Immunology 1986 59 195-201

Biochemical characterization of the T-cell alloantigen RT-6.2

H.-G. THIELE, F. KOCH, A. HAMANN & R. ARNDT* Abteilungfuir Immunologie, Medizinische UniversitdtsklinikHamburg-Eppendorf, Hamburg, West Germany

Acceptedfor publication 3 June 1986

SUMMARY

This study presents the partial molecular characterization ofthe alloantigenic rat T-cell marker RT-6.In contrast to most lymphocyte surface membrane proteins, RT-6 proved to be resistant to short-term (15 min) solubilization by T X- 100 at 4°, but could be efficiently solubilized by long-term (16 hr)exposure at 4° or short-term (15 min) exposure at 37°. SDS-PAGE analyses of RT-6.2immunoprecipitates under non-reducing conditions revealed two bands with apparent molecularweights of 21,000 and 24,000 (25,000 and 28,000 under reducing conditions). Radiolabelled sugarsfailed to be incorporated metabolically into RT-6.2, neither endoglycosidase-F and 0-glycanasedigestion norNaOH treatment caused any detectable decrease in the molecular weight of RT-6.2, andRT-6.2 failed to bind to any of a series of agarose-coupled lectins with a wide range of sugar-bindingspecificities. These observations suggest that RT-6.2 may lack carbohydrate moieties.

INTRODUCTION

The rat T-lymphocyte alloantigen system, formerly referred toas ART-2 (Lubaroff et al., 1979a, b), Pta (Butcher & Howard,1977), Ag-F (DeWitt & McCullough, 1975) and RT-Ly-2(Wonigeit, 1979), is now designated RT-6 (Lubaroff et al.,1983). This alloantigenic system is well defined in serologicalterms. The antigen exists in at least two allelic haplotypes, whichare defined by the reference strains BHRT-62 and LEWRT-6 I andtheir congenic counterparts BHRT-6 I and LEWRT-62 Theseantigens have been shown to be expressed selectively on thesurface membranes of the majority, if not all, peripheral T cells,but not on thymic lymphocytes (Wonigeit, 1979, and our ownunpublished observations). The antigens are coded for by genesmapped in linkage group 1 (Gasser, 1981). In a preliminarypaper (Thiele et al., 1979b), we suggested that the RT-6determinants were expressed by a 21,000 peptide.

The present study provides further evidence to indicate thatRT-6 determinants are found on a non-glycosylated protein(Thiele, Arndt & Wonigeit, 1983).

* Present address: Labor Dr Keeser, Poppenbuttler Bogen 25, 2000Hamburg 65, West Germany.

Abbreviations: Con A, concanavalin A; DTBP, dimethyl-3-3-dithio-bis-propionimidiate-hydrochloride; DTE, di-thiothreithol; Endo-F,endoglycosidase F; gp, glycoprotein; LNLy, lymph node lymphocytes;NaDOC, sodium deoxycholate; PAGE, polyacrylamide gel electro-phoresis; PMSF, phenylmethylsulphonfluoride; SDS, sodium dodecylsulphate; TBS, Tris-buffered saline; ThyLy, thymic lymphocytes; T X-100 and T X-1 14, Triton X-100 and Triton X-1 14.

Correspondence: Prof. Dr H.-G. Thiele, Abteilung fur Immunolo-gie, Medizinische Universitiitsklinik, Martinistrale 52, 2000 Hamburg20, West Germany.

MATERIALS AND METHODS

Antisera and monoclonal antibodiesAn allo-anti-RT-6.2 serum (a-RT Ly-2.2) was a gift from Dr K.Wonigeit, Hannover. A monoclonal rat antibody Gy 1/12(IgG2c) reacting with RT-6.2 as well as the corresponding

I hybridoma were kindly provided by Dr G. W. Butcher,I Cambridge, U.K. The following monoclonal antibodies

(ascites) were kindly provided by Dr A. F. Williams, Oxford,U.K.: W3/25 recognizes the 48,000-53,000 gp marking rat T-helper cells; MRC OX-7 recognizes the 25,000 Thy-1.1; MRCOX-18 recognizes the MHC class I gp; MRC OX-19 recognizesa 70,000 gp expressed by all peripheral rat T cells and thymiclymphocytes.

