lectin methods and protocols volume 9 || t-cell receptor purification

T-Cell Receptor Purification

Kelly P. Kearse

1. Introduction The antigen receptor expressed on most T-lymphocytes (T-cell antigen

receptor [TCR]) is a multisubumt complex conststmg of at least SIX dtfferent polypeptrdes (a, j3, y, 6, a, and 0, several of which are modified by addition of N-linked oligosaccharlde chains Ollgosacchartde side chains on TCR glycoproteins undergo well-characterized processing events wtthm the endoplasmic reticulum (ER) and Golgl systems, including removal of mannose residues and subsequent addition of galactose and siahc acid ohgosaccharrdes. In the current chapter, a method is described for the tsolatton of immature and mature TCR complexes from murme T-lymphocytes using lectm affinity chromatography This techmque IS rapid, sensitive, and does not disrupt the integrity of assembled TCR complexes.

1.1. Assembly, Intracellular Transport, and Posttranslational Modification of Murine T-Cell Receptor Proteins

The TCR complex is composed of three different famtltes of proteins: clonotyptc ap polypeptides, invariant CD3-y,S,s subunits, and invariant < chains (I). Assembly of the multtsubumt TCR complex takes place wrthm the ER and is initiated by formation of noncovalently associated pans of CD36a and CD3ya proteins (2-5). Nondisulfide-linked a and p polypeptides then assemble with CD3 chains to form intermediate TCR complexes consisting of a& and Pys subunits, which, in turn, assemble to form incomplete c@&ye TCR complexes (6,7), clonotypic @3 proteins are subsequently disulfide-linked to each other, and & proteins are added to yield complete c@sys&~ TCR complexes (6,7). The intracellular transport of TCR proteins IS directly related to then assembly status. Unassembled individual TCR proteins and partial complexes of CD3 components are retained within the ER and, depending on the

From Methods in Molecular Me&me Vol 9 Lectm Methods and Protocols Edlted by J M Rhodes and J D Mllton Humana Press Inc , Totowa, NJ

291

292 Kearse

Table 1 Glycosylation of Murine T-Cell Receptor Proteins

Ohgosacchande side chains

TCR protems High mannose-type” Complex-typeh Total

a 0 34 354 P 1 2-3 34 ii 0 2 1 1 3 1

0 0 0 0 0 0

Wumber of N-linked ohgosaccharlde side chains on mature protein susceptible to digestion with Endoglycosldase H

“Number ofN-linked ollgosaccharlde side chains on mature protein resistant to digestion with Endoglycosldase H, containing complex-type sugars

particular protein, degraded (8.9) Incomplete a@&ya TCR complexes and complete a@~ye<c complexes egress the ER and transit through the Golgl apparatus (8,9) Incomplete TCR complexes are targeted to lysosomes for degradation; only complete TCR complexes are effectively transported to the plasma membrane (I 0-l 2)

Several members of the TCR complex are posttranslatlonally modified by the addition of N-linked ohgosaccharlde side chains clonotyplc a$ proteins and invariant CD3-y,F chains (Table 1) On egress from the ER and transit through Golgl system, some, but not all, N-linked sugar chains of TCR glycoproteins are converted from high mannose-type ollgosaccharldes to complex- type sugar chains. Indeed, mature TCRa and CD3y proteins contam exclusively complex-type N-lmked ollgosaccharlde chains, whereas mature TCRP and CD36 chains contam both high mannose and complex-type ohglosaccharlde chains (Table 1) Because of then- unique bmdmg specificity for ohgosaccha- rides, lectms have been widely used m the purification of glycoproteins (23) Regarding the TCR complex, lectms have been utilized m the purlficatlon of TCR proteins from immature CD4+CD8+ thymocytes and splemc T-lymphocytes (24, I5), murme T hybrldoma cells (16), and human T-lymphocytes (17) In the current chapter, a method 1s described for the lsolatlon of immature and mature TCR complexes from murme T-lymphocytes using nnmoblhzed lectms specific for ollgosaccharldes added m the trans Golgl compartment of the cell

