Biosynthesis of glycophospholipid bound and secreted murine class I Qa-2 polypeptides

Biosynthesis of glycophospholipid bound and secreted murine class I Qa-2 polypeptides

Molecular Immunology, Vol. 28, No. 11, pp. 1299-1310, 1991 Printed in Great Britain. 0161-5890/91 $3.00+ 0.00 © 1991 PergamonPress pie BIOSYNTHESIS ...

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Molecular Immunology, Vol. 28, No. 11, pp. 1299-1310, 1991 Printed in Great Britain.

0161-5890/91 $3.00+ 0.00 © 1991 PergamonPress pie

BIOSYNTHESIS OF GLYCOPHOSPHOLIPID B O U N D A N D SECRETED M U R I N E CLASS I Qa-2 POLYPEPTIDES GREGORY P. EINHORN,* LU QINt and MARK J. SOLOSKIt~ *BRB/NIAID/NIH, Bldg 4, Bethesda, MD 20892, U.S.A. and tDivision of Molecular and Clinical Rheumatology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A. (First received 6 June 1990; accepted in revised form 16 January 1991)

Abstraet--Murine T cells synthesize and express a cell-surface glycophospholipid anchored 40 kDa and a secreted water-soluble 39 kDa Qa-2 polypeptide. We have examined the biosynthetic pathways which lead to the production of the membrane-bound and water-soluble isoforms of the Qa-2 molecule. Using the detergent TX-114, both detergent (membrane)-bound and soluble Qa-2 polypeptides can be identified in cell lysates and can be distinguished by charge and molecular weight. Two membrane-bound forms, a 40-kDa Endo H resistant cell-surface form and a 38 kDa-Endo H sensitive form can be identified, both of which can be biosynthetically labeled with 3H-ethanolamine and can be converted to water soluble forms by digestion with a phosphatidylinositolspecific phospholipase C. In addition, several water soluble polypeptides at 39, 37, 35 kDa, and a minor species at 33 kDa were identified, none of which radiolabel with 3H-ethanolamine. While the 39-kDa polypeptide was Endo H resistant, the other isoforms were sensitive to Endo H digestion. Pulse chase experiments and molecular weights of the deglycosylated core polypeptides suggest a precursor to product relationship between the intracellular water-soluble species and the mature 39-kDa secreted Qa-2 molecule. This relationship is supported by the observation that murine L cells transfected with the Qa-2 encoding class I gene Q7 fail to express membrane-bound Qa-2 molecules yet synthesize both intracellular water-soluble and secreted Qa-2 molecules. These findings argue for a pathway in which secreted soluble Qa-2 molecules are derived from intracellular precursors.

INTRODUCTION

Gene products of the major histocompatibility complex have been shown to serve key roles in the function and regulation of the immune system. Murine class I H-2K, H-2D and H-2L molecules function in T cell recognition of foreign antigen and as potent stimulators of tissue graft rejection (Zinkernagel and Doherty, 1974). Molecular genetic analyses have identified up to 35 distinct murine class I genes (Weiss et al., 1984; Steinmetz et al., 1982; Flavell et aL, 1986). The majority of these genes are located telomeric to the H-2D loci, in the Qa and TL regions of the murine 17th chromosome (Weiss et aL, 1984; Winoto et al., 1983). Serological and biochemical studies have demonstrated six distinct Qa/TL molecules; expressed on the cell surface are the Qa-1, Qa-2, TL and "37" molecules (Stanton and Hood, 1980; Michaelson et aL, 1977; Vitetta et al., 1972; Cochet et al., 1989), and two secreted molecules; Qb-1, and Q10 (Robinson, 1987; Cosman et al., 1982) encoded within this chromosomal region. All the Qa/TL molecules exhibit typical class I structure, consisting of a heavy chain polypeptide, non:~Author to whom correspondence should be addressed. Abbreviations: fl2m,//-2 microglobulin; B6, C57BL/6 mice; Con A, concanavalin A; Endo H, endoglyeosidase H; Endo F, endoglycosidase F; NMS, normal mouse serum; PIPLC, phosphatidylinositol specific phospholipase C; TX-II4, Triton X-I14.

covalently associated with fl-2 microglobulin (/~2m), and are predicted to fold forming a peptide binding groove (Prochnicka-Chalufour et al., 1989; Stroynowski, 1990). The cell surface Qa-1, Qa-2, TL and "37" molecules differ significantly from H-2K, D and L as they have not been shown to serve as T cell restriction elements, in some cases are expressed primarily on tissues of hematopoietic origin and display limited genetic polymorphism (Flaherty, 1981). The function(s) of class I molecules encoded within the Qa/TL subregion are not known. The Qa-2 molecule is a cell surface protein of 40,000 mol. wt which is non-covalently associated with f12m and has been shown to be membraneanchored via a glycophospholipid (Stiernberg et al., 1987; Stroynowski et al., 1987; Waneck et al., 1988; Soloski et al., 1988b). Thus Qa-2 joins a growing list of mammalian proteins similarly anchored, including decay accelerating factor (DAF), neural and cell adhesion molecules (N-CAM and LFA-3), scrapie prion protein (PrP), Thy-1 and T cell activating protein (TAP) (reviewed in Low and Saltiel, 1988; Low, 1989; Fergnson and Williams, 1988). Activated T cells can express both a 40-kDa cell surface and a 39-kDa secreted Qa-2 polypeptide (Soloski et al., 1986; Robinson, 1987). Based on reculturing studies, it was proposed that solublereleased Qa-2 molecules are derived from cell-surface molecules that were processed to a soluble form (Robinson, 1987). However, the bicohemical proper-

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ties of the secreted Qa-2 molecule are inconsistent with the secreted Qa-2 molecule being derived from cell-surface forms via release by an endogenous phospholipase (Soloski et al., 1988b). In addition L cell fibroblasts, when transfected with the Qa-2 encoding class I genes Q7 or Q9, fail to display any Qa-2 molecules on the cell surface yet synthesize a soluble polypeptide biochemically indistinguishable from the secreted Qa-2 molecule synthesized by activated T cells (Stroynowski et al., 1987; Soloski et al., 1988a). Based on these observations we have re-examined the biosynthetic pathways which lead to the production of membrane-bound and water-soluble intracellular Qa-2 polypeptides. These studies identify both watersoluble and membrane-bound intracellular isoforms which we propose are direct precursors for the mature secreted and cell-surface Qa-2 polypeptides.