CellsSuspensions of thymic lymphocytes (ThyLy) and lymph nodelymphocytes (LNLy) of rats (strains LEWRT-62 Han, and DaRT-62/Han) were prepared as described previously (Thiele et al.,1979a).

RadiolabellingCell surface iodination with Na'25I (Amersham International,Amersham, Bucks, U.K.) was catalysed by lactoperoxidaseaccording to Marchalonis, Cone & Santer (1971).

For metabolic incorporation of radiolabelled carbo-hydrates, Con A-induced LNLy-blast cells (2 x 107) were incu-bated in 10 ml RPMI (Gibco, Grand Island, NY) containing 2mm glucose, 15 mm HEPES, 100 U penicillin/ml, 100 pgstreptomycin/ml, 50 pM mercaptoethanol and a mixture oflabelling sugars [1 pCi of '4C-galactose, 14C-glucasomine, '4C-

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mannose (all 60 mCi/mM, Amersham), 10 pCi of 3H-fucose (5 4Ci/mM, Amersham) per ml] according to van Eijk & Muhlradt(1977). For metabolic peptide labelling, Con A-induced LNLyblast cells (2 x 106/ml) were incubated in RPMI (Gibco selecta-mine kit) in which leucine, lysine, alanine, cysteine and methio-nine were replaced by 15 pCi/ml of 3H-leucine (130 Ci/mM), 3H-lysine (87 Ci/mM), 1 5 pCi 14C-alanine (165 mCi/mM) or 150 pCi/ml 35S-cysteine (59 Ci/mM) and 35S-methionine (1 130 Ci/mM) (allfrom Amersham) for 5 hr at 370 in 5% Co2.

For metabolic lipid labelling, Con A-induced LNLy blastcells (3 x 106/ml) were incubated for 22 hr at 370 in 5% CO2 inDulbecco's MEM with 800 pCi/ml 3H-palmitic acid (23 5 Ci/mm, New England Nuclear, Boston, MA).

Solubilization proceduresThe following detergents were used for solubilization: T X-100(Riedel de'Haen), T X-1 14 (Serva), Lubrol PX (Sigma, Poole,Dorset, U.K.) and sodium deoxycholate (Merck, Darmstadt).Surface radioiodinated or metabolically labelled cells werewashed in PBS and resuspended (107 cells/ml) in solubilizationbuffer: TBS pH 8-0, 0-2% or 0 5% (v/v) of the respectivedetergent, 1 mm EDTA, 1 mm phenylmethansulphonylfluoride(PMSF) and/or other protease inhibitors (pepstatin, antepain,bestatin, chymostatin). Twenty-five pg DNAse and 25 pgRNAse/ml were included during solubilization with deoxycho-late. Cells were disrupted by incubation at 4° or 37° for 15 min.Nuclei and other insoluble material were removed by centrila-gation for 10 min at 600 g. The pellets were resuspended insolubilization buffer and incubated either for 15 min at 370 orovernight at 0°. In some experiments a two-step solubilizationprocedure was performed in which cells were first solubilizedwith T X-100 at 0° for 10 min. The detergent-resistant materialwas pelleted (600g for 10 min at 00) and resolubilized with 0 2%sodium deoxycholate for another 10 min at 0°. The solubilizedmaterial ofeach step was cleared by centrifugation at 10,000g or100,000 g for 30 min and subjected to immunoprecipitation andanalysis by SDS-PAGE. All solubilization procedures werecarried out in the absence or presence of 1 mm Ca2+ and Mg2+.