2. Materials

2.7. Lecfin Affinity Chromatography

1 The principle behind using lectm affinity chromatography for the separation of immature and mature TCR complexes 1s shown m Fig 1 Immoblllzed lectms are

T-Cell Receptor Punficatlon 293

Lectin-Matrix I

I I Gala&se, Sialic Acid Add~tma

Endo H Resistance

Fig. 1. Separation of Immature and mature TCR complexes by lectm affitnty chromatography TCR complexes locahzed wrthm the ER contam Immature high mannose ohgosaccharide chains and are, therefore, not bound by lectm matrices specific for complex-type sugars (galactose, sialic acid). TCR complexes whtch effectively egress the ER and transit to the Golgi system undergo processmg by Golgr glycosrdases and glycosyltransferases (e g , galactosyltransferase, stalyltransferase) and are therefore effectively retained on lectm matrices specific for complex-type sugars

available from numerous commercral suppliers Wheatgerm agglutnnn (WGA) and Rzczn communzs agglutm (RCA) conJugated to agarose matrices (EY Labora- tories, San Mateo, CA) were utrhzed m our studres (14-l 6)

2 Trrs-buffered saline (TBS). 20 mMTrrs-HCl, 150 mMNaC1, pH 7 2, with 10 mM rodoacetamide, 20 pg/mL leupeptin, and 40 pg/mL aprotmm

3 Drgitonm solutron. Drgttonm (Wako, Kyoto, Japan) IS prepared as a 2% stock solutron (w/v) and boiled for 10 mm Solutron is allowed to cool at room temperature at least 30 mm before use

4. Lectin wash buffer: TBS containmg 0 5% dtgttonm 5. Precrpttatron wash buffer TBS containing 0 2% drgrtonm 6 Elutron buffer. precrprtatron wash buffer containing appropriate competmg oh-

gosaccharrde, typically at concentrations of 100-500 n&I For WGA, affimty chromatography, 500 mMN-acetylglucosamme (GlcNAc) is used, for RCA, 150 mM P-lactose IS used Ohgosacchartdes were purchased from either Sigma (St Louis, MO) or EY

294 Kearse

7 PBS,pH72 8 20% Bleach solution 1s required m experiments using RCA (see Note 1)

2.2. Immunoprecipitation, Glycosidase Digestion, and Gel Electrophoresis

1 Cell lysls buffer* TBS containing 1% dlgltonin 2 TCR specific antlbodles conjugated to protein A-sepharose (Pharmacla, Uppsala,

Sweden) 3 Preclpltatlon wash buffer. TBS containing 0 2% dlgltonm 4 PBS,pH 72 5 1% SDS solution 6 Glycosldase digestion buffer 75 mM sodium phosphate, pH 6 1, 75 mA4 EDTA,

0 1% NP-40 7 Endoglycosldase H (Genzyme, Cambridge, MA) 8 For analysis of TCR proteins, 13% polyacrylamlde gels are typically used Stan-

