Fractionation and identification of pig lymph node plasma membrane glycoproteins

0161-51(90~82;020201-17s03.~/0 0 1982 Pwgamon Press Ltd

FRACTIONATION AND IDENTIFICATION OF PIG LYMPH NODE PLASMA MEMBRANE GLYCOPROTEINS BRIAN

H. BARBER*

and

SUDHA

ARYA

~epartrnel~t of Microbiology and Parasitology, University of Toronto. FitzGerald Building. Toronto, Ontario, Canada M5S lA8 (Rewired

19 March

1981:

mepted 28 Ma>, 1981)

Abstract-Plasma membrane vesicles purified from pig mesenteric lymph node tissue were solubilized in 1’: (w/v) sodium deoxycholate and the fractionation of both membrane proteins and glycoproteins assessed by gel filtration on AcA 34. Milligram quantities of the glycoprotein fraction, consisting of 12 distinct bands on SDS-PAGE ranging in apparent mol. wt from 12,000 to 250,000, were purified by affinity chromatography on lentil &tin-Sepharose. Identified among these by specific immunoprecipitation were the major histocompatibility antigen, SLA (band 9), in association with pz-microglobulin (band 12); as well as the d( (band 10) and fi (band 11) subunits of the la-like antigens. A tentative identification of the membrane-bound immunoglobulins (IgM, IgG and IgA) was also proposed on the basis of their known affinities for protein A. Thus, the domestic pig represents an inexpensive, abundant source of defined IympbocyIe plasma membrane glycoproteins suitable for further structural analysis.

ficient quantities of purified PM in the genetically refined small-animal models, such as the The development and control of an immune mouse, are considerable, and make it necessary response are known to be cooperative phenomfor this type of study to explore the utility of ena at the cellular level (Golub, 1977). larger-animal models. The principal sensory organelle in the recepOne such large-animal system which has tion and transduction of the regulatory signals been exploited for this purpose in the past is is the lymphocyte PM.? In terms of underthe domestic swine. The mesenteric lymph standing immunological phenomena at the node tissue of domestic swine slaughtered for molecular level, it would, therefore, be very commercial purposes represents an abundant, useful to understand a great deal more about inexpensive supply of raw material for structhe structural and organizational features of tural studies. Methods have been developed for the lymphocyte PM proteins and glycothe convenient isolation of large quantities of proteins. One approach to realizing this objecextensively purified PM vesicles from this tive involves the isolation. in large quantity, of source (Allen & Crumpton, 1970; Snary et ai., highly purified lymphocyte PM vesicles. With 1976). Extending these fractionation prosuch a vesicle system it would then be possible cedures to the isolation of inside-out and rightto probe organizational parameters of the PM side-out PM vesicles has greatly facilitated the components at both the external and cytoplastopographical analysis of individual membrane mic interface, and potentially relate these strucproteins (Walsh et nl., 1976; Walsh & Crumptural features to the molecular mechanism of ton, 1977). The pig PM system has also proved transmembrane information transfer. However, useful in defining the parameters of interaction the problems associated with generating sufbetween cytoskeletal components, such as actin, and the lymphocyte PM (Barber & * To whom correspondence should be addressed. Crumpton, 1976). t Abbreviations used: DOC. sodium deoxycholate; A considerable advance in the utility of the NP-40, Nonidet-40: SDS-PAGE, sodium dodecyl sulfatepolyacrylamjde gel clcctrophoresis: .S. IWWS, S~~~~z~~~- pig as a large-animal model for studies of the c~cctls uurrrrs; PM, plasma membrane; NEM, N-ethyl immune system has been provided by Sachs et maieimide; TBS, Tris-buffered saline; LcH, lentil lectin; al. (1976) with the development of miniature a-MeMan, a-methylmannoside; BSA. bovine serum albumin; PSA, pig serum albumin; TCA, trichloroacetic swine herds inbred for their MHC. Among acid ; PMSF, phenylmethylsulfonyl fluoride; b2-m, numerous advantages, the availability of these /3*-microglobulin; SLA, swine leukocyte antigen; MHC, inbred strains enables one to generate large major histocompatib~lity complex. INTRODUCTION

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BRIAN

H. BARBER

amounts of alloantisera specific for gene products of the MCH, thus facilitating their identification, characterization and isolation in quantity from animals of known genotype (Lunney & Sachs, 1978, 1979). In this report, we have extended our studies of the domestic swine system by examining the fractionation properties of the detergent-solubilized PM vesicles purified from the mesenteric lymph node tissue. In part, we have demonstrated that LcH affinity chromatography can be used to purify large quantities of intact PM glycoproteins; a preparation which can be shown to include the major histocompatibility antigen SLA (Ivanyi, 19773, as well as the n- and 1_3-polypeptides of the Ia-like antigens. Thus, the domestic swine system represents an inexpensive and reproducible means of isolating relatively large (i.e. milligram) quantities of numerous important membrane molecules for further structural and functional analyses.

MATERIALS

AND

METHODS

PM vesicles were prepared from abattoirderived pig mesenteric lymph node tissue according to the procedure of Snary et al. (1976). The purified PM vesicles were incubated with 7 mM NEM (Calbiochem) in 10 mM Tris, 0.15 M NaCl, pH 7.4 (TBS) for 30 min on ice prior to solubilization with 1% (w/v) DOC. The alkylation of free sulfhydryl groups by NEM was found to be essential in order to prevent detergent-induced artifactual disulfidebonding (Allore & Barber, manuscript in preparation). DOC solubilization of the PM vesicles was achieved by mixing an equal volume of PM vesicles in TBS (at 5-8 mg of membrane protein/ml) with 2Y;/ (w/v) DOC in 10 mA4 Tris, pH 8.2. After incubation for 30min on ice, the mixture was centrifuged at l~,OOO g for 1 hr and the supernatant used for gel filtration or affinity chromatography.

Gel fi ftration Five millilitres of the DOC supernatant fraction (approximately 2 mg of protein/ml) was loaded onto an AcA 34 Ultrogel (LKB) column (2.5 x lOOcm, equilibrated in 10 mM Tris, 0.57; DOC, pH 8.2) using an upward flow rate of 30 ml/hi-. The column was developed at the

and SUDHA

ARYA

same flow rate with 0.5% DOC, 10 11% Tris, pH 8.2, collecting 3-ml fractions and monitoring protein by measurement of OD at 280 nm.