Immunoprecipitation and SDS-PAGEDetergent extracts were preabsorbed for 2 hr at 4° with proteinA-Sepharose (Pharmacia, Uppsala, Sweden) and normal ratserum. The supernatants were incubated for 2 hr at 40 with theappropriate antibody and protein A-Sepharose as describedpreviously (Thiele et al., 1979b). Immunoprecipitates werewashed, extracted with sample buffer, and analysed by electro-phoresis on 12% polyacrylamide slab gels according to Maizel(1971) under reducing or non-reducing conditions. 3H-, l4C-,and 35S-radiolabelled bands were traced by fluorography ofamplify (Amersham)-impregnated gels by exposing Kodak X-Omat AR film to the dried gel at - 70°-and '251-labelled bandswere traced by direct autoradiography of the gels.

Density gradient centrifugationLysates (0-2 ml) from '251-labelled LNLy cells were centrifugedon a linear sucrose gradient (5-25%, 0 2% T X-100, 1 mMEDTA, TBS, pH 8-0, volume 4-8 ml) for 15 hr at 100,000 g(Beckman rotor SW 50). Parallel calibration runs were per-formed with 0 8 mg each of trypsin inhibitor (MW 21,000),Fractions of 0 3 ml were collected, immunoprecipitated with

anti-RT-6.2 plus protein A-Sepharose and analysed by SDS-PAGE.

Cross-linking with DTBPCross-linking experiments were performed according to Snaryet al. (1977). Briefly, cell lysates (from 2 x 106 cells, solubilizedfor 18 hr at 00 with 0 5 Triton X- I00), were reacted with varyingamounts (0 1-5 mg/ml) ofDTBP for 60 min at 20°. The reactionwas stopped by the addition of 0 1 volumes of 1 M ammoniumbicarbonate for 15 min.

Lectin- and sugar-binding experimentsLysates from 2 x 106 '25l-labelled solubilized LNLy (0-5% T X-100, 18 hr, 00) were preabsorbed with Sepharose and incubatedfor 60 min at room temperature in binding buffer (TBS, 0 2% TX-100, 1 mM Ca2+, 1 mMMg+, 1 mM Mn2+) with 50p1 of one ofthe following lectin- (medac) or sugar-agarose preparations: D-mannose (D-glucose)-binding lectins: concanavalin A, lentil; N-acetyl-D-glucosamine-binding lectins: wheat germ agglutinin,potato, gorse, GS II; N-acetyl-D-galactosamine-binding lectins:edible snail, lima bean, Japanese pogoda tree; D-galactose-binding lectins: peanut, castor bean; L-fucose-binding lectins:gorse; sialic acid-binding lectins: horseshoe crab. carbo-hydrates: fucose, N-acetylgalactosamine, N-acetyl-glucosa-mine, galactose, mannose, fl-glucuronide, thiomannopyrano-side, fl-D-lactose, thiofucopyranoside, melibiose, sialic acid.After washing in TBS containing 0 1% T X- 100, the beads wereeluted for 40 min at room temperature with 200 p1 of theappropriate sugar (0 5 M) in TBS supplemented with 0-1% T X-100 as well as I mm Ca2+, Mg2+ and Mn2+. Non-bound andeluted material was subjected to immunoprecipitation andanalysis by SDS-PAGE.

Endoglycosidase-F and 0-glycanase digestionT X-100 solubilisates (50 p1) of I x 106 surface radioiodinatedLNLy were immunoprecipitated with Gy 1/12, anti RTIA, W3/25 and protein A-Sepharose. For Endo-F-treatment, immuno-precipitates were washed twice with TBS-0 1% T X-100 andonce with 0-1 M Na-phosphate, 50mM EDTA, pH 6 1, boiled for2 min in 0 05% SDS, 1% mercaptoethanol in 01 M Na-phosphate, 50 mm EDTA, pH 61. Samples were adjusted to0.5% T X-100 and digestion with endoglycosidase-F (NewEngland Nuclear) was carried out for 4 hr at 370 under constantshaking. For 0-glycanase treatment, immunoprecipitates werewashed three times in 0 02 M Tris-maleate buffer containing 1mm calcium acetate, 0-6% T X-100 and 0 1% SDS (p11 7-0).Samples were then reacted for 45 min with neuraminidase(Behring, Marburg, FRG) (dialysed before usage against theTris-maleate buffer) and thereafter exposed for 4 hr at 370 to 0-glycanase (Genzyme, Boston, MA).