Cell culture, isolation of lymphoid cell subpopulations and radiolabeling

Activated T cells were prepared by culturing spleen cells for 72hr in RPMI-1640 medium containing 5 % heat inactivated FCS, 50/z M 2-mercaptoethanol, 1-5/tg/ml concanavalin A (Con A), glutamine and antibiotics. These cells were purified to > 90% viability by centrifugation over Ficoll-Metrizoate. Thymidine kinase negative L cells (C3H fibroblasts, H-2K k) designated as L cells transfected with the Q7 b (C57BL/10) class I gene and Hepa/OVA cells transfected with either the Q7 b or Q9 b Class I genes were established previously (Stroynowski et al., 1987; Soloski et al., 1988a). L cell transfectants were maintained in alpha MEM containing hypoxanthine, aminopterin, thymidine, 5% FCS, glutamine and antibiotics while Hepa/OVA transfectants were maintained in the same medium with containing G418. MATERIALS AND M E T H O D S Activated T cells, L cells and Hepa/OVA cells were metabolically labeled with 35S-methionine (35SMaterials translabel (ICN), averaging 1000Ci/mmol or 35SAlpha MEM, RPMI-1640, Hanks Balanced Salt methionine (NEN), averaging 1100 Ci/mmol) at a solution were obtained from Whittaker MA. Bio- concentration of l 0 7 cells/ml in MEM without products (Walkersville, MD); Spinners Balanced Salt methionine. All 3SS-biosynthetic labelings were solution and MEM non-essential amino acids from carried out at a concentration of 300/~Ci/ml. For Inland Laboratories (Austin, TX). Ultrapure protein pulse-chase and pulse experiments cells were incuA was from Genzyme (Boston, MA). 35S-L- bated in media without methionine for 20 min before methionine, Endo F (700 units/ml) and Endo H adding radiolabel. For pulse-chase experiments cells (45 #g/ml) were from N E N (Boston, MA). Phos- were pulsed for 20 min with radiolabel before adding phatidylinositol specific phospholipase C (PIPLC) excess cold methionine. purified from B. thuringiensis was a kind gift from Dr Activated T cells were labeled with 3H-ethanolMartin Low, Columbia College of Physicians and amine by adding 0.50/~Ci/l& cells to 48 hr Con A Surgeons, New York, NY. 14C-methylated mol. wt. cultures. After 18 hr of labeling, cells and media were protein standards, [1-3H]ethan-l-OL-2-amine hydro- harvested. Ethanolamine-labeled cells were purified chloride (123mCi/mg) and Na 125I were from over Ficoll-Metrizoate and 1.1 x l0 s cells, 85-90% Amersham (Arlington Heights, IL). Trans:5S-label vital, were recovered. Ten percent of this purified was from ICN Radiochemicals (Irvine, CA). FCS population was recultured with 0.225 mCi 35S-methwas from Hyclone Laboratories Inc (Logan, UT). ionine for an additional 5 hr. Both cells and media Triton X-114 (TX-114) was from Fluka AG (Switzer- were also harvested after the methionine labeling. land). All other common chemical reagents were from Transfected Hepa/Ova and L cell lines were harSigma (St Louis, MO). vested by light trypsinization, washed in Hanks' Balanced Salt Solution with 10% FCS, and radioSerological reagents and mice labeled with 35S-methionine at a cell density of 107 Adult male C57BL/6 (B6) (H-2 b, Qa-2 ÷) and the cells/ml for 5 hr. For cell-surface analysis, activated congenic strains B6.K3 (H-2 k, Qa-2 ÷) and B6.KI T cells were labeled by lactoperoxidase-catalysed (H-2 b, Qa-2-) mice were used in this study. B6 and iodination as described by Goding (1984). B6.K3 strains were used to generate activated T cells; both have identical Qa-2 subregions. Mice were Lysis and phase separations with the detergent Triton either purchased from the Jackson Laboratory (Bar X 114 Harbor, ME) or bred at the Johns Hopkins UniverMetabolically labeled cells were lysed with 1% sity School of Medicine. Normal mouse serum (NMS) was purchased from Pel-Freeze (Rogers, TX-114 (Bordier, 1981) in the presence of protease AK). Rabbit anti-mouse immunoglobulin was pre- inhibitors ( l m M phenylmethyl sulfonyl fluoride pared by immunization of rabbits with purified IgG (PMSF), 0.01 M EDTA, 0.01 M benzamidine hydroand IgM myeloma proteins (Vitetta et al., 1976). chloride, 0.05 e-amino caproic acid, 20 mM iodoacMAbs used were 34-1-2 (IgG2a; anti-Qa-2) (Sharrow etamide, 10/~g/ml leupeptin, 1 #g/ml pepstatin and et al., 1984), and Hll.4.1 (IgG2a; anti-H-2K ÷) (Oi 100/zg/ml soybean trypsin inhibitor) in Tris-buffered et al., 1978). Affinity purifications of MAb were saline (TBS) pH 7.4 (lysis buffer). The amphiphile effected with Protein A coupled to Sepharose 4B Bromophenol Blue was added (40#M) to aid in isolating the micellar pellet as it distributes exclu(Pharmacia, Uppsala, Sweden).