Lectin ujiinity chromatography The purification of LcH and the coupling of LcH to CNBrSepharose (Pharmacia) have been described elsewhere (Barber & Arya, manuscript submitted for publication). In general, 4-5 ml of DOC-solubilized PM protein (approximately 2 mg/ml) were loaded onto a 5-ml column of LcH-Sepharose (3-4mg of LcH per millilitre of gel) at a flow rate of 10-20 ml/hr in 0.576 DOC, 10 mM Tris, pH 8.2. After washing the column with 10 column volumes of the same buffer, and ensuring that the OD at 280 nm returned to a background level, the column was eluted by the addition of 0.1 M @-MeMan (Sigma) in 0.5% DOC, 10 mM Tris, pH 8.2. In some cases, the eluted glycoprotein fraction was concentrated by pressure ultrafiltration using an Amicon apparatus (Model 75) with UM 10 diaflo membranes (Amicon). In order to ensure that all available LcH-binding glycoproteins had been removed by the earlier procedure, the unretarded fraction was run over a second fresh LcH-Sepharose as described earlier. In this case, a negligible amount of material was eluted upon the addition of 0.1 M a-MeMan.

Labelling proteins

qf‘ membrane proteins

and glyco-

The Bolton-Hunter reagent (Bolton & Hunter, 1973) [N-succinimidyl-3-(4-hydroxy, 5-[ I2 “I]iodophenyl)propionate~ (Amersham, product No. IM.861) was used to label the proteins in individual fractions from the gel filtration column. Twenty-five microcuries of the ’ 251-Bolton-Hunter reagent was evaporated to dryness in a small glass test tube by a stream of N, and 0.5 ml of the AcA 34 column fractions added to the dried ester. The mixture was incubated on ice for 30min with periodic shaking, and then the sample was prepared for SDSPAGE by the addition of SDS to 2% (w/v), glycerol to 10% (w/v) and Tris to 0.1 M, pH 6.8. LcH-Sepharose-purified glycoproteins were resimilarly labelled with i2’T-Bolton-Hunter agent. Two hundred to two hundred and fifty microcuries of the reagent was evaporated to dryness as before, and then I ml (0.8-1.0 mg) of

Fig Lymph

Node Plasma

the glycoprotein fraction added. After 30min on ice the labelled glycoprotein was dialysed against 0.5”/, DOG, 1 mM Tris, pH 8.2 (3 x 200 ml), prior to use as labelled antigen in the immunoprecipitation analyses. immunoprrcipitution

A stock solution of protein A-Sepharose (,Pharmacia) was prepared by placing 0.5 g of dry gel in 5 ml of PBS containing 0.5% (w/v) NP-40 and 1 mg/ml BSA. This solution, once prepared, was washed 3 times with the same buffer and resuspended in a final volume of 5 ml. Prior to use for immunopre~ipitation, the l 251-labelled glycoprotein fraction (approximately 1 mg/ml) purified in DOC from the LcH-Sepharose column was made 0.5% (w/v) in NP-40 and incubated with an equal volume of the protein A-Sepharose stock solution on ice for 30 min. The protein A--Sepharose was then pelleted by a 3-min spin in the Eppendorf microcentrifuge (Model 5412; 12,000 9). The supernatant was saved for immunoprecipitation analysis, and the pellet resuspended in a small volume of SDS-PAGE buffer for subsequent examination of the adsorbed components. A second adsorption of the same supernatant was found not to remove any further protein bound lz51. Adsorption of the ‘251-labelled glycoprotein fraction with fixed S. u~eus (Pansorbin, Calbiochem) was performed by mixing equal volumes of the glycoprotein fraction (approximately 1 mg/ml) and the fixed S. uureus suspension (10% suspension in 0.5% NP-40, 1 mgjml BSA, phosphate-buffered saline), incubated for 30 min on ice as before, and then centrifuged for 1 min in the Eppendorf microcentrifuge to recover the supernatant for immunoprecipitation. For immunoprecipitation, a 50-~1 aliquot fraction of the ‘251-labelled glycoprotein (2-4 x 10’ TCA-precipitable cpm) was incubated with 10-20/d of antiserum for 15 hr at 4°C. The immune complexes were then collected by the addition of protein A-Sepharose [S-fold excess (v/v) for the mouse. rabbit or goat antisera, and 20-fold excess for the swine alloantisera (see Fig. X)]. After incubation for 30 min on ice, the protein A-Sepharose was collected by a I-min spin in the Eppendorf microcentrifuge. The pellet was washed 3 times with 1.5 ml of the NP-4OjBSA/Tris buffer, and after the final wash was transferred to a clean

Membrane

203

Glycoproteins

Eppendorf microcentrifuge tube and resuspended in SDS-PAGE buffer containing 5% (w/v) 2-mercaptoethanol (Sigma Chemical Co.). After a 5-min incubation at room temperature, the solubilized radioactivity was recovered in the supernatant by a 3-min spin in the Eppendorf microcentrifuge. Samples were stored at -20°C prior to analysis by SDS-PAGE.

SDS-PAGE was carried out using a slab gel apparatus and the Laemmli Tris/glycine buffer system (Laemmli, t970) as described in detail elsewhere (Allore & Barber, manuscript submitted for publication). Autoradiography of the fixed, stained and dried gels was carried out by exposure to Kodak X-Omat H-l X-ray film in a Kodak X-Omatic enhancing screen cassette (regular) at - 70°C for various lengths of time. The reaction of fixed and stained SDS-poIyacrylamide gels with ‘251-L~H in order to visualize the glycoproteins was carried out according to the methodology described by Burridge (1976). and visualized by autoradiography as outlined earlier. RESULTS Gel

jltrtrtion

of the DOC-soluhilized

PM pro-

teins

The ability of various ionic and non-ionic detergents to solubilize the purified pig lymph node PM vesicles has recently been described elsewhere (Allore & Barber, manuscript submitted for publication). Very briefly, 1% DOC has been shown to solubilize 6~70~ of the total protein associated with the purified vesicles and SO-90% of the PM glycoproteins. Because DOC has a relatively small micelle size. it is possible to fractionate membrane proteins dissolved in this detergent by means of gel filtration (Helenius & Symons, 1975). Therefore, as a preliminary step towards the purification of individual membrane proteins, and to assess indirectly the nature of intermolecular associations among the membrane proteins, we have examined the fractionation of the DOC supernatant by chromatography on crosslinked agarose gels. Fractionation was achieved by ascending chromatography on columns of AcA 34. In general, 10 mg of membrane protein (2 mg/ml) in the presence of 1% DOC, 10 mM Tris, pH 8.2, was loaded onto the

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Volume (ml) Fig. 1. Profile AcA 34 in the right indicate encircled

PM protein fractionated by gel filtration on of the OD at 280 nm for l”,, DOC-solubilired presence of OS”,, DOC as described in Materials and Methods. The arrows from left to the elution positions of blue dextran. aldolase. BSA and cytochrome c, respectively. The numbers indicate representative fractions taken from the column for further analysis.