dard gel electrophoresls reagents and gel equipment are also needed

3. Methods 3.1. Lectin Affinity Chromatography

A flow diagram of this procedure 1s presented in Fig. 2 Resuspend cells (approx 1 x 10s cells for murme thymocytes or splemc T-lymphocytes, 5 x lo7 cells for murme T-cell hybndomas) m 800-1000 pL cell lysls buffer, and Incubate at 4°C for 20 mm Remove msoluble material by centrlfugatlon and transfer lysate (Sn) to a new tube Mix lysate with an equal volume of TBS to yield a final detergent concentration of 0 5% Place half of the sample at 4°C until further analysis (unfractlonated material), mix the other half with approx 200-400 pL of immoblhzed lectm slurry that was previously washed three times with 1 mL PBS and once with 1 mL lectm wash buffer Incubate material for 34 h at 4°C with rocking Centrifuge sample, remove Sn (unbound material), and transfer to a new tube Recentrlfuge material, remove supernatant, and transfer to a new tube Repeat this process twice more (four times total) to ensure that no carryover of lectm beads has occurred Place samples at 4°C until further analysis Wash lectm beads containing bound maternal five times in 1 mL preclpltatlon wash buffer, resuspend m 1 mL elutlon buffer containing appropriate ohgosacchande, and incubate at 4°C for 60 mm Centrifuge lectm beads, remove supernatant (contaimng previously bound material) transfer to a new tube Repeat this process three more times to ensure that no carryover of beads has occurred. Place samples at 4°C until further analysis. Note. All materials that come m contact with RCA must be decontaminated with 20% bleach solution, as this material 1s extremely toxic. Also, proper ventllatlon and protective clothmg must be used to avoid inhalation or contact with this material (see Note 1)

T-Cell Receptor Purification 295

LYSATE (1% DIGITONIN)

7 DILUTE 1:2 TBS

UNFRACTIONATED MATERIAL

LECTtN-AGAROSE

Y

3-4 HRS.@ 4C

t

SPIN, REMOVE Sn y UNBOUND MATERIAL

SPIN, REMOVE Sn 34X

7

WASH 5X IN PPT WASH BUFFER

ELUTION BUFFER \4 1 HR. @4C

f SPIN, REMOVE Sn y

BOUND MATERIAL SRN’ REMoVE ‘” 3-1

Fig. 2. Isolatton of immature and mature TCR complexes by lectm affimty chromatography. Flow diagram of Isolation of tmmature and mature TCR complexes by lectin-agarose matrices The reader IS referred to Section 3 1 for details

3.2. Immunoprecipitation, Glycosidase Digestion, and Gel Electrophoresis

1 MIX unfractlonated material, unbound maternal, and bound maternal wtth TCR- specific MAb preabsorbed to protein A sepharose (approx 60 pL of slurry), and incubate for 3 h at 4°C

2 Wash lmmunoprecrpttates three trmes wrth 1 mL prectpltatton and once wtth 1 mL PBS Prior to the last wash with PBS dlvtde precipitates m half for glycostdase digestion

3. Add 5 pL of 1% SDS to washed beads, and boll samples for 5-10 mm 4 Allow samples to cool for 5 mm at room temperature and then add 35 uL of

glycosrdase digestion buffer. 5 Add 5 pL (10 mu) of Endo H (Genzyme, Cambridge, MA) to Endo H drgested

groups; mock treated samples recetve 5 pL of glycosldase dtgestton buffer 6 Samples are incubated overnight at 37’C 7 Stop glycostdase digestton by addition of 60 pL of 3X gel electrophoresrs sample

buffer

296 Kearse

d, Trap - E-

r-

5-

30 minute Pulse 60 minute Chase

- dR. YR

- dsr YS

Fig. 3. Separation of immature and mature TCR complexes from splenic T lymphocytes by RCA matrices: Radiolabeled lysates of splenic T-cells were either unfractionated or incubated with Rich communis agglutin (RCA) conjugated to agarose beads. Unfractionated and RCA-fractionated material was precipitated with MAb to CD3c; precipitates were either mock treated or digested with Endo H and analyzed by one dimensional SDS-PAGE under reducing conditions. The positions of ct$,y,F, TRAP (TCR associated protein), E, and 1; chains are indicated. The mobilities of TCR glycoproteins containing immature, Endo H sensitive oligosaccharide chains (a&, 6s, and ys) and mature, Endo H resistant oligosaccharide chains (apa, ija, and yR) are indicated.

8. Run precipitates on SDS-PAGE gels using standard electrophoretic techniques and analyze by appropriate biochemical methods: immunoblotting, autoradiography, and so on.