Biosynthesis of Qa-2 sively in the detergent fraction. Metabolically radiolabeled culture supernatant was equilibrated to final concentrations of 1% TX-114, 1 mM PMSF, 0.01 M EDTA, 0.05 M E-amino caproic acid, 40/~M Bromophenol Blue. Iodinated cells were lysed as above. Cell lysates were centrifuged 3000g for 15min to remove nuclei and nuclei free cell lysates and cell free media were centrifuged at 10,000 g, 15 min. Detergent partitioning was effected as described by Goding (1984). Briefly, cell and media fractions were underlayed with 6% sucrose, 0.05% TX-114 in: 20mM Tris, pH7.4, 0.15M NaC1 with 0.01M EDTA and 0.05 M e-amino caproic acid (Buffer A). Cell and media fractions were warmed to 37°C until supramicellar aggregates formed. Fractions were then centrifuged 1000g for 5min and the detergent and aqueous fractions were harvested. The aqueous fraction was re-equilibrated with 1% TX-114 and repartitioned. This aqueous phase was harvested as the "water soluble fraction". Both detergent pellets were pooled, resuspended in buffer A, re-phase partitioned, and this detergent pellet recovered and resuspended in Buffer A (now designated the "membrane fraction").

Immunoprecipitations The procedures for preclearing of non-specific material with NMS and rabbit anti-mouse immunoglobulin have been described in detail (Soloski et al., 1986). Precleared cell and media fractions were immunoprecipitated with saturating amounts of the appropriate control (Hl l.4.1) or anti-Qa-2 (34-1-2) MAb. Immune complexes were removed with Protein A-Sepharose, washed three times with TBS containing 0.25% NP-40, 0.1% SDS and 0.5% deoxycholate pH 7.4, and once with TBS. Washed immune precipitates were either prepared for twodimensional analysis as described by O'Farrell (1975) with modifications suggested by Jones (1984) or reduced and denatured by boiling for 2 min in 0.06 M Tris, 10% w/v glycerol, 5% 2-meraptoethanol (2-ME) and 2.3% SDS (SDS sample buffer) in preparation for one-dimensional SDS-PAGE. Insoluble material was removed by centrifugation for 5 min at 10,000 g.

Enzymatic treatments Immunoprecipitates to be digested with endoglycosidases were solubilized by boiling in 0.2% SDS and 0.2% 2-ME. Samples to be mock treated or digested with Endo F were adjusted to 0.10 M sodium phosphate, pH 6.1, 0.05 M EDTA, 1% NP-40, 0.1% SDS and 1% 2-ME, while samples to be mock treated or digested with Endo H were adjusted to 0.10M sodium citrate, pH 5.5, 0.075% SDS and 0.2% 2mercaptoethanol. All samples were preheated to 95°C for 4min, cooled and PMSF and aprotinin were added as protease inhibitors before the addition of 1 Unit of Endo F or l#g/cc of Endo-H to the "digest" fractions. All fractions were incubated for

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5hr at 37°C. Following digestion, samples were dialysed with distilled H20 overnight, lyophilized and resuspended in SDS or IEF sample buffer for electrophoretic analysis. Phospholipase digests of precleared and TX-114 phase partitioned cell lysates was effected with PIPLC from B. thuringiensis (Low et al., 1988). Detergent soluble phases were split evenly in half for mock digest or PIPLC digestion with a 1/1000 dilution of a 300 unit/ml stock solution. Both mock and digest fractions were shaken for two consecutive rounds of: 1 hr at 37°C, 1 hr at 4°C; with a final 30-rain 37°C incubation after which the samples were centrifuged 1000g for 5min. The detergent pellet (membrane fraction) and aqueous (water soluble) phases were harvested without further treatment.

Polyacrylamide gel analysis and fluorography For one-dimensional analysis, 10-15% linear gradient or 12.5% acrylamide SDS gels were used (Laemmli, 1970). For gels containing 125I-labeled protein, molecular weight markers (BioRad, Richmond, CA) were run in parallel. Immunoprecipitates prepared for two-dimensional electrophoresis were run using isoelectric focusing in the first dimension followed by 10-15% linear gradient or 12.5% acrylamide gels as described (Soloski et al., 1986). 14C-methylated mol. wt. standards were run in parallel in the second dimension. After electrophoresis, gels containing all- or aSS-labeled protein were treated with Fluoro-hance (RPI Corp., Mt. Prospect, IL). Gels containing t25I-labeled proteins were stained with Coomassie Blue and extensively destained to localize mol. wt. standards. Gels were dried and exposed at - 7 0 ° C on Kodak XRP-5 films using Cronex lightening plus intensifying screens (Dupont, Wilmington, DE). Densitometric analysis of radiographs was effected with an LKB 2222-010 UltraScan XL (LKB Instruments, Gaithersburg, MD). RESULTS

Identification of membrane-bound and soluble Qa-2 polypeptides in cell lysates Based on previous studies outlined above we reasoned that secreted Qa-2 molecules are likely to be derived from intracellular precursors. We utilized phase partitioning with the detergent TX-114 to determine if water-soluble intracellular Qa-2 molecules were present and to evaluate their biochemical relationship to the secreted 39-kDa Qa-2 molecule. Activated T cells were radiolabeled with 35S-methionine for 5 hr or vectorially radiolabeled by lactoperoxidase catalysed iodination. Radiolabeled cells were lysed in TX-114, phase partitioned into aqueous (water soluble) and detergent (membrane bound) fractions and the distribution of Qa-2 molecules in each phase identified by immunoprecipitation. Cellfree media were similarly treated following addition of TX-114. Two-dimensional gel analysis of the Qa-2

