Sialic Acid Capping of CD8beta Core 1-O-Glycans Controls Thymocyte-Major Histocompatibility Complex Class I Interaction*

Anne Marie MoodyDagger §, Simon J. North||, Bruce ReinholdDagger §, Steven J. Van Dyken**, Mark E. RogersDagger Dagger , Maria Panico||, Anne Dell||, Howard R. Morris||§§, Jamey D. Marth**¶¶, and Ellis L. ReinherzDagger §||||

From the Dagger  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, Dagger Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bidentate interaction of a T-cell receptor and CD8alpha beta heterodimer with a peptide-MHCI complex is required for the generation of cytotoxic T-lymphocytes. During thymic development, the modification of CD8beta 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 CD8beta -glycopeptides during T-cell development. Several threonine residues proximal to the CD8beta 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 CD8beta stalk by ST3Gal-1 sialyltransferase creates a molecular developmental switch that affects ligand binding.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha  and beta , whose polypeptide products are expressed in one of two forms, CD8alpha alpha homodimers or CD8alpha beta heterodimers (8, 9). Most T-cells mature within the thymus and express cell surface CD8alpha beta 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 CD8alpha beta -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 CD8alpha beta -MHCI avidity after induction of ST3Gal-1 (10). Given that CD8beta glycans change during thymic development (10, 11), we examined the physical nature of CD8beta O-glycosylation. Through the application of recent advances in mass spectrometry (12), we have been able to identify a developmental change in CD8beta stalk glycosylation, which functions as a molecular switch to critically affect ligand binding.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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CD8beta 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-CD8beta 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) CD8beta bands were excised. The gel slices were digested with either trypsin or N-glycanase (PNGase) followed by trypsin using conditions described previously (13).

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Peanut Agglutinin (PNA) Lectin Detects Glycosylation Differences on Developing Thymocytes and Binds CD8beta -- 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 (Galbeta 1-3GalNAcalpha 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 alpha 2-3 linkage to terminal galactose (Siaalpha 2-3Galbeta 1-3GalNAcalpha 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 CD8beta 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 CD8beta -/- mice showing that CD8beta is a major binder of PNA in the cortex.

Mass Spectrometry Analysis of CD8beta Glycans-- To identify N-and O-linked glycan sites on CD8beta and define glycosylation changes associated with the DP to CD8 SP thymocyte transition, mass spectrometry was used to analyze tryptic peptides of CD8beta prepared from immunoprecipitates. CD8alpha beta proteins were immunoprecipitated from lysates of cell surface-labeled DP and CD8 SP thymocytes sorted by MoFlo, using Sepharose-coupled anti-CD8beta mAb and separated on two-dimensional non-reducing/reducing SDS-PAGE gels as described previously (10). Whereas three distinct pairs of CD8alpha beta heterodimers (alpha 38Kd beta 30Kd, alpha 38Kd beta 29Kd, and alpha '33Kd beta 29Kd) are evident on DP thymocytes, CD8beta heterogeneity is reduced upon DP to SP maturation (Fig. 2a). By the CD8 SP stage, a single 38-kDa CD8alpha subunit is paired with a major 30-kDa CD8beta glycoform. Note that aside from the 33-kDa CD8alpha ' cytoplasmic RNA splice variant found in DP thymocytes, CD8alpha 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 CD8alpha beta immunoprecipitation pattern is most similar to that of the isolated DP thymocytes. The two-dimensional gel pattern of CD8beta 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 CD8alpha beta molecules. a, surface biotin-labeled CD8 proteins immunoprecipitated with CD8beta 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 CD8beta are marked in the inset to the right.

An early comparative study of CD8beta 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 CD8beta 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 CD8beta 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 CD8beta -(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 CD8beta stalk region glycopeptide-(112-126). A tryptic digest of CD8beta 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.   CD8beta glycosylation sites. a, CD8alpha beta heterodimer schematic. Amino acid numbers show the start of the mature CD8beta 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, CD8beta 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 CD8beta 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 CD8alpha are marked by open circles.

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 CD8beta SP/DP glycoproteins.

Because of the complexity of the nanospray MS spectra of the total CD8beta digests, a further preparation of SP and DP CD8beta 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 CD8beta 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 CD8beta -(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+.

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 CD8beta -(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 CD8beta -(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 alpha -galactose-capped antenna (m/z 13633+) or two alpha -galactose-capped antennae (m/z 13153+).

Key Differences in CD8beta 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 CD8beta 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 CD8beta -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.

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 CD8beta -(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 CD8beta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD8alpha beta rather than CD8alpha alpha was previously shown to be the critical co-receptor on thymocytes for MHCI binding (10, 22). In CD8beta knock-out mice, for example, CD8alpha alpha 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 CD8beta at different thymic developmental stages correlates with the noted change in the CD8alpha beta 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 CD8alpha beta co-receptor MHCI ligand-binding function (10, 23-25). First, the sialylation of the CD8beta stalk may affect the orientation of the co-receptor globular head domains relative to the T-cell membrane and/or CD8alpha beta domain-domain association strength, modulating the ability of the distal binding surface of the CD8alpha beta Ig-like domain to clamp MHCI (10). Second, sialic acid residues might reduce the clustering of CD8alpha beta 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 CD8alpha beta given the previous lack of direct evidence for developmentally controlled sialic acid addition to the CD8 co-receptor itself.

The current biochemical analysis favors reorientation of the globular head domains of CD8. We find that on the noted stalk threonines, CD8beta 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 CD8alpha beta in the plasma membrane by virtue of larger hydrodynamic radii. Consistent with this view, we observed no differences in the distribution of CD8alpha beta co-receptors on the surface of DP and CD8 SP thymocytes (10). Furthermore, CD8beta remains constitutively concentrated in cholesterol-sphingolipid-rich plasma membrane microdomains due, at least in part, to CD8beta cytoplasmic tail palmitoylation (28).

The structure of O-glycan adducts revealed herein also limits the likelihood that galectins are operating to cross-link galactose residues on neighboring CD8beta stalk adducts. Galectin-1 has been implicated in thymocyte apoptosis through the recognition of core O-glycans on CD43 and CD45 (27, 29). Galectin-3 binding to beta 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 CD8beta stalk confirms that the addition of sialic acid per se modulates CD8alpha beta co-receptor function.

Our findings show that the genetically programmed alteration of CD8beta 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 CD8beta 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 CD8beta homologues sequenced to date, residing within or adjacent to the lysine-rich segment that is unique to the CD8beta stalk (10). In view of both the weak association between CD8alpha and CD8beta head regions (10), an uncharacteristic feature for Ig-like domain heterodimers and the almost certain requirement for participation of CD8alpha and CD8beta CDR-like loops in the binding to the MHCI alpha 3 domain (by extension from crystallographic analysis of two CD8alpha alpha ·pMHCI complexes) (33, 34), sialylation in this specific region may impact significantly on CD8alpha beta binding to MHCI. The addition of sialic acid to the CD8beta 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 CD8alpha beta domain-domain association and/or disposition of the CD8 globular headpiece relative to the cell surface, CD8beta 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 CD8beta 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.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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