9. The separation of immature and mature TCR complexes of splenic T-lymphocytes by RCA matrices is shown in Fig. 3. In this experiment, purified splenic T- cells were metabolically labeled with [35S]methionine for 30 min and chased in medium containing excess unlabeled methionine for 60 min to allow movement of metabolically labeled proteins from the ER to the Golgi apparatus. Cells were solubilized in 1% digitonin and lysates were either unfractionated or exposed to RCA matrices. Material was separated into RCA-unbound and RCA-bound fractions. TCR complexes present in unfractionated material, RCA-unbound, and RCA-bound fractions were immunoprecipitated with anti-CD3c specific MAb and precipitates digested with Endo H. Samples were analyzed on 13% SDS- PAGE gels under reducing conditions, and gels processed for autoradiography. It can be seen that RCA effectively separated immature and mature TCR complexes of splenic T-cells in these studies as Endo H-sensitive TCR glycoproteins were only present in RCA-unbound fractions and Endo H-resistant TCR chains existed exclusively in RCA-bound fractions (Fig. 3).

4. Notes 1. Caution: Rick communis agglutin is extremely toxic. Therefore, appropriate

safety measures must be used when handling this material. It is recommended

T-Cell Receptor Put-/f/cation

that commercial supphers and local supervlsors be consulted prior to use of this reagent for specific safety gmdelines

2 DigestIon of isolated material with Endo H glycosldase 1s a useful means for determining the efficiency of separation of immature and mature TCR complexes usmg WGA and RCA lectins, TCR glycoprotems containing immature (Endo H sensitive) ohgosaccharldes are localized within the unbound fraction, whereas mature (Endo H resistant) species are restricted to the bound fraction (Fig. 3) AlternatIvely, separation of immature and mature TCR complexes by lectin matrices may be assessed by measuring addition of slahc acid residues to ohgosaccharlde chains using neurammldase dlgestlon.

3 Although lectm bmdmg IS compatible with many detergent solutions, the use of dlgitomn is recommended m these studies to maintain the integrity of assembled TCR complexes

4. The binding activity of many lectms (e.g , Concanavalin A) IS cation-dependent and thus requires addition of Ca 2+, Mg2+, Mn2+, to buffer solutions However, the lectms WGA and RCA do not require cations for their bmdmg actlvlty and, thus, can be used with most standard laboratory buffer solutions

5 It should be noted that the lectm of choice for lsolatlon of TCR complexes 1s dependent on the cell type being studied For example, TCR complexes expressed on murine 2B4 T-cell hybridomas are efficiently bound by WGA matnces, whereas TCR complexes expressed on immature thymocytes and mature splemc T-cells are not The reason for this difference IS unclear, but may reflect dlfferen- tial processing of ollgosaccharlde chains of TCR complexes upon T-cell actlva- tlon In contrast to these findings, TCR complexes expressed on all of the abovementloned T-cell types are efficiently retained on RCA matrlces Thus, the use of RCA seems best suited for isolation of TCR complexes of any cell type, although it IS conceivable that other, less hazardous, lectms may also be used, for example Sambucus nzgra (SNA) or Lzmax flavus (LFA), specific for slahc acid containing ollgosaccharldes

References 1 Klausner, R D , Llppmcott-Schwartz, J., and Bomfacino, J S. (1990) The T-cell

antigen receptor: insights mto organelle biology. Ann Rev Cell Blol 6,403-43 1 2. Ohashi, P. S,, Mak, T W , van den Elsen, P., Wangal, Y , Yoshikal, Y., Calman,

A. F , Terhorst, C., Stobo, J D., and Weiss, A (1985) Reconstitution of active surface T3/T-cell antigen receptor by DNA transfer. Nature 316, 606-609.