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polypeptides recovered in the various fractions are shown in Fig. 1. All of the cell-surface 40-kDa Qa-2 molecule radiolabeled by lactoperoxidase catalysed iodination partitions in the membrane bound (detergent) phases (Fig. 1, panels E and F). In addition, all 35S-methionine radiolabeled secreted Qa-2 molecules found in cell-free medium can be recovered in the water soluble (aqueous) phase as a single 39-kDa species (Fig. 1, panels C and D). These observations agree with previously published data (Soloski et al., 1988b). Interestingly, when the 35S-methioninelabeled polypeptides found in cell lysates are examined, molecules reactive with anti-Qa-2 reagents can be recovered in both phases. Two major species of 40 and 38 kDa are recovered exclusively from the membrane phase (Fig. 1, panel A). The 40-kDa species appears similar to the cell surface Qa-2 molecule (compare panels A and E) while the 38-kDa species is a more basic intracellular form. A minor species at 37kDa is also observed in the membrane phase, (Fig. 1, panel A). Several polypeptides reactive with anti-Qa-2 reagents are recovered in the cell associated/water soluble phase (Fig. 1, panel B). The more acidic 39-kDa species is identical in its isoelectric focusing pattern to the secreted molecule (compared Fig. 1, panels B and D). In addition, three more basic

isoforms are seen exclusively in the cell-associated water-soluble fraction. These include a major species of 37 kDa, two minor species at 35 and 33 kDa and a 41~12-kDa species. This latter species is variably seen in different experiments and can be recovered in both the membrane bound and water soluble fractions. All of the Qa-2 species identified in the B6 cell lysates are also detected with anti Qa-2 serum. They were not observed when immunoprecipitates with irrelevant (H.11.4.1, anti H-2K ÷) antibodies were analysed nor were they identified in cell lysates from B6.K1 (Qa-2-) mice (data not shown). The membrane-bound and soluble Qa-2 polypeptides found in activated T cells are the products o f the Q7 b and Q9 b class I genes

Several Q-region class I genes have been shown to encode molecules reactive with anti Qa-2 reagents (Mellor et al., 1985; Straus et al., 1985; Flaherty et al., 1985). The finding that both membrane-bound and soluble polypeptides reactive with anti-Qa-2 reagents can be identified in the same cell lysates suggested the possibility that multiple class I genes may individually encode for these membrane-bound and/or soluble forms. To address this possibility, cell lines transfected with either the Qa-2 encoding class I genes Q7 b

DETERGENT PHASE (membrane bound)

AQUEOUS PHASE (soluble)

cell associated

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Fig. 1. Two-dimensional gel electrophoresis analysis of Qa-2 molecular isoforms. Mitogen activated T cells from B6 mice were metabolically radiolabcled with 35S-methionineor surface radiolabeled by lactoperoxidase catalyscd iodination. Cells and cell-free media were harvested, phase separated into detergent and water-soluble fractions and the Qa-2 molecule in each phase recovered by immunoprecipitation with MAb 34-1-2 and analysed by two-dimensional gel elcctrophoresis. Each panel represents equal cell equivalents and displays an identical area of the gel. Displayed are the Qa-2 polypcptides recovered from the detergent (panels A, C, E) and water soluble phases (panels (B, D, F) from: 35S-methionine-radiolabeled cell lysates (panels A and B) and cell-free medium (panels C and D); or 125I-labeledcell lysates (panels E and F), The isoelectric focusing dimension was run from left (basic) to right (acidic). The positions of known molecular weight markers run in parallel are shown on the right. The arrows and arrowheads denote Qa-2 polypeptides uniquely found in the detergent or aqueous phases, respectively. The asterisk denotes a ~ 4 lkDa species which is variably seen in both fractions. The ~44-kDa species found most prominently in the aqueous phase is likely actin, a common contaminate of immunoprecipitates.

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Biosynthesis of Qa-2 or Q 9 b were metabolically radiolabeled and similarly analysed. Two-dimensional gel analysis of cell-associated Q 7 and Q 9 gene products arc compared with those of activated T lymphocytcs in Fig. 2. Both membrane-bound and soluble polypeptidcs reactive with anti Qa-2 reagents can be readily identified in cell lysatcs of Q 7 b or Q 9 b transfectcd cells. The isoclectric profiles of the 40-kDa membrane-bound Q 7 b and Q 9 b gcnc products are similar (Fig. 2, panels c and e). In contrast, the charge profiles of the 38-kDa

membrane-bound Q7 and Q9 products are distinguishable. While each 38-kDa product has two major spots, the Q7 b species has a unique basic species while the Q9 b has a unique acidic species (Fig. 2, panels c and e). Importantly, if the membrane-bound Q7 and Q9 polypeptides are mixed and analysed, the pattern is identical to the species identified in the membrane-bound fraction derived from C57BL/6 activated T cells (compare Fig. 2, panels a and g). A similar scenerio is observed with the

DETERGENT PHASE

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B6 I

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Q7 Q9 HEPA Q7 (cell)

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31 Fig. 2. Two-dimensional gel analysis of the polypcptides encoded by the class I genes Q7 b or Q9 b. Hepa-OVA or L cellstransfectcdwith the class I genes Q7 b or Q9 b and activatedT cellswere radiolabclcd with 35S methioninc, lysed with TX-114 and celllysatesor cell-freemedium partitioned into membrane and water-soluble phases. Each phase was immunoprccipitated with M A b 34-I-2 and analysed by two-dimensional gel elcctrophoresis.Equal cellequivalents are represented in all panels with identical areas of the gel represented. Displayed are the polypeptides recovered from the detergent (panels a, c, e, g, i,k) and water-solublephases (panels b, d, f,h, j,I)of celllysatesderived from activatedT cells(panels a and b), Q9 b transfccted Hepa-I cells(panels c and d), Q7 b transfcctedHcpa-I cells(panels c and f), a mixture of Q7 b and Q9 b (panels g and h) and Q7 b transfcctedL cells(panels i and j) or from cellfrcc media from Q7 b transfcctedL cells(panels k and I).The isoelcctricfocusing dimension was run from left (basic) to right (acidic).The positions of known molecular weight markers run in parallelare indicated on the right. M I M M 28/1 l - - I

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water-soluble Q7 and Q9 proteins identified in the cell lysates. The isoelectric profiles of the water-soluble 37 kDa and 35 kDa Q7 b and Q9 b polypeptides are distinguishable (Fig. 2, panel d and f). Again when mixed and analysed, the Q7 b and Q9 b charge profiles reconstitute the charge profiles of the C57BL/6 35kDa and 37kDa water-soluble cell associated polypeptides (Fig. 2, panels b and h). The minor Qa-2 reactive species identified in the membrane fraction at 37 kDa and the aqueous fractions at 33 kDa are not identified in the Q7/Q9 transfected cell lines. Based on these observations we conclude that the major membrane-bound and soluble Qa-2 polypeptides identified in cell lysates of B6 activated T cells are derived from the Q-region class I genes, Q7 b and Q9 b.