Samples were heated for 2 min at 950 and analysed by SDS-PAGE. Control samples were incubated with 6% or 25%glycerol instead of Endo-F and 0-glycanase, respectively.

Mild alkaline hydrolysisMild alkaline degradation was carried out according to Gahm-berg (1983). Samples of 2 x I07 surface radioiodinated LNLywere disrupted by T X-100 (0 5%, 15 min) at 37°. Solubilisateswere preabsorbed (60 min, 40) with normal rat serum coupled toprotein A-Sepharose and then immunoprecipitated with Gy 1/12 (60 min, 40) and W3/25 (60 min, 40) and protein A-

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Sepharose. Bound material was dissociated in 2% SDS (2 min,950) and reacted either with TBS (pH 7-2) or with 50 mm NaOH,0-05% SDS for 30 min at 800. The hydrolysed material wascooled to 00 and neutralized with 1 M HC1. After dialysis againstH20 (5 hr, 40), samples were lyophilized, boiled in 1% SDS (3min, 950) and subjected to SDS-PAGE (12% gel) underreducing (DTE) and non-reducing conditions, respectively.

Phase separation in T X-114Phase separation of integral membrane proteins of LNLy in TX-1 14 was performed according to Bordier (1981). Briefly, T X-114 lysates of surface radioionidated cells (200 Ml) were layeredon 300 p1 6% (w/v) sucrose containing 0-06% T X-1 14, 1 mmEDTA in TBS, pH 7 2, warmed up to 300 for 3 min, andcentrifuged for 30 min at 300 g. The upper aqueous phase wasadjusted to 0 5% T X-1 14 (with 10% T X-1 14) and cooled to 00,placed on the same sucrose cushion that had been used for thefirst run, warmed to 300 for 3 min and centrifuged at 250. Theaqueous phase was cooled in an ice-bath and adjusted to 2% TX- 1 14. After rewarming to 300 and another centrifugation (300g), the aqueous phase (200 Ml) was mixed with 200 p10 5% T X-114. The detergent phase (4 5 pl) was mixed with 400 p1 TBS at4°. The aqueous, sucrose and detergent phases were monitoredfor radioactivity and subjected to immunoprecipitation andanalysis by SDS-PAGE.

RESULTS

Solubilization behaviour of RT-6.2

When '25l surface-labelled LNLy (lymph node lymphocytes) ofThyLy (thymus lymphocytes) were extracted with 0 2% or 0 5%T X-100, T X-1 14, Lubrol PX, or sodium-deoxycholate for 15min at 0-40, more than 95% of the plasmalemma-boundradioactivity was found in the 10,000 g supernatants, indicatingthat most of the radiolabelled cell surface proteins weresolubilized. Following precipitation with the appropriate anti-bodies, analyses by SDS-PAGE showed that all of OX19,OX1 8, most of W3/25, but only traces of Thy- 1.1 (ThyLy) andRT-6.2 (LNLy) were solubilized by this procedure. When thedetergent-resistant pellets were reincubated for 16 hr at 00 in thepresence of one of these detergents, the bulk of RT-6.2 (LNLy)and the rest of Thy- 1.1 (ThyLy) were found to be solubilized(Fig. 1). Detergent extraction in the absence or presence ofCa2+or Mg2+ did not affect the solubilization pattern. In contrast toThy-1.1 (Letarte-Muirhead, Acton & Williams, 1974), RT-6.2was not solubilized more efficiently by Lubrol PX than bydetergents of the T X series.