column and eluted with 0.5% DOC, 10mM Tris, pH 8.2. In each case, the purified PM vesicles were treated with NEM prior to detergent solubilization. This was found to be necessary in order to prevent artifactual disulfide bond formation at the time of detergent solubilization (Allore & Barber, manuscript in preparation). The profile of OD at 280 nm for a typical fractionation is presented in Fig. 1. The observation that the majority of the protein is not excluded from the gel indicates that the solubilized proteins are not extensively aggregated and that fractionation according to molecular size and shape has taken place. The fact that relatively little protein is distributed throughout a large column volume facilitates fractionation, but presents problems in assessing the protein content of individual fractions. Various methods were tried to circumvent this problem. Prelabelling of the purified PM vesicles with millicurie amounts of lz51 either via the lactoperoxidase procedure (Morrison & Boyse, 1976), or by means of the chloramine T reaction (Hunter & Greenwood. 1962). was found to yield insufficient radioactivity in a single fraction to enable convenient detection of the proteins by autoradiography of SDS-polyacrylamide gel tracks. Pooling or concentration of radiolabelled fractions by means of precipitation with either cold acidified acetone or ethanol at -2O’C, was also unsatisfactory, as these procedures often lead to aggregates of the radiolabelled proteins which would not enter the SDS-polyacrylamide gel (10% acrylamide). Satisfactory results were achieved, however, by the labelling of individual fractions with the Bolton-Hunter reagent, an lz51-containing N-hydroxy-succinimide

ester of iodinated p-hydroxyphenyl propionic acid (Bolton & Hunter. 1973). Thus, the Bolton-Hunter reagent (approximately 20 FCi) was reacted with a l-ml sample of the column fraction of interest for 30 min on ice in the presence of OS”/;; DOC, 10 mM Tris, pH 8.2, and subsequently prepared for SDS-PAGE by the addition of SDS to 22, (w/v), glycerol to 10% (v/v) and Tris to 0.1 M, pH 6.8. Approximately l&20’%, of the added radioactivity was incorporated into TCA-precipitable material for each of the various fractions across the column. Therefore, SDS-PAGE followed by autoradiography provided a convenient means of assessing both the qualitative and quantitative distribution of protein in a given fraction. The results of this type of analysis for the AcA 34 fractionation are presented in Fig. 2 for seven representative fractions across the column profile shown in Fig. 1. Both 10% (Fig. 2A) and 15x, (Fig. 2B) acrylamide gels were run on each fraction in order to resolve adequately the high and low mol. wt components. Examination of the constituent proteins in each gel track confirms that fractionation according to molecular size has indeed occurred during the column elution. In tracks l-3 of Fig. 2A, one can observe fractionation among the four high mol. wt components in the 150,00~200,000 mol. wt region. However, in addition to these high mol. wt components, tracks 2 and 3 also contain a low mol. wt protein in the region of 13,000. Its appearance at this point on the gel profile suggests that in its native form it either exists as a high mol. wt homopolymer of numerous subunits. or represents a subunit of a much higher mol. wt

Pig Lymph Node Plasmlt Membrane Glycoproteins

205

206

BRIAN H. BARBER and SUDHA

complex. The same argument can be applied to a number of other lower mol. wt components as well. The major component of both tracks 4 and 5, which comigrates with BSA, is known to be PSA. Previous studies have indicated that PSA remains tightly adherent to the PM vesicles during their isolation (Owen et ul., 1980) and then fractionates exclusively in the supernatant fraction upon solubilization with DOC (Barber & Allore, 1979). Its localization exclusively in tracks 4 and 5, corresponding to the position of the BSA marker on the AcA 34 column, provides a convenient internal control on the fidelity of the fraction procedure. In contrast to the examples cited thus far, there are some proteins which appear to be present in each of fractions l-6 across the column profile. Actin, another protein which has been previously shown to associate with the purified PM vesicles (Barber & Crumpton, 1976), is an example of this class. Although track 4 in Fig. 2A appears to contain the greatest amount of actin, a comigrating band is also apparent in each of tracks l-6. It is well known that actin possesses the ability to form homopolymers of varying size, and the distribution of this component across the entire column profile may reflect this type of self-association. On the other hand, it may be that actin appears in the different fractions by virtue of its participation in intermolecular complexes of varying mol. wt. In this regard, it is interesting to note that at least two other proteins with apparent mol. wt of approximately 85,000 and 38,000, also appear in each of the fractions in a manner similar to that of actin. Whether their distribution parallels that of actin by coincidence, or by virtue of a non-covalent interaction with actin remains to be established. Finally, in track 4 of Fig. 2B there appears a prominent low mol. wt band which has been shown to comigrate with a sample of human flz-m (data not shown). In all probability this represents the flz-m which has been shown to associate with the major histocompatibility antigen of swine, SLA (Chardon et al., 1978). The appearance of this component in track 4, and to a lesser extent in track 5 (Fig. 2B), correlates well with the appearance of a broad band migrating just ahead of actin (as observed in Fig. 2A). As will be demonstrated later, this corresponds to the migration position of the heavy chain (‘3) of the SLA complex. Collectively these indications suggest that the SLA complex elutes from the AcA 34 column in a

ARYA

position corresponding approximately to that of BSA, consistent with a dimeric structure composed of a single copy each of the SLA heavy chain and f12-m, in association with a small amount of bound detergent (Snary rt Al., 1975). As an alternative to labelling all of the proteins with the BoltonHunter reagent. 1251labelled LcH ( 1251-L~H) can be used to focus on the distribution of glycoproteins across the gel profile. In so far as it has been previously demonstrated that LcH binds to a large fraction of the protein-bound carbohydrate in the pig lymph node PM preparation (Hayman & Crumpton, 1972) this method should provide a fair estimate of the total glycoprotein distribution across the gel profile. The results obtained overlay procedure for the for the r2’I-LcH same seven fractions depicted in Figs 1 and 2 are presented in Fig. 3. The glycoproteins most readily visualized by this technique are grouped in the 150,00~200,000 mol. wt region (Fig. 3A). Longer exposure times were required to visualize the lower mol. wt components (Fig. 3B). Fraction 1, the void volume peak, contains ‘251-L~H reactive material, relatively little whereas fraction 2 contains a prominent doublet (better resolved at lower exposures) which stains intensely with ’ 251-L~H. By comparison of the patterns for fractions 1 and 2 in Figs 2A and 3A, it is clear that the bulk of the protein in these fractions is not reactive with 1251-L~H (i.e. probably not glycosylated), and that the glycoproteins present are high mol. wt components (i.e. > 130,000). Thus the data suggest that the high mol. wt glycoproteins could be extensively purified from the bulk of the membrane protein by subjecting fraction 2 from the AcA 34 column to affinity chromatography on a column of LcH-Sepharose. The extent to which the LcH-Sepharose affinity chromatography would be successful in providing highly purified glycoprotein would also be a reflection of the non-covalent interactions between these high mol. wt glycoproteins and the non-glycosylated components of fraction 2. It is apparent on the longer exposure autoradiogram (Fig. 3B) that fraction 4 contains a number of ‘2sI-LcH-reactive components. Most prominent among these is a glycoprotein with an apparent mol. wt of 55,OOt&60.000. There is also a cluster of labelled bands in the 38,00@42,000 mol. wt range and a prominent doublet in the region of 26,000-32,000. From

Fig. 3. Autoradiograms ‘2”I-LcH as described

3

4

5

67

of the SDS-PAGE patterns for fractions 1-7 and the loaded supernatant (S) for the AcA 34 column depicted in Materials and Methods. A and B represent lower and higher exposures of the same 15”, gel. The mol. wt markers are: phosphorylase a (94,000); WA (68,000); actin {42,ooO); DNase I (31,CUH); and cytochrome c (12,500).