Endoglycosidase isoforms

sensitivities

of

Qa-2

The 39-kDa secreted molecule is sensitive to Endo F and resistant to Endo H digestion (Fig. 3, a, b, c). The mol. wt of the completely deglycosylated secreted Qa-2 polypeptide is approximately 33 kDa. In the cell-associated water-soluble phase the major isoform of 37 kDa, the less abundant 41-kDa isoform, as well as a minor, poorly resolved, 39-kDa species (Fig. 3, lane d) is observed. Following Endo H digestion, the 39-kDa species is well resolved as an Endo H resistant form while the 41 and 37-kDa forms are Endo H sensitive (Fig. 3, lane f). All of these species are sensitive to Endo F (Fig. 3, lane e). Digestion by Endo H or F results in a major species at 33 kDa and a second species at 36 kDa (Fig. 3, lanes e and f). Additional endoglycosidase does not effect the recovery of the 36- and 33-kDa species (data not shown). Therefore, it is likely that the 36-kDa species represents completely deglycosylated 41-kDa polypeptide while the 33-kDa species are derived from the 39- and 37-kDa polypeptide.

molecular

To further characterize membrane-bound and water-soluble Qa-2 polypeptides, and to understand their biochemical interrelationships, we examined the sensitivities of these forms to digestion with Endoglycosidase H or F. The 38- and 40-kDa membranebound forms were both sensitive to Endo F digestion (Fig. 3, lane h), but only the 38-kDa form was sensitive to Endo H digestion (Fig 3, lane i). All membrane-bound polypeptides, whether digested by Endoglycosidases F or H, are converted to a core polypeptide mol. wt. of 34 kDa.

Cell

Media

b

The 40-kDa Qa-2 expressed on the extracellular surface is anchored into the membrane bilayer by a glycophospholipid structure which is covalently attached to the carboxy-terminus of the polypeptide (Stiernberg et al., 1987; Stroynowski et aL, 1987;

Associated

Aq

Aq a

Membrane-bound Qa -2 polypeptides are solubilized by PIPLC and soluble Qa-2 lacks components of the glycolipid anchor

c

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Fig. 3. Endoglycosidase sensitivities of Qa-2 molecular isoforms. Activated T cells from B6 mice were metabolically radiolabeled, cells and media harvested, TXII4 phase separated and immunoprecipitated with saturating amounts of MAb 34-1-2. Immunoprecipitates were mock digested or digested with Endo H or Endo F as described in Materials and Methods. Shown are Qa-2 polypeptides recovered from cell-free medium (lanes a--c), cell-associatedwater-soluble fraction (lanes d-f) or cell-associateddetergent fraction (lanes g-i) that had been mock digested (lanes a, d and g), digested with Endo-H (lanes b, e and h) or digested with Endo-F (lanes c, d and i). ~4C-methylatedmolecular weight markers co-run with experimental samples flank each side of the gel, with their molecular weights in kDa are indicated to the fight. Arrows inset into the panel identify: on the left, 39-kDa secreted Qa-2 and the 33-kDa water-soluble core polypeptide; to the right, the 40-, 38-kDa and detergent-soluble 34-kDa core polypeptide. The asterisk denotes a ~41-kDa species which is variably seen in both fractions while the arrowhead marks the position of the 39-kDa isoform found in the aqueous phase.

Biosynthesis of Qa-2 Soloski et al., 1988b; Waneck et al., 1988). Therefore, we wished to determine if the 38-kDa cell-membrane associated polypeptide was also anchored via this glycophospholipid structure. Our experimental strategy was based on the observation that proteins that are membrane bound via this glycolipid anchor are substrates for PIPLC and upon digestion are converted to water soluble forms. Qa-2 molecules synthesized by activated T cells and associated with TX-114 detergent micelles (membrane associated) were mock treated or treated with a PIPLC purified from B. thuringiensis and repartitioned to evaluate any changes in molecular hydrophobicity caused by phospholipase digestion. After digestion with PIPLC, over 90% of the 38- and 40-kDa species as well as the noncovalently associated fl2-microglobulin are lost from the detergent phase (Fig. 4, lanes 1 vs 2) and

Lipase Sensitivity of Biosynthetic Precursors 1234

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-14

Fig. 4. Lipase sensitivity of cell-associated detergent-soluble Qa-2 polypeptides. Activated T cells from B6.K3 mice were radiolabeled with 35S-methionine, lysed in TX114 and phase partitioned as described in Materials and Methods. The detergent phase was then mock treated or digested with PIPLC purified from B. thuringiensis, re-partitioned and immunoprecipitated with the anti-Qa-2 MAb 34-1-2 and immunoprecipitates analysed by SDS-PAGE. Displayed are the Qa-2 polypeptides recovered in the detergent (lanes 1 and 2) and aqueous phases (lanes 3 and 4) after digestion with PIPLC (lanes 1 and 3) or mock digestion (lanes 2 and 4). The positions of molecular weight markers run in parallel are indicated on the right of the panel. Arrows highlight the 38- and 40-kDa Qa-2 polypeptide.

