCp interactions during
the very earliest stages of CD8+ T cell activation and
immunological synapse formation. In this, O-linked sugars would
appear to be key.
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FOOTNOTES |
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b Both authors contributed equally to this work.
e Recipient of a Royal Society traveling fellowship award.
g Supported by the Wellcome Trust.
i Present address: Avidex Ltd., 57c Milton Park, Abingdon, OX14 4RX, UK.
j
To whom correspondence may be addressed. E-mail:
pmr{at}glycob.ox.ac.uk.
k
To whom correspondence may be addressed. E-mail:
sdavis{at}molbiol.ox.ac.uk.
1 The abbreviations used are: TCR, T cell receptor; CHO, Chinese hamster
ovary; MS, mass spectrometry; IgSF, immunoglobulin superfamily; NP-HPLC,
normal phase-high performance liquid chromatography; MALDI-TOF MS,
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry;
MHC, major histocompatibility complex; MHCp, MHC-peptide complex; Q-TOF,
quadrupole time-of-flight mass spectrometry; sCD8, soluble form of CD8;
sCD8K1, murine sCD8
expressed in CHO K1 cells;
sCD8
Lec, murine sCD8
expressed in Lec3.2.8.1
cells; sCD8
E, human CD8
expressed in E.
coli cells; 2-AB, 2-aminobenzamide; HexNAc, N-acetylhexosamine;
PNGaseF, peptide N-glycosidase F; S, Svedberg units.
2 P. Stanley, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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