2

in Fig. 1 overlaid with indicated by the arrows

208

BRIAN

H. BARBER

and SUDHA

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Fig. 4. Autoradiograms of the SDS--PAGE patterns for the ““I-Bolton-“Hunter-labelled DOC supernatant (S), the unretarded fraction (U). and the retarded and 0.1 ,V cl-MeMan-&ted fraction (R). from the LcH-Sepharose fractionation of the DOC-soiubiIized PM proteins: ia) IO”,, gel: and (b) IS’,, gel of the same fractions. The numbers 1-12 arbitrarily designate the bands consistently found in the retarded and eluted fraction. LcH designates the labelled band comigrating with the LcH subunit. The mol. wt markers are: (a) 94,000; 68,000; 42,000 and 31,000; and (b) 94,000; 68,000: 42,000 and 12,500.

Pig Lymph Node Plasma Membrane Glycoproteins

209

in order to provide resolution of components over the range 1O,OOO-300,000.For convenient reference, the major bands, or groups of bands, have been numbered in descending order of apparent mol. wt [Fig. 4(a) R and Fig. 4(b) R]. The 12 bands thus identified were consistently represented from one glycoprotein preparation to another. The ‘2SI-labelled band designated ‘LcH’ was aIso co~lsistently found in the 0.1 M ~-MeMan-eluted glycoprotein fraction, and was observed to comigrate with the 18,000 mol. wt subunit of purified LcH. If the 2% DOC supernatant was labelled with the r2’I-BoltonHunter reagent prior to loading on the column (as opposed to labelling the ~-reman-el~lted fraction), then this band was no longer detected in the autoradiogram of the glycoprotein preparation (Barber & Arya, manuscript submitted for publication). On the basis of this evidence, we conclude that this band represents LcH eluted from the LcH-Sepharose column upon the addition of a-MeMan. The presence of the LcH subunit in the column eluate has significant implications for subsequent manipulation of the purified glycoprotein fraction and is a point which should be kept in mind by those using LcH-Sepharose chromatography in the presence of DOC. By loading DOC supernatants which have LcH-Sepharose qfiinity chromatography been pre-labelled with “‘1 either by the Lectin affinity chromatography is now exten- chloramine T procedure (Hunter & Greensively used as a means of glycoprotein purifica- wood, 1962), or by use of the Bolton~Hunter tion from detergent-solubilized extracts of cells reagent (Bolton & Hunter, 1973), it is possible to quantitate that recovery of protein in both or membrane vesicles (Lotan & Nicolson 1979). One of the earliest applications of the the unretarded and the retarded and eluted lectin derived from Lens culinaris (i.e. LcH) was fractions. In general, it was found that apin the purification of glycoproteins from a puri- proximately SO-857; of the loaded protein fied pig lymph node PM preparation solubil- passed unretarded through the LcH-Sepharose ized in DOC (Hayman & Crumpton, 1972). We column, and &-loo/, was recovered by the addition of 0.1 M ar-MeMan. have re-examined this fractionation procedure The loss of 5-10% of the total TCA-precipifor the pig lymph node system with the purtable counts, presumably due to either specific pose of more precisely defining the constituent glycoproteins. The polypeptide profiles of the or non-specific high-affinity binding sites, 2% DOC supernatant (S) loaded onto the means that one can only estimate the glycoLcH-Sepharose column, the material which protein content of the DOC supernatant as passed unretarded through the column (U), being 6150/, of the total protein. On the basis of OD measurements at 280nm, Hayman & and the components eluted from the column Crumpton (1972) reported the yield of glycowith 0.1 M a-MeMan (R), have been presented in Fig. 4. The unretarded and the eluted frac- protein as 12% of the total protein added to the column. Our findings would indicate that tions were labelled via the “‘1-Bolton-Hunter reagent after fractionation, and compared by their values may be a somewhat inflated estimate due to the likely presence of the LcH SDS-PAGE with the starting supernatant labelled in the same way. The gel profiles for subunit in the eluted fraction. Assuming, thereboth 10 and 15% acrylamide slab gels have fore, a yield of 10% one can calculate that approximately 15 mg of membrane glycoprotein been presented [Fig. 4(a) and (b) respectively]

the data of Lunney & Sachs (1978) in the miniature swine system, it is shown that the heavy chain of the major histocompatibility antigen, SLA, and the dimer of la-like polypeptides are reactive with LcH. Thus we can speculate that at least one of the LcH-reactive components in the 38,00&42,000 mol. wt region corresponds to SLA. The major histocompatibility antigens of other species are known to be sensitive to proteolytic degradation; thus all three bands observed in this region may be related to SLA. The pair of bands in the 26,OOO-33,000 mol. wt region correspond well with the mol. wts cited for the CI- and ~-subunits of the la-like dimer in the miniature swine system (Lunney & Sachs, 1978). As will be illustrated later this pair of bands also corresponds to the Ia-like antigens of the domestic swine as defined by specific immunoprecipitation. Thus it is interesting to note that r2%LcH react with both the a- and P-subunits of the la-like dimer, indicating that each polypeptide possesses covalently bound carbohydrate. In concert with this finding, the cr- and P-subunits of the human la-like antigens have both been shown to be sialoglycoproteins (Springer et al., 1977).

MIMM. 19!2--8

210

BRIAN

H. BARBER

can be purified by LcH-Sepharose affinity chromatography of the DOC-solubilized PM vesicles for each 150 g of wet weight mesenteric lymph node tissue used in the PM preparation (Allan & Crumpton. 1970). For reference. 150 g of tissue can be readily obtained from the mesenteric lymph nodes of 224 B-month-old domestic pigs and this represents the amount routinely processed in each PM preparation. Thus one can appreciate the potential to generate, both economically and in a relatively short space of time, milligram quantities of the major glycoproteins depicted in Fig. 4R. By comparing the total supernatant polypeptide profile with that of the unretarded and eluted fractions (Fig. 4) it is clearly possible to see the extensive homology between the starting supernatant [Fig. 4(a) S] and the components which do not interact with LcHSepharose [Fig. 4(a) U]. This, of course, is consistent with the glycoproteins representing only a small fraction (i.e. approximately 10%) of the total protein loaded onto the column. As expected, the PSA present in the solubilized PM passed unretarded through the LcHSepharose, with only a trace amount of comigrating protein visible in the eluted fraction [Fig. 4(a) R]. Similarly, actin, another protein known not to be glycosylated, was equally unretarded by the LcH-Sepharose column and is very poorly represented, if at all, in the glycoprotein fraction [Fig. 4(a) R]. As indicated earlier, the 157; acrylamide gels were utilized to extend the observable mol. wt range to the region where one could expect to see PI-m. It is well known that in the pig (Chardon rt al., 1978) the major histocompatibility antigen occurs as a dimer in association with a protein extensively analogous to serum P2-m. More definitive evidence indicating by means of immunoprecipitation that band 12 corresponds to the Pz-m component of the SLA complex will be presented later. It is also noteworthy that the glycoproteins purified by LcH-Sepharose affinity chromatography were isolated in the absence of added proteolytic inhibitors. When the purified PM vesicles were solubilized by 2% DOC in the presence of a proteolytic inhibitor, 1 mM PMSF and the subsequent LcH-Sepharose chromatography carried out in buffers containing 1 mM PMSF, essentially the same glycoprotein profile illustrated in Fig. 4 was obtained. In view of this observation, and because the proteolytic inhibitor was found to