3 Salto, T , Weiss, A , Guner, K C., Shevach, E M., and Germam, R. N (1987) Cell surface T3 expression requires the presence of both a-P-chains of the T-cell receptor. J Immunol 139,625-628

4 Alarcon, B., Berkhout, B., Breitmeyer, J , and Terhorst, C (1988) Assembly of the human T-cell receptor-CD3 complex takes place m the endoplasmlc reticulum and mvolves mtermedlary complexes between the CD3-gamma, delta, and epsl- lon core and single T-cell receptor alpha or beta chains. J Blol Chem 263,2953- 2961

298 Kearse

5

6

7

8

9

10

11.

12

13

14

15

16

17

Bomfacmo, J S, Chen, C , Llppmcott-Schwartz, J , Ashwell, J D , Klausner, R D (1988) Subunit interactions wlthm the T-cell receptor clues from the study of partial complexes Proc Nat1 Acad Scz USA 35,6929-6923 Kearse, K P , Roberts, J L , and Singer, A. (1995) TCRa-CD3& association is the mmal step m c+ dlmer formatlon m murme T-cells and is hmltmg m lmma- ture CD4+CDS+ thymocytes Zmmuruty 2,391-399 Minaml, Y., Weissman, A M , Samelson, L. E , and Klausner, R D (1987) Build- mg a multlcham receptor synthesis, degradation, and assembly of the T-cell antl- gen receptor. Proc Nat1 Acad Scz USA 84,2688-2692 Chen, C., Bomfacmo, J S., Yuan, L , and Klausner, R D (1988) Selective degradation of T-cell antigen receptor chains retained m a pre-Golgl compartment J Cell BloI 107,2 149-2 16 1 Llppmcott-Schwartz, J , Bomfacmo, J S , Yuan, L , and Klausner, R D (1989) Degradation from the endoplasmlc reticulum dlsposmg ofnewly synthesized proteins Cell 54,209-220 Wlleman, T , Carson, G R , Concmo, J , Ahmed, A , and Terhorst, C (1990) The y and E subumts of the CD3 complex inhibit pre-Golgl degradation of newly synthesized T-cell receptors J Cell Bzol 110,973-986 Sussman, J. J., Bomfacmo, J S , Llppmcott-Schwartz, J , WeIssman, A. M , Salto, T , Klausner, R D , and Ashwell, J D (1988) Failure to synthesize the T-cell 1; chain: structure and function of a partial T-cell receptor complex Cell 52,85-95 Hall, C. Berkhot, B , Alarcon, B , Sancho, J., Wlleman, T , and Terhorst, C (199 1) Requirements for cell surface expresslon of the human TCRXD3 complex m non- T-cells Int Immunol 3,359-368 Lotan. R , Beattle, G , Hubbell, W , and Nlcolson, G L (1977) Actlvltles of lectms and their lmmoblllzed derlvatlves m detergent solutions Imphcatlons on the use of lectm affimty chromatography for the purficatlon of membrane glycoprotems Blochemzstry 16, 1787-l 794 Kearse, K P , Wiest. D L , and Singer, A (1993) Subcellular locahzatlon of T- cell receptor complexes contammg tyrosme-phosphorylated < proteins m lmma- ture CD4+CD8+ thymocytes Proc Nat1 Acad Scz USA 90,2438-2442 Kearse, K P., Roberts, J L., Mumtz, T., Wlest, D L., Nakayama, T , and Singer, A (1994) Developmental regulation of c@ T-cell antigen receptor expression re- sults from differential stability of nascent TCRa proteins wlthm the endoplasmic reticulum of immature and mature T-cells EMBU J 13,4504-45 14 Kearse, K. P. and Singer, A. (1994) Isolation of immature and mature T-cell receptor complexes by lectm affinity chromatography J Zmmunof Methods 167, 75-8 1 Chllson, 0 P and Kelly-Chllson, A E (1989) Mltogemc lectms bmd to the antl- gen receptor on human lymphocytes. Eur J Immunol. 19,389-396

lectin methods and protocols volume 9 || t-cell receptor purification

Documents