Consecutive 10-min exposures ofLNLy cells to T X-100 andsodium deoxycholate at 00 effectively released RT-6.2 as did a10-min exposure to any of the detergents at 370 (not shown).

Since T X-1 14 exhibits a low cloud point at about 200(Goldfarb & Sepulveda, 1969), this detergent allows for phaseseparation of integral and non-integral membrane proteins(Bordier, 1981). After solubilization with T X- 1 14 at 00 for 16 hrand subsequent phase separation at 300, RT-6.2 was foundexclusively within the detergent phase (Fig. 2), suggesting thatRT-6.2 is an integral membrane protein.

Lymph node cells Thymic lymphocytesOX19 OX18 W3/25 GyV12OX-7 OX19

NS a b a b a b a b a b o b NS M

Figure 1. Comparison of the solubilization behaviour of surfacemembrane proteins of DA-rat lymphocytes as recognized by monoclon-al 0X19, 0X18, W3/25 and Gy 1/12 (lymph node cells) as well as 0X7and OX19 (thymic lymphocytes). i251 surface-labelled cells were dis-rupted by exposure to 0-5% T X-100 for 15 min at 40 (a). Non-solubilized material was resolubilized in 0 5% T X-1I00 for 15 min at 370(b). Aliquots of solubilisates (a) and (b) were first reacted with normalDA-rat serum (NS) and subsequently immunoprecipitated with one ofthe respective antibodies and subjected to SDS-PAGE (non-reduced)autoradiography. 'IC marker proteins (M): lysozyme 14,000, carboan-hydrase 30,000, ovalbumin 46,000, bovine serum albumin 69,000.phosphorylase 92,000, myosin 200,000 (Amershamn)

Total Aqueous Detergentsolubilisate phase phase

-69,000

46000

.30,000

& ~~,,~~ -14,000

Figure 2. Phase separation of DA-rat lymph node cell surface membraneproteins in T X-l 14. i251 surface-labelled cells were disrupted byexposure to 0-5% T X-l1 14 for 16 hr at 40. Solubilisates were warmed to300, layered on a 6% sucrose cushion and centrifuged (30 min) in thepresence of T X-1 14. After separation, aliquots of the aqueous anddetergent phases as well as of the unseparated primary solubilisates werecooled to 40 and reacted with either normal rat serum (Tracks 1, 4 and7), Gy 1/12 (Tracks 2, 5 and 8) or Lewis anti-DA-rat serum (Tracks 3, 6and 9).

Analysis by SDS-PAGESDS-PAGE analyses of solubilized and immunoprecipitatedRT-6.2 from radioiodinated cells revealed two bands withapparent molecular weights of21000 and 24,000 (non-reduced)and 25,000 and 28,000 (reduced), indicating that RT-6.2

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H.-G. Thiele et al.

consists of polypeptide chains with intrachain disulphidebridge(s) (Fig. 3). Even when a density gradient centrifugationstep was intercalated between solubilization and immunopreci-pitation, RT-6.2 appeared as a 21,000/24,000 doublet uponSDS-PAGE (not shown). The molecular forms of RT-6.2 couldnot be cross-linked to each other or to other membraneous orsubmembraneous constituents by varying amounts of the cross-linker DTBP.

Metabolic incorporation studies

Biosynthetic studies using radiolabelled amino acids revealed a

low but significant incorporation into RT-6.2. In correspondingstudies with radiolabelled sugars no incorporation into RT-6.2could be detected, while significant labelling of RT-IA (MHCclass I) with carbohydrates was found (Fig. 4). Moreover, RT-6.2 could not be labelled metabolically with 3H-palmitic acid(not shown). These results suggest that RT-6.2 may not beglycosylated and may not be covalently linked to palmitic acid.

14,000-

®Front -

Figure 4. Metabolic incorporation studies of3H- and '4C-labelled sugars

(Tracks 1 and 2) or amino acids (Track 3) in Con A-stimulated (66 hr at370) lymph node cells. T X- 100 solubilization: 16 hr at 40, immunopreci-pitation with anti-RT Ly-2.2 (Tracks 1 and 3) or Lew anti-DA-rat serum(Track 2).