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Fig. 5. An autoradiogram of the SDS-PAGE pattern (IO”,, gel) for the 1251-labelled LcH-Sepharose-purified glycoprotein fraction before (A) and after (B) adsorption with fixed S. aureus (Pansorbin), The components eluted by SDS-PAGE buffer from the washed S. critreus used in the adsorption are depicted in C. The mol. wt markers designated by the arrows are. in descending order: 94,000; 68,000; 55.000 (pig y-chain); 42,000: 31,000 and 25,000 [pig light chain (L.C.)].

cause some precipitation of the purified glycoprotein fraction, exogenous proteolytic inhibitors were not used in any of the purification procedures.

Pig Lymph

Node Plasma

Membrane

Glycoproteins

211

Ident$cation qf individual PM glJ’coproteins ha inzmunopwcipitation or interaction \$-ithprotein A

The IgG binding properties of protein A derived from S. aureus have been well documented (Goding, 1978). As a result, fixed S. aureus organisms are now commonly used to facilitate the collection of immunoprecipitates in preparation for SDS-PAGE. Therefore, as a prelude to reacting the LcH-Sepharose-purified glycoprotein fraction with specific antisera, the glycoproteins were adsorbed with fixed S. aureus or with protein A coupled to Sepharose (protein A-Sepharose). The results of these adsorptions are depicted in Figs 5 and 6. Comparison of tracks A and B in Fig. 5 indicates that the major glycoproteins remain in the supernatant after exposure to an excess of fixed S. aureus. However, by extracting the bacterial pellet resulting from this adsorption with SDSPAGE buffer, it is possible to elute those components which are adherent to the immunoadsorbent (i.e. Fig. 5C). Expected among these components would be the PM-associated immunoglobulins by virtue of their capacity to interact with the protein A on the fixed S. aureus. Thus, it is interesting to note that a closely spaced pair c, bands was found to migrate at the position of the pig serum IgG heavy-chain marker, and another pair of bands was observed corresponding in apparent mol. wt with the IgG light-chain marker. In addition, two higher mol. wt S. aureus-binding components were observed, one comigrating with band 4 in the glycoprotein preparation, and another migrating somewhat above the bulk of band 7 in the mol. wt region expected for the p-chain of membrane-bound IgM (Singer & Williamson, 1980). In order to differentiate between the components which were interacting directly with protein A on the fixed S. aureus and those which might be binding in some other fashion to the bacterial surface, a comparison was made between adsorptions of the glycoproteins with fixed S. aureus and protein A-Sepharose (Fig. 6). By comparing tracks C and E of Fig. 6 it is possible to distinguish those components which are bound both to S. aureus and to protein A-Sepharose (indicated by arrows) from those which appear to interact only with the fixed bacteria (indicated by an ‘X’). Among those which appear to interact only with the fixed bacteria is the high mol. wt band 4 comigrating component. In contrast,

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>

X

x

Fig. 6. A comparison by SDS-PAGE (15:); gel) of the adsorption of ‘251-labelled LcH-Sepharose-purified glycoproteins with fixed S. aureus and with protein A-Sepharose: (A) the unadsorbed “‘1-glycoprotein fraction; (B) the glycoprotein fraction adsorbed with fixed S. aureus; (C) the components bound to the fixed S. aureus used in the adsorption: (D) the glycoprotein fraction adsorbed by protein A-Sepharose; and (E) the components bound to the protein A-Sepharose used in the adsorption. The small arrows indicate components thought to be reactive with protein A and the ‘X’ components which appear to react with non-protein A sites on fixed S. aurcus. The markers are: 94,000; 68,000; 42,000; 31,000 and 12,500. LcH indicates the position of migration of the 18,000 mol. wt LcH subunit.

the polypeptides which migrate in the mol. wt region of the immunoglobulin heavy chains p and y, and in the vicinity of the light-chain marker, appear to be bonafide protein A-binding components (indicated by arrows).

212

BRIAN

H. BARBER

It has recently been demonstrated that when pig serum is passed over a column of protein A-Sepharose, IgG binds almost quantitatively to the column (Milton et al., 1978). IgM and IgA were also detected in the low pH elution of the same protein A-Sepharose column, indicating that at least three of the immunoglobulin isotypes in the pig are capable of interacting with protein A. Based on this illformation and the data presented in Figs 5 and 6, we tentatively conclude that the protein A-binding polypeptides correspond to the membranebound immunoglobulins. Thus, we suggest that the component migrating just above band 7 represents the [l-chain of membrane-bound IgM, and the ~-chain-comigrating component in the region of band 8 represents the heavy chain of membrane-bound IgG. The appearance of a doublet at the position of the y-chain marker in track C of Fig. 5 raises the possibility that both the c(- and ;I-chain are present in the glycoprotein fraction. It is known from studies of pig IgA (Rejnek & Prokesova, 1973), that the apparent mol. wt of the x-chain falls in the range of 61,000, only marginally greater than that of y. The doublet may, therefore. represent closely spaced P*-and ~I-polypeptides derived from membrane-bound IgA and IgG respectively. Having adsorbed the Bolton-Hunterlabelled glycoprotein preparation with protein A---Sepharose, this fraction was then reacted with specific antisera in an attempt to characterize further the remaining glycoproteins. Previously, Lunney & Sachs (1979) demonstrated that alloantisera specific for the k-haplotype of the I-region of the mouse MHC would crossreact with the la-like antigens in a number of different species, including man, the rat and miniature swine. Utilizing one of the alloantisera described in their study, A.TH. anti-A.TL., we examined whether or not the same crossreactivity could be observed with the lymph node glycoproteins isolated from the domestic swine population. The adsorbed glycoprotein preparation was also reacted with a goat antipig f12-rn antiserum in an attempt to precipitate the SLA complex. Autoradiograms of the SDS-PAGE profiles obtained for these immun~~precipitations are presented in Fig. 7. The immunoprecipitates were collected by means of protein A Sepharose utilizing a minimum of 50 ~1 of the standard protein A-Sepharose suspension for each 10 ~11of rabbit, mouse or goat serum present in the precipitation mixture. As

and SUDHA

ARYA

ABCD

LcH*

Fig. 7. An autoradiogram of the SDS--PAGE pattern (IS’,, g,el) for the immune complexes recovered by protein A-Sepharose upon reaction of the “sl-labelled LcH-Sepharosc-puri~ed glycoprotein fraction with: (A) nonimmune A.TH. mouse serum: (B) A.TH. anti-A.TL. mouse alioantiserum; (C) normal goat serum: and (D) goat antipig jj2-m antiserum The putative Ia r- and /i-polypeptides, as well as the SLA r-chain and fi,-m have been indicated. LcH refers to the position of migration of the 18,000 mol. wt subunit of LcH.