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quantitatively recovered in the aqueous phase (Fig. 4, lanes 3 vs 4). To biocbemically confirm that both the 38- and 40-kDa Qa-2 isoforms have this glycolipid anchor and to investigate whether the 39-kDa media/water soluble Qa-2 could be derived from phospholipase digestion of membrane-associated polypeptides, metabolic labeling with 3H-ethanolamine was performed. It was reasoned that if the water soluble Qa-2 found in the media was generated by phospholipase digestion of membrane-bound polypeptides, then the secreted Qa-2 would be radiolabeled by 3H-ethanolamine, the moiety that covalently links the glycolipid anchor to the COOH terminus (Medof et al., 1986; Fatemi and Tartakoff, 1988; He et al., 1987; Takami et al., 1988). When activated T cells were radiolabeled with 3H-ethanolamine, only the 38- and 40-kDa cell membrane Qa-2 polypeptides incorporated the isotope (Fig. 5, lane a). No cell-associated water-soluble or secreted Qa-2 polypeptides were radiolabeled with ethanolamine (Fig. 5, lanes b, c). Precursor to product relationships between Qa-2 isoforms The 37- and 35-kDa water-soluble cell-associated Qa-2 isoforms are sensitive to Endo H and have the same deglycosylated core molecular weight as the mature, Endo-H resistent, secreted 39-kDa polypeptide. In addition, two-dimensional gel analysis reveals that the 33-kDa core polypeptide derived from either the water-soluble cell-associated or secreted Qa-2 polypeptides display identical isoelectric profiles (Fig. 6, panels b, d and f). These data argue that the cell-associated water-soluble Qa-2 species are precursors of the mature Qa-2 polypeptides. To confirm a precursor to product relationship between these Qa-2 molecular isoforms, pulse-chase studies were performed. Activated T cells were pulsed for 20 min with 35S-methionine and chased with excess cold methionine. At each time point, cells were harvested, lysed in TX-I14, phase partitioned, and Qa-2 molecules recovered by immunoprecipitation and analysed (Fig. 7). Qa-2 isoforms of 41, 39, 37 and 35 kDa were recovered in the cell associated/water soluble phases following the 20-min pulse (0 time chase, Fig. 7, panel B). The 37- and 35-kDa cellassociated water-soluble polypeptides diminish in intensity throughout the duration of the chase (Fig. 7, panels B, E, H and K). Small amounts of the secreted 39-kDa polypeptide are detected after 30 min of chase and increases in intensity throughout the duration of the chase (Fig. 7, panels C, F, I and L). The loss of water-soluble Qa-2 from the cell-associated fraction and the corresponding increase in "mature" Qa-2 isoforms strongly suggests a precursor to product relationship between the water-soluble intracellular isoforms and secreted 39-kDa polypeptides. This relationship is supported by studies which identified only cell-associated water-soluble forms in L cell

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GREGORY P. EINHORN et al.

Biosynthetic

Labeling

Qa2 Glycolipid C B A

methionine

of the

Anchor a

b c

ethanolamine

Fig. 5. Biosynthetic labeling of activated T cells with 3H-ethanolamine. Activated T cell blasts from B6 mice were radiolabeled with 3H-ethanolamineor 35S-methionineand both cells and media harvested. Cells were lysed with 0.5% TX-114 while medium was adjusted to 0.5% TX-114. Phases were separated, immunoprecipitated with saturating amounts of the anti-Qa-2 MAb 34-1-2 and immunoprecipitates analysed by SDS-PAGE. Each methionine-labeled fraction has one tenth as many cell equivalents as the ethanolamine-labeled fraction. Both ethanolamine- and methionine-labeledfractions were analysed on the same gel. The ethanolamine autoradiograph was exposed for 60 days while the 35S-methioninefilm was exposed for 6 days. Displayed are Qa-2 polypeptides radiolabeled with 35S-methionine(lanes AqS) or 3H-ethanolamine(lanes a--c)recovered from the cell-associateddetergent phase (lanes A, a), cell-associated aqueous phase (lanes B, b) or cell-freemedium (lanes C, c). ~4C-methylatedprotein standards were co-run and are seen flanking the experimental lanes, with their sizes in kDa noted on the right. The arrows denote the 38- and 40-kDa species recovered in the detergent phase. transfected with Q7 b (Fig. 2, panels i and j) or Q9 b (data not shown). L cells transfected with Q7/9 genes synthesize only a secreted 39-kDa Qa-2 polypeptide (see Fig. 2, panels k and 1, and Stroynowski et al., 1987; Soloski et al., 1988). The 38-kDa membrane-bound isoform is clearly detected after the 20-min pulse (Fig. 7, panel A) while both the 38- and 40-kDa forms are seen at the 30-min chase (Fig. 7, panel D). Surprisingly, during longer chases, the 38- and the 40-kDa membrane-bound species coordinately increase in intensity. In addition to the above-mentioned forms, the 41-kDa species can be identified in both the water-soluble and membrane-bound fractions (Fig. 7, panels A and B). This species is detected after the 20-min pulse and its level remains constant throughout the chase (Fig. 7, panels A, B, D, E, G, H, J and K). DISCUSSION

Utilizing the detergent TX-114 we identified several distinct cell-associated polypeptides reactive with anti Qa-2 reagents partitioning either in detergent (membrane bound) or aqueous (water soluble) phases. Previous studies have identified the Q-region class I genes Q7 and Q9 as encoding a 40-kDa PI anchored cell surface molecule as well as a secreted 39-kDa molecule. Both are biochemically indistinguishable from the Qa-2 isoforms synthesized by activated T

cells (Stroynowski et al., 1987; Waneck et al., 1988; Sherman et al., 1988; Soloski et al., 1988b). Based on these studies it was reasoned that the bulk of cellsurface or secreted Qa-2 molecules expressed by C57BL/6J activated T cells were encoded by the Q7 and/or Q9 genes (Soloski et al., 1988b). In the studies described herein, a comparison of the cell-associated polypeptides synthesized by cell lines transfected with the Q7 or Q9 genes identified all the major watersoluble and membrane-bound Qa-2 polypeptides found in the cytoplasm of activated T cells. Analysis of a mixture of Q7 and Q9 polypeptide creates a pattern identical to that of Qa-2 species derived from activated T cells. These observations are consistent with the notion that the major membrane-bound or water-soluble Qa-2 polypeptides identified in the cytoplasm of activated T cells from B6 mice are encoded by either the Q7 or Q9 Q-region class I genes (Soloski et al., 1988b). Since the minor 37- and 33-kDa species were not identified in Q7 or Q9 transfected cell lines, it remains a possibility that these polypeptides are encoded by other Q-region class I genes. The predicted primary sequences of Q7 and Q9 gene products are almost identical, with only one amino acid change: Q9 polypeptides have a glutamic acid instead of a glutamine at position 165 (Devlin et al., 1985). While the Endo H insensitive 40-kDa membrane bound and the 39-kDa secreted forms