Lectin- and sugar-binding studies

For lectin-binding experiments, RT-6.2 was immunoprecipi-tated from lysates of radioiodinated LNLy cells and allowed toreact with a series of agarose-coupled lectins with a widespectrum of sugar-binding specificities, including those for D-

mannose, L-fucose, N-acetyl-galactosamine, D-galactose, N-acetyl-glucosamine, N-acetyl neuraminic acid, and glucuronicacid. Of all the lectins tested, only Con A was found to bindtraces of '25I-labelled, solubilized RT-6.2. This lectin, however,is known to contain-in addition to its specific carbohydratebinding site-a hydrophobic cleft by which it may interact non-

specifically with hydrophobic moieties. Moreover, non-specificcharge-charge interactions between this lectin and variousmacromolecules have been observed (Davey et al., 1974; Doyle,Woodside & Fishel, 1968; Podder, Surolia & Bachhawal, 1974).Since on the one hand only traces of RT-6.2 are retained by ConA agarose beads, and on the other hand the bound RT-6.2 can

be dissociated not only by the appropriate sugar but also non-

specifically by glucuronic acid, it seems unlikely that the traces

2

Figure 3. Apparent molecular weights of RT-6.2 under reducing andnon-reducing conditions. I251-labelling, immunoprecipitation and SDS-PAGE autoradiography as in Fig. 1 (T X- 100 16 hr at 40). Track 1, non-reducing conditions; Track 2, reducing conditions.

of RT-6.2 actually observed to bind to Con A do so by means ofspecific carbohydrate-lectin interaction. The results of theseexperiments lend additional support to the suggestion that RT-6.2 is not glycosylated.

Sugar-binding experiments revealed that RT-6.2 has no

binding capacity to any of the sugars tested.

Endoglycosidase-F and 0-glycanase treatment

In order to determine whether RT-6.2 contains any N- or o-

glycosylation sites, the molecules were subjected to digestionwith endoglycosidase-F, which cleaves N-linked sugars of boththe high mannose and the complex type (Elder & Alexander,1982) or to 0-glycanase known to hydrolyse the Gal-f (1-3)-Gal-Nac disaccharide linked to either serine or threonine inmucin type glycoprotein (Umemoto, Bhavanandan & David-son, 1977). Lysates of radioiodinated lymph node cells were

immunoprecipitated with Gy 1/12, W3/25 or anti-RT-IA andincubated for 4 hr at 370 in the presence of one of theendoglycosidases or glycerol and analysed by SDS-PAGE.Figure 5a shows that Endo-F treatment does not cause a shift inthe apparent molecular weight of RT-6.2, whereas it does cause

a significant reduction in the apparent molecular weight of thegp RT-1A. Similarly, O-glycanase treatment does not affect themolecular weight of RT-6.2 but causes a shift in the molecularweight of the gp recognized by W3/25 (Fig. 5b).

Mild alkaline treatment

Comparable results were obtained after mild alkaline treatment,which is known to hydrolyse the o-glycosidic linked oligosac-charides from glycoproteins. This treatment causes a decrease inthe molecular weight of gp W3/25 but not to RT-6.2 whenanalysed under reducing conditions in SDS-PAGE (Fig. 5c).Interestingly, under non-reducing conditions the molecularweight of RT-6.2 shifted from 21,000 to 25,000 after alkalinetreatment (Fig. 5c). This phenomenon is most easily explainedby disulphide exchange as occurring in slightly alkaline solu-tions (Ryle & Sanger, 1954).

)Stort -

69,000-46,000-

30,000-

198

1 2 3

21,000

Rat T-cell alloantigen RT-6

(a) 2 3 4

30,l

(c) 1 2 3 4 5 6 7 8

-67,00

-46,000

-30,000

-14,000

_ - + _-+ - +

-14,000

Reduced Non-reduced

Figure 5. Sensitivity of RT-6.2 to endoglycosidase-F, 0-glycanase and mild alkaline hydrolysis. For experimental protocol, see text.