indicated in Fig. 8, protein A-Sepharose is less efficient in the collection of pig IgG than it is for rabbit or mouse IgG, and therefore, 2004 of the protein A-Sepharose stock suspension was used for each lo-$-input of pig alloantiserum. A similar increase in the amount of

Pig Lymph

Y 50

100 {,I Pmt.

150 A-Sepharose

200

Node Plasma

250

susp.

Fig. 8. A plot of the percentage of the input “‘1-labelled IgG recovered by varying amounts of the stock protein A-Sepharose solution (0.5 g protein A--Sepharose dissolved in 5 ml of 0.5”,, NP-40, 1 mg,‘ml BSA. PBS) when incubated for 30 min at 22 C with lOOb(g of: mouse IgC (&-); rabbit IgG (-0 --): and pig IgG (- A - ): and then centrifuged for 1 min in an Eppendorf microcentrifuge.

fixed S. aclreu.s required for the optimal collection of pig immunoglobulin was noted previously by Lunney & Sachs (1978). As can be seen by a comparison of tracks A and B in Fig. 7, the A.TH anti-A.TL antiserum precipitated a pair of closely spaced bands migrating just above and below the 31,OOOmol. wt DNase I marker protein. These bands were completely absent in the precipitation with non-immune A.TH mouse serum. By analogy with the results of Lunney & Sachs (1979), these bands have been designated as the c(- and fl-polypeptides of the domestic swine Ia-like antigens. These components correspond with bands 10 and 11 in the LcH-Sepharose glycoprotein fraction. Comparison of tracks C and D in Fig. 7 indicates that the goat anti-p,-m immunoprecipitated two prominent polypeptides. These are a human p,-m-comigrating polypeptide (i.e. pig &m) just in front of the 12,500mol. wt cytochrome c marker, and a second major component migrating very near the 42,000 mol. wt actin marker. Again, by analogy with the data of Lunney & Sachs (1978) in the miniature swine system, and consistent with the known association of &m, we have designated the higher mol. wt band as the heavy chain (a) of SLA. By co-electrophoresis on the same SDS-polyacrylamide slab gel, SLA-x and &rn have been shown to comigrate, respectively, with bands 9 and 12 of the LcH-Sepharose retarded and eluted glycoprotein fraction (Fig. 4).

Membrane

Glycoproteins

213

Note that in each immunoprecipitate there appears a prominent band comigrating with the major subunit of LcH (designated as LcH). As indicated previously. the LcH subunit can be detected in the LcH-Sepharose-purified glycoprotein fraction (Barber & Arya, manuscript submitted for publication). The appearance of 1251-labelled LcH in the immunoprecipitates collected by protein A-Sepharose suggests that at least some of the eluted LcH has the capacity to interact with the carbohydrate portions of the serum IgG bound to the protein A. Thus considerable caution should be exercised when designing experiments with glycoproteins purified by means of LcHSepharose in the presence of DOC. The presence of functional LcH, and the possibility of LcH-glycoprotein complexes or LcH-mediated cross-linking of glycoprotein, could lead to artifactual results in many different types of experiments.

Cross-rractiuity of miniature swine ulloantisera with glycoproteins dericed from thu domestic sbcine population Utilizing the inbred miniature swine system, Lunney & Sachs (1978) have demonstrated that by means of the appropriate cross-immunization, alloantisera can be generated which can be used to identify the gene products of the miniature swine MHC region. In order to determine whether or not the miniature swine alloantisera would recognize determinants in the MHC antigens of the domestic swine population at our disposal, the various alloantisera were reacted with the Bolton-Hunter ‘251-labelled lymph node glycoprotein fraction. The results found for five of the alloantisera [AA c( DD, AA r CC, CC x AA, AD s( AC and AC x AD (Lunney & Sachs, 1978)] are depicted in Fig. 9. Weak but detectable crossreactivity was observed using the antisera derived from allo-immunizations between animals which were homozygous at their MHC loci. Tracks A-C indicate precipitation of the SLA cc-chain, with the pz-m component less prominent but clearly visible on the original autoradiogram. Tracks B and C also contain visible bands at the positions corresponding to the E- and p-chains of the Ia-like molecules, indicating a degree of cross-reactivity for these sera with the pool of Ia-like antigens in the domestic swine glycoprotein preparation. The AD x AC and AC c( AD alloantisera depicted

214

BRIAN

H. BARBER

ABCDE

and

SUDHA

ARYA

LcH represented a prominent the collected immunoprecipitates.

component

of

DlSCUSSION

LcH>

*

P2nl’

”

. ,*

-

I-‘I~. 9. An autoradiogram of the SDS-PAGE pattern (15”,, gel) for the immunoprecipitates recovered by protein A-Sepharose after interaction of the ““I-labelled domestic pig glycoprotein fraction with various miniature swine alloantisera: (A) alloantiserum AA anti-DD; (B) AA anti-CC; (C) CC anti-AA: (D) AD anti-AC; and (E) AC anti-AD. The positions of migration on the same gel of the SLA heavy chain, Ia r- and /&polypeptides, the LcH 18.000 mol. wt subunit and /iz-m have been indicated. The small arrows indicate faint bands in tracks B and C which comigrate with the la r- and fi-polypeptides.

tracks D and E. respectively, demonstrated a much lower, barely detectable, degree of reactivity with the same purified glycoprotein fraction. Results similar to those depicted in Fig. 9 were obtained with three independently purified glycoprotein preparations, each derived from a pool of four to six abattoir-derived mesenteric lymph node tissues on different occasions. As with the previous immunoprecipitations utilizing the LcH-Sepharose-purified glycoproteins as the antigen. the ‘2sI-labelled in