Biosynthesis of Qa-2

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ENDO-F (+)

ENDO-F (-)

-4 5

CELL AQUEOUS

a

. -31

m

;i

¸

-45

MEDIA

(SECRETED)

-31

C

45 MIXTURE e.

m

,31

Fig. 6. Two-dimensional gel analysis of deglycosylated Qa-2 polypeptides. Activated T cell blasts from B6 mice were radiolabeled with 35S-methionineand the Qa-2 polypeptides found in the aqueous phase of TX-114 solubilized cell lysates or from cell-free medium were recovered by immunoprecipitation. Immunoprecipitates were either digested or mock digested with Endo F followed by analysis by two-dimensional gel electrophoresis. Displayed are the fully glycosylated (panels a, c, e) or Endo F deglycosylated (panels b, d, f) Qa-2 polypeptides recovered from the cell-associatedaqueous phase (panels a and b), the cell-free medium (panels c, d) or a mixture of cell-associated aqueous phase and cell-free medium (panels e and f). The isoelectric focusing dimension was run form left (basic) to fight (acidic). The positions of molecular weight markers run in parallel are displayed on the fight. encoded by the Q7 and Q9 genes are indistinguishable by charge and molecular weight, the Endo-H sensitive forms found in either the membrane-bound or water-soluble fractions display slight charge differences consistent with their primary sequence differences. Endo H sensitive Q7-encoded species display a more basic profile than Q9 species of the same mol. wt. These data suggest that the processing to an Endo H insensitive mature form masks a charge difference caused by differences in amino acid sequence. This implies that a single amino acid difference can signal alterations in glycosylation patterns. In this regard, it has been reported that variations in primary sequence amongst MHC encoded class I polypeptides can alter the sialylation patterns of N-linked carbohydrates (Powell et al., 1987). Detergent partitioning studies identified two hydrophobic membrane associated Qa-2 isoforms of 40 and 38 kDa which are membrane bound via a glycolipid anchor since (1) they can be converted to aqueous solubility by digestion with PIPLC and (2) are both radiolabeled with 3H-ethanolamine. The cell-associated 40-kDa molecule is resistant to Endo H digestion and has a similar isoelectric profile to the 40-kDa molecule expressed on the cell surface. In contrast, the more basic 38-kDa Qa-2 polypeptide is

not found on the cell surface and is sensitive to Endo H. These data indicate that the 38-kDa molecule contains high mannose N-linked carbohydrates characteristic of immature intracellular forms and is the intracellular precursor to the mature 40-kDa cell surface molecule. This relationship is supported by the observation that both membrane-associated forms digest down to a "core" polypeptide of approximately 34 kDa upon Endo F or (for the 38 kDa Qa-2) Endo H digestion. Interestingly, although the 38-kDa species appears first in the pulse-chase studies, both species proportionally accumulate during the chase. This would suggest that the 38-kDa species may derive from another precursor pool. This pool could be membrane-bound precursors not recognized by the serological reagents employed or from species identified in the aqueous phase. Previous studies have determined that 50-60% of the Qa-2 molecules expressed on the surface of activated T cells are resistant to lipase release (Soloski et al., 1988b). In contrast, 90-95% of the Qa-2 molecules found in the TX-114 detergent phase recovered from cell lysates are lipase sensitive (Fig. 4). This would suggest that PI-PLC resistance is not due to the presence of a peptide anchor. One possibility is that intermolecular interactions on the cell surface,

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GV,EC_,ORYP. EINHORN et al.

CELL ASSOCIATED

DETERGENT PHASE

CELL ASSOCIATED AQUEOUS PHASE

SECRETED

0 hr

0.5 hr

2.5 hr

5.0 hr

Fig. 7. Pulse-chase analysis of Qa-2 polypeptides from activated T lymphocytes. Activated T cells from B6 mice were metabolically radiolabeled for 20 min with 35S-methionineand chased with excess cold methionine for up to 5 hr. At the indicated time points cells and media were harvested, cells were lysed in TX-114, phase partitioned and all the phases and cell-free medium immunoprecipitated with anti-Qa-2 MAb 34-1-2 and analysed by two-dimensional gel electrophoresis. Displayed are the Qa-2 polypeptides recovered in the cell associated aqueous phase (panels A, D, G, J), cell associated detergent phase (panels B, E H, K) and cell-free medium (panels C, F, I, L) at time 0 (panels A-C), 0.5 hr (panels D-F), 2.5 hr (panels G-I) and 5.0 hr (panels J-L) of the chase. The isoelectric focusing dimension was run from left (basic) to right (acidic). The positions of molecular weight standards co-run in parallel are indicated on the right-hand side of the panels. The arrows and arrowheads denote Qa-2 polypeptides uniquely found in the detergent or aqueous phases respectively. The asterisk denotes a ~41 K species which is variably seen in both fractions.

disrupted by detergent lysis sterically inhibit enzyme action. Several PI-anchored proteins have been found to exist in both membrane-bound and a soluble form (Low and Saltiel, 1988; Low, 1989; Ferguson and Williams, 1988). It has been proposed that the soluble forms of the molecule are produced either by alternative splicing or by release of cell-surface forms via an endogenous hydrolase (Barbas et al., 1988; Low, 1989). In our study, we have identified several intracellular water soluble Qa-2 isoforms of 39, 37 and 35 kDa. The 37 and 35 kDa forms were sensitive to both Endo H and Endo F and both were reduced in mol. wt to 33 kDa after removal of N-linked carbohydrate. A 39-kDa water-soluble isoform was also found to be cell associated. It is identical in size, isoelectric profile and Endo H insensitivity to the 39-kDa water soluble isoform found secreted into the culture supernatant. Following removal of N-linked carbohydrates, the 35, 37 and 39 kDa polypeptides recovered from either the cell-associated or secreted fractions all have 33-kDa core polypeptides with identical isoelectric profiles. These studies, as well as the pulse-chase studies, suggest that there is a precursor to product relationship between the intracellular