(a) Endoglycosidase-F, Tracks 1 and 2: RT-6.2, Tracks 3 and 4: RT-1 A (Endo-F +, glycerol -). (b) O-glycanase. Tracks 1 and 2: W3/25; Tracks 3 and4: RT-6.2 (0-glycanase+, glycerol -). (c) NaOH hydrolysis: solubilisates were immunoprecipitated first with Gy 1/12 (Tracks 1, 2, 5 and 6), then withW3/25 (Tracks 3, 4, 7 and 8) (NaOH +, neutral pH -). Tracks 1-A under reducing conditions, Tracks 5-8 under non-reducing conditions.

DISCUSSION

The present study shows that approximately 95% of the'ectoproteins' of 1251 surface-labelled sIg- rat lymph node cellscan be solubilized by a 15-min exposure at 40 to 0 2% or 0 5% TX-100, T X-1 14 Lubrol PX, or NaDOC, an observation inaccord with data recently presented by others (Brown, Lewin-'son & Spudich, 1976; Crumpton, Davies & Wigglesworth, 1981;Mescher, Jose & Balk, 1981; Schliwa & van Blerkom, 1981). Incontrast, most of the RT-6.2 and, when using thymic lympho-cytes, the Thy-1.1, proved to be resistant to this solubilizationprocedure, but can be solubilized either by a short-term (15-min)detergent exposure at 37° or by a long-term (overnight)detergent exposure at 4°.

The resistance of the RT-6.2 molecule to short-term expo-

sure to 0-5% detergents of the T X series and sodium deoxycho-late raises some questions concerning its anchorage within theplasmalemma. Thus, it may be anchored to the so-called 'tritoncytoskeleton' (Brown et al., 1976; Osborne & Weber, 1977;Schliwa & van Blerkom, 1981). However, it seems unlikely thatRT-6.2 is linked to microtubules since neither low temperaturenor Ca2+ concentrations as high as 1 mm, which are known todepolymerize microtubules (Osborne & Weber, 1977; Schliwa et

al., 1981) altered the solubilization behaviour of RT-6.2.There is now indeed a good deal of evidence to suggest that

some of the membrane lipids remain selectively associated withthe T X-100-insoluble fraction (Mescher et al., 1981; Yu,Fischmann & Steck, 1973) and, moreover, that in some cases

essential lipid may be so tightly bound to a particular proteinthat it is not removed even when an excess of detergent isemployed (LeMaire, Moller & Tanford, 1976; Tanford &Reynolds, 1976). In contrast to the transferrin receptor, whichcan be labelled biosynthetically with 3H-palmitic acid (Omary &Trowbridge, 1981), 3H-palmitic acid could not be incorporatedinto RT-6.2. This, however, does not exclude the association ofRT-6.2 with fatty acids. Interestingly, evidence has been putforward recently (Low & Zilversmit, 1980; Low & Kincade,1985) to suggest that some membraneous proteins, including theThy-I glycoproteins, are covalently bound to phosphatidyli-

nositol residues within the membrane. Corresponding experi-ments with respect of RT-6.2 are presently in preparation (seeNote added in proof).

Our results show that solubilized RT-6.2 migrates in SDS-PAGE under non-reducing conditions as a doublet withapparent molecular weights of 21,000 and 24,000. Underreducing conditions, the apparent molecular weights of theimmunoprecipitable RT-6.2 doublet are shifted to 25,000 and28,000, indicating that both forms of RT-6.2 contain intrachaindisulphide links.