Detailed structural analysis of individual eukaryotic surface membrane proteins is most often limited by a lack of highly purified material, and in this regard the lymphoid system is no exception. Although the mouse represents the most sophisticated model with which to study cell surface interactions in the immune system (Klein, 1975). the limited availability of lymphoid tissue from these animals places major constraints on the amount of PM material available for biochemical analysis. For example, Abney et (I/. (1976) were required to use 1750 mouse spleens in order to prepare 130 mg of purified lymphocyte PM as the first step in the purification of mouse membrane IgD. In contrast, the same amount of purified PM material can be isolated from the mesenteric lymph node tissue of two 6-month-old domestic pigs, using the methods outlined earlier. Therefore, in terms of developing new methodology, isolating individual membrane assessing general structural proteins, or features of the purified PM vesicles, the economic and strategic advantages of the pig system are worthy of consideration. It should also be noted that Sachs ef ~1. (1976) have recently developed three herds of miniature swine inbred with respect to their MHC. Thus the opportunity now exists to exploit many of the genetic advantages which were previously the domain of the inbred rodent systems, while at the same time retaining the considerable size advantage of the pig model in terms of biochemical analysis. As well, the immunological cross-reactivity between the miniature swine alloantisera and the domestic swine antigens suggests that considerable homology can be expected between the two systems. Gel filtration in the presence of detergents has been successfully applied to the isolation of several lymphoid cell surface antigens, most notably the gene products of the mouse and human MHC @nary et al., 1975: Springer rt ul., 1977; Freed et lrl., 1979). The results outlined in this report confirm the utility of gel filtration in the presence of a small micelle detergent such as DOC as a effective means of fractionating solubilized membrane proteins. In particular it was found that the ‘251-BoltonpHunter reagent in conjunction with SDS-PAGE provided a

Pig Lymph

Node Plasma

Membrane

Glycoproteins

215

the CI-and /?-polypeptides of the Ia-like anticonvenient method of monitoring the distribugens (Fig. 4, bands 10 and 11 respectively). tion of proteins in individual fractions, without Having established this correlation, it is having to develop efficient means of precipinow possible to relate the properties of these tating small amounts of protein and glycoprotein. Similarly, it was shown that ‘251-L~H polypeptides to the structural and functional could be used to localize the glycoproteins in features of their counterparts in other species. the SDS-PAGE patterns across the same A case in point is the question of an interaction between histocompatibility antigens and memchromatographic profile, and thus provide valuable additional information from each gel brane-associated actin. Koch & Smith (1978) filtration analysis. described a P815 murine mastocytoma system in which they were able to demonstrate a It has also been demonstrated that LcHnon-covalent association Sepharose affinity chromatography of the detergent-resistent, DOC-solubilized PM vesicles provided an between H-2 and actin. If this were a general feature of histocompatibility antigens, one effective and reproducible method of preparing might expect that it would be possible to detect milligram quantities of the PM glycoproteins. A numbering system has been proposed to actin in the LcH-Sepharose bound and eluted identify the 12 most prominent bands in the fraction of the detergent-solubilized PM. In LcH-Sepharose-retarded and z-MeMan-eluted fact, actin passed unretarded through the LcHfraction. These range in apparent mol. wt from Sepharose column [Fig. 4(a)], and was not approximately 250,000 (band 1) to 11,700 (band detected to an appreciable extent in the 12). The challenge now is to correlate these retarded and eluted fraction. Thus no evidence was found to support a pre-existing stoichiomolecules with specific cell surface functions, and, by analogy, to relate them to similar mol- metric interaction between actin and SLA, or ecules which have been identified in the lym- between actin and any of the other prominent glycoproteins. Koch & Smith (1978) also stated phoid system of other species. For example, Standring et al. (1978) have identified an anti- that they were unable to demonstrate an intergenie determinant which is expressed on action between actin and H-2 in lymphocytes. It is also noteworthy in terms of the relationprominent high mol. wt glycoproteins of thymocytes, T-lymphocytes and B-lymphocytes of ship between actin and histocompatibility antigens, that on the loo/(, SDS-PAGE pattern the rat. The T-lymphocyte molecule bearing [Fig. 4(a)] the mobility of SLA exceeds that of this antigen (termed the leukocyte-common antigen) has an apparent mol. wt of 170,000, actin, and on the 150/, gel their mobilities are and the B-lymphocyte component one of approximately the same [Fig. 4(b)]. Hermann 200,000. Trowbridge er ul. (1977) have de- & Mescher (1979) demonstrated in a 12% gel scribed a very similar marker in the mouse sys- that H-2 migrated with a distinctly greater apparent mol. wt than that of the actin marker, tem referred to as T200. Although one expects analogous structures to be expressed on pig whereas HLA possessed an apparent mol. wt lymphocytes, the relationship between these lower than that of actin on the same gel. This difference in mobility of the two histodeterminants and the group of glycoproteins in compatibility antigens was attributed to the the 150,000-250,000 mol. wt region (i.e. bands fact that H-2 possesses two covalently bound l-4) of the pig mesenteric lymph node prepcarbohydrates side chains (Ewenstein et al., aration remains to be established. At present 1978) and HLA only one (Parham et al., 1977). the pig system lacks well-defined surface antiThus one can speculate, by analogy in terms of genie markers which can be used to identify specific T-lymphocyte subpopulations (Binns, their mobility on SDSPAGE, that the SLA 1980). Hopefully by isolating these glyco- polypeptide also possesses only a single covalently attached carbohydrate side chain. This is proteins in quantity and preparing monospecifit antisera against individual components, a point which we hope to clarify by a chemical it will be possible to identify and characanalysis of SLA isolated from the glycoprotein terize subpopulation-specific surface glycoprofraction by means of preparative SDS-PAGE. Another set of glycoproteins which has been teins. identified in the LcH-Sepharose-eluted fraction One set of components in the purified glycoprotein fraction that has been identified are the are the protein A-reactive components. For principal gene products of the MHC. These in- reasons outlined earlier, we have suggested that clude the SLA heavy chain (Fig. 4, band 9) and these correspond to the heavy and light chains

216

BRIAN

H. BARBER

of membrane-bound IgM, IgA and IgG. The fact that they are not major components of the total glycoprotein profile is presumably related to the limited numbers of B-cells in the mesenteric lymph node tissue. Recent data suggest that approximately 20% of the pig mesenteric lymph node lymphocytes are surface Ig positive (Baxi et al., 1980). Chavin et ul. (1975) previously reported that it was possible, by Ouchterlony analysis, to detect IgG and IgM determinants in cholate-solubilized PM purified from pig mesenteric lymph nodes; however, comparable assays for IgA were not performed. Thus it is currently possible to isolate individual glycoproteins such as SLA, the Ia polypeptides, or the membrane Ig chains in milligram quantities by means of preparative SDSPAGE. Although this material will be very useful in studying the primary structure of these molecules, in order to best exploit the pig as a model system for the study of lymphocyte membrane proteins, it will be necessary to develop the means of isolating individual glycoproteins under non-denaturing conditions. Towards this end, we are presently involved in the preparation of monoclonal antibodies for use as affinity chromatography reagents, using spleen cells -from mice-i&m&zed with the complete glycoprotein fraction. Ackno~~Irdge,,le,~ta We wish to thank Swift Eastern (Toronto) for their generous cooperation in the provision of the pig mescnteric lymph node tissue. We also wish to thank Drs D. H. Sachs, J. K. Lunney and T. L. Delovitch for their enthusiastic cooperation in making available the miniature swine alloantisera. the g.oat anti-pig pz-m and the mouse A.TH anti-A.TL, respec :tively. This work was supported by the Medical Researc..,h Collrlcil ____. of Canada. B.H.B. is a Scholar of the Medical Researcl 1 Council.