35- and 37-kDa polypeptides and the secreted 39-kDa molecule. Furthermore, in Q7 transfected L cells, the cell-associated water soluble polypeptides but no membrane-found forms were synthesized. Therefore, a 39-kDa Qa-2 polypeptide is secreted into the media of L cell cultures without the concurrent synthesis of membrane-associated isoforms. In addition, it is important to note that neither-cell associated nor secreted water-soluble Qa-2 polypeptides incorporate 3H-ethanolamine. This observation argues further against the water soluble forms being derived from membrane-bound molecules via endogenous or serum lipases since molecules generated by solubilization of PI anchored structures with phospholipase A~, A 2, C or D would be predicted to yield a water-soluble polypeptide containing ethanolamine (Davitz et al., 1987; Low and Prasad, 1988). Collectively these data argue that the 35- and 37-kDa Q7 b polypeptides are the direct intracellular precursors of the water-soluble 39-kDa molecule. The origin of the soluble intracellular Qa-2 polypeptides remains unclear. It is possible that these species are generated via alternative splicing of Q7/Q9 mRNA or perhaps represent nascent Qa-2 polypeptide chains in which the COOH terminus

Biosynthesis of Qa-2 hydrophobic segment has been removed but the PI-glycan structure has yet to be added. In the latter case this water-soluble species could either be processed for secretion as a soluble molecule or be a substrate for subsequent PI-glycan addition and transport to the cell surface. If true, the water-soluble 35- and 37-kDa species could be precursors of both the secreted and cell-surface Qa-2 molecules, a point consistent with the pulse-chase studies described above. Currently, models for assembly of PIanchored polypeptides argue that a preformed glycolipid anchor structure is attached en bloc to nascent polypeptide chains bearing the appropriate signal structures at the C O O H terminus (Bangs et al., 1985; Ferguson et al., 1986; Conzeimann et al., 1987; Low and Saltiel, 1988). Pulse-chase studies in a variety of systems reveal that this attachment occurs virtually co-translationally (within 1-2 min) and it has been proposed to occur via a single transamidase reaction which simultaneously facilitates peptide removal and PI-glycan addition (reviewed by Low and Saltiel, 1988; Low, 1989; Ferguson and Williams, 1988). Such a model would preclude the presence of a watersoluble species lacking a hydrophobic transmembrane segment. However, there are several indications that the biosynthesis of Qa-2 polypeptides may be unique. First, in contrast to Qa-2, radiolabeling studies analysing Thy-1 biosynthesis found no evidence for significant intracellular water-soluble species (Conzelmann et al., 1987, 1988; Fatemi and Tartakoff, 1988). Secondly, in studies examining a mutant T cell l y m p h o m a defective in PI-anchor assembly, it was observed that while this mutant fails to express Thy-l.2 or Ly-6e, the expression of Qa-2, although diminished, is nevertheless present (Teng et al., 1989). Assuming that the Qa-2 expressed in the mutant cell line is PI-anchored, this would imply the existence of alternative pathways for assembly and expression of PI-anchored proteins. Clearly further investigations into the structure of the C O O H terminus on both the water-soluble and PI-anchored Qa-2 molecules will resolve these issues. Acknowledgements--The authors would like to thank Drs Dan Conrad and John Vernachio for their helpful discussions during the course of these studies and Drs Iwona Stroynowski and Lee Hood for the use of the transfected cell lines. This work was supported by NIH grants R01-20922 and T32-07247 as well as grant 871132 from the American Heart Association. REFERENCES

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Cochet M., Casrouge A., Dumont A., Transy C., Baleux F., Maloy W., Coligan J., Cazenave P. and Kourilsky P. (1989). A new cell surface molecule closely related to mouse class I transplantation antigens. Eur. J. lmmun. 19, 1927-1931. Conzelmann A., Spiazzi A. and Bron C. (1987). Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation. Biochem. J. 246, 605-610. Conzelmann A., Spiazzi A., Bron C. and Hyman R. (1988) No glycolipid anchors are added to Thy-1 glycoprotein in Thy-1 negative mutant thymoma cells of four different complementation classes. Molec. cell. Biol. 8, 674-678. Cosman D., Kres M., Khoury G. and Tay G. (1982) Tissue-specific expression of an unusual H-2 (class I) related gene. Proc. natn. Acad. Sci. U.S.A. 79, 4947--4951. Davitz M. A., Hereld D., Shak S., Krakow J., Englund P. T. and Nussenzweig V. (1987) A glycan-phosphatidylinositol-specific phospholipase D in human serum. Science 238, 81-84. Devlin J., Weiss E., Paulson M. and Flavell R. (1985) Duplicated gene pairs and alleles of class I genes in the Qa-2 region of the murine major histocompatibility complex: a comparison. E M B O J. 4, 3203-3207. Fatemi S. H. and Tartakoff A. M. (1988) The phenotype of five classes of T lymphoma mutants. Defective glycophospholipid anchoring, rapid degradation, and secretion of Thy-1 glycoprotein. J. biol. Chem. 263, 1288-1294. Ferguson M. A. J., Duszenko M., Lamont G. S., Overath P. and Cross G. A. M. (1986) Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phsophatidylinositol membrane anchor. J. biol. Chem. 261, 356-362. Ferguson M. and Williams A. (1988) Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. A. Rev. Biochem. 57, 285-320. Flaherty L. (1981) The Tla region antigens. In The Role o f the Major Histocompatibility Complex in Immunobiology

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