These results differ from the data previously published byWilliams & DeWitt (1976), who reported the isolation of a

35,000-40,000 polypeptide from rat lymphocyte membranesthat was proposed to carry the AgF-1 determinant. Williams &DeWitt (1976) raised their alloantiserum by immunizing Lewisrats with Fisher rat lymphocytes. After they had published theirdata, evidence was put forward to suggest that this combinationof strains differs not only in RT-6 allotypes but also in their RT-1C class I MHC determinants (Lew rats = RT- IC1, Fi rats = RT-1Cvlv (Haustein, Stock & Gunther, 1982; Stock & Gunther,1981). The latter antigen is a heterodimeric glycoproteincomposed of 40,500-43,000 and 12,500 chains, and is exposedby the majority of B and peripheral T lymphocytes (Haustein et

al., 1982). Based on these observations, it is plausible thatWilliams & DeWitt (1976) isolated the heavy chain of the RT-IC product. Our results suggest that the short-term solubiliza-tion procedure (0-5% NP 40, 20 min at 40) employed byWilliams & DeWitt (1976) would solubilize the heavy chain ofMHC antigens, but not RT-6.

Based on some preliminary observations, we had previously(Thiele et al., 1983) suggested that RT-6.2 may not be glycosy-lated. The data presented here strongly support this suggestion.Thus, RT-6.2 did not bind to any of a wide range of lectins

tested, and its relative molecular weight was affected neither bydigestion with endoglycosidase-F or 0-glycanase nor by treat-ment with NaOH.

In biosynthetic labelling studies with an assortment ofradiolabelled sugars, the class I MHC RT-IA product was

distinctly labelled, while no incorporation of these sugars into

199

-67X000

-46,000

-30,000

-14 cYm=vw

200 H.-G. Thiele et al.

RT-6.2 could be detected. It cannot be excluded, however, thatthe lack of sugar incorporation into RT-6.2 is due to a low rateof synthesis in rat lymphocytes, since the incorporation ofradiolabelled amino acids into RT-6.2-although unequivo-cally detectable-appears low in comparison to other mem-brane proteins. In order to study more effectively the meta-bolism of RT-6.2, we have constructed a rat T-cell hybridomaexpressing RT-6.2 (Koch & Thiele, 1985).

Additonal examples of exceptions to the generally acceptedrule that most, if not all, integral membrane proteins areglycosylated have recently been described, e.g. the humanRho(D) antigen of erythrocytes (Gahmberg, 1983) and the c-chain of the human T-3 complex (Borst et al., 1983). Like theRho(D) protein, RT-6.2 was not affected by trypsin treatment(our unpublished observations). Preliminary experiments con-ducted in our laboratory revealed that RT-6.2-in contrast tothe T-3 molecule (Brenner, Trowbridge & Strominger, 1985)-could not be cross-linked in situ to any other membraneconstituent. Nevertheless, it is of interest to study whether RT-6.2 is related to the a-chain of T-3. Molecular cloning experi-ments presently under way in our laboratory should answer thisquestion. Moreover, it will be of interest to determine whetherthe non-glycosylation of these membrane proteins is of any(common) functional significance.

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BORST J., ALEXANDER S., ELDER J. & TERHORST C. (1983) The T3complex on human T lymphocytes involves four structurally distinctglycoproteins. J. biol. Chem. 258, 5135.

BRENNER M.B., TROWBRIDGE I.S. & STROMINGER J.L. (1985) Cross-linking of human T cell receptor proteins: association between the Tcell idiotype subunit and T3 glycoprotein heavy subunit. Cell, 40, 183.

BROWN S., LEWINSON W. & SPUDICH J.A. (1976) Cytoskeletal elementsof chick embryo fibroblasts revealed by detergent extraction. J.Supramol. Struc. 5, 119.

BUTCHER G W. & HOWARD J.C. (1977) An alloantigenic system on ratperipheral T cells. Rat News Letter, 1, 12.

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Note added in proof

After the submission of this report, we found that RT-6.2 can bereleased from lymphocytic cells by a highly purified phosphati-dylinositol-specific phospholipase-C, indicating that RT-6 is

anchored within the cell membrane by a covalent linkage tophosphatidylinositol [Koch, Thiele & Low, J. exp. Med (inpress)].