REFERENCES Abner E. R., Hunter I. R. & Parkhouse R. M. E. (1976) Preparation and charactcrizatlon of an antiserum to the mouse candidate for IgD. Nature, Lnnd. 259, 404406. Allen D. & Crumpton M. J. (1970) Preparation and characterization of the plasma membrane of pig lymphocytes. Biochcvn. J. 120, 133-143. Barber B. H. & Allore R. J. (1979) In T/X, hfokwlur Bu.sis of Irnmurze Cell Function (Edited by Kaplan J. G.), pp. 127-137. Elsevier/North-Holland Biomedical Press. Amsterdam. Barber B. H. & Crumpton M. J. (1976) Actin associated with purified lymphocyte plasma membrane. FEBS ht. 66, 215 220. Baxl P. U.. Milan A., Fran/ J. & Metrger J. J. (1980) Use of Ruorescein conJu&ated staphylococcal protein A as a labelling agent for porcine lymphocytes. J. Immun. Meth. 35, 249-258. Binns R. M. (19X0) Pig lymphocytes mmbehaviour. distributlon. and classification. ,Monoyr. AI/cry!. 16. 19-37.

and

SUDHA

ARYA

Bolton A. E. & Hunter W. M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a ‘251-containing acylating agent. Biochem. J. 133, 529-539. Burridge K. (1976) Changes in cellular __ glvcoaroteins after . transformation: identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Pro<. nurn. Acud. Sci. U.S.A. 13, 4457-4461. Chardon P., Vaiman M., Renard C. & Arnoux B. (1978) Pig histocompatibility antigens and /j,-microglobulin. Trunspluntation 26, 107-l 12. Chavin S. I., Johnson S. M. & Holliman A. (1975) Plasma membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes. Biochrm. J. 148, 417-423. Ewenstein B. M., Nisizawa T., Uehara H., Nathenson S. G., Coligan J. E. & Kindt T. J. (1978) Primary structure of murine major histocompatlbility complex alloantigens: isolation, biochemical characterization, and preliminary alignment of CNBr fragments from the H-2Kh glycoprotein. Proc. nun. Acad. Sci. U.S.A. 75, 2909-29

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Freed J. H., Sears D. W., Browne J. L. & Nathenson soluS. G. (1979) Biochemical purification of detergent bilized H-2 alloantigens. M&c. Inlmun. 16, 9-21, Goding J. W. (1978) Use of staphylococcal protein A as an immunological reagent. J. Immun. Meth. 20, 241-253. Golub E. S. (1977) The Cellular Basis qf thr Immune Re.sponse. Sinauer Associates, Sunderland, Massachusetts. U.S.A. Hayman M. J. & Crumpton M. J. (1972) Isolation of glycoproteins from pig lymphocyte plasma membrane using Lrns culinuris phytohemagglutinoin. Bioc~hem. hiophys. Res.

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Helenius A. & Simons K. (1975) Solubilization of membranes by detergents. Biochim. hmphys. Actu 415, 29 79. Herrmann S. H. & Mescher M. F. (1979) Purification of the H-2Kk molecule of the murine major histocompatibility complex. J. hiol. Chem. 254, 8713 8716. Hunter W. M. & Greenwood F. C. (1962) Preparation of iodine-131 labelled human erowth hormone of hieh specific activity. Noture. Londr 194. 495-496. Ivanyi P. (1977) The major histocompatibility system of the pig. In The Major Histocompatihilit~ System in Man und A,limtrls (Edited by Gotze D.). pp. 168-174. Springer, New York. Klein J. (1975) Biology of the Mouse Histocompatihility-2 Complrr. Springer, New York. Koch G. L. E. & Smith M. J. (1978) An association between actin and the major histocompatibility antigen H-2. Nuturr. Lond. 213, 274-278. Kvist S.. Klareskog L. & Peterson P. A. (1978) Identification of H-2 and la-antigen analogues in several species by immunological cross reactions of xenoantisera. Sumd. J. Immun. 7, 447-452. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacte;iophage T4.

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J. (1980) Albumin associated with purified pig lymphocyte plasma membrane. Eiochem. J. 192, 49957. Parham P., Alpert B. N.. Orr H. T. & Strominger J. L. (1977) Carbohydrate moiety of HLA antigens: antigenic properties and amino acid sequences around the site of glycosylation. J. hiol. Chem. 252, 755557567. Rejnek J. & Prokesova (1973) Immunoglobulins and antibodies in pigs. Contemp. Topics Molec. Immun. 2, 117-153. Sachs D. H., Leight G., Cone J.. Schwarz S., Stuart L. & Rosenberg S. (1976) Transplantation in miniature swine. 1. Fixation of the major histocompatibility complex. Ttxnsplunrution 22, 559-567. Singer P. A. & Williamson A. R. (1980) Cell surface immunoglobulin p and )’ chains of human lymphoid cells are of higher apparent molecular weight than their secreted counterparts. Eur. J. Immun. 10, 180-186. Snary D., Goodfellow P., Bodmer W. F. & Crumpton M. J. (1975) Evidence against a dimeric structure for membrane-bound HLA antigens. Nature, Lond. 258, 24&242. Snary D.. Woods F. R. & Crumpton M. J. (1976) Disruption of solid tissue for plasma membrane preparation. Anolyf. Biockem. 74, 457-465.

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217

Springer T. A., Kaufman J. F., Terhorst C. & Strominger J. L. (1977) Purification and structural characterization of human HLA-linked B-cell antigens. Nature, Lond. 268, 213-218. Springer T. A., Mann D. L., DeFranco A. L. & Strominger J. L. (1977) Detergent solubilization, purification and separation of specificities of HLA antigens from a cultured human lymphoblastoid cell line, RPM1 4265. J. biol. Chem. 252, 4682-4693. Standring R., McMaster W. R., Sunderland C. A. & Williams A. F. (1978) The predominant heavily glycosylated glycoproteins at the surface of rat lymphoid cells are differentiation antigens. Eur. J. Immun. 8, 832X339. Trowbridge I. S., Nilsen-Hamilton M., Hamilton R. T. & Bevan M. J. (1977) Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes Biochem. J. 163, 21 I-217. Walsh F. S., Barber B. H. & Crumpton M. J. (1976) Preparation of inside-out vesicles of pig lymphocyte plasma membrane. Biochemistry 15, 355773563. Walsh F. S. & Crumpton M. J. (1977) Orientation of cellsurface antigens in the lipid bilayer of lymphocyte plasma membrane. Nuturr, Land. 269, 3077311.