Transplantation antigens in a high molecular weight form — I Mouse H-2 antigens

lmmunochemistry. PergamonPress 1971. Vol.8, pp. 7-16. Printedin Great Britain

T R A N S P L A N T A T I O N ANTIGENS IN A H I G H MOLECULAR W E I G H T F O R M - I MOUSE H-2 ANTIGENS U. I-IAMMERLING*, D. A. L. DAVIES and A. J. MANSTONE Institute of Virology, University of Giessen, West Germany, and Searle Research Laboratories, High Wycombe, England

(First received 11 May 1970; in revisedform 29July 1970)

Abstract-By extraction of membrane fragments of mouse spleen cells with sodium dodecylsulphate in the presence of starch stearate, a soluble fraction may be obtained which carries the H-2 specificities defined by the particular mouse genotype used. These specificities reside on a substance having a molecular size in the region of several million, as indicated by Sephadex G-200 exclusion and Sepharose 4B inclusion. This is a complex that degrades to H-2 active units of molecular weight about 50,000 where each molecule generally carries only a limited number of the H-2 specificities of the parent cell. Some of these derived smaller molecules can be separated from the larger complex by DEAE ion exchange chromatography as well as by gel filtration. INTRODUCTION H-2 antigens and m a n y other alloantigens that have been studied are integrated into cell membranes in a m a n n e r not yet understood, but such that solubilization has presented a major problem hindering attempts at purification and characterization. Enzymic methods of degradation of crude membranederived material have been the most successful and have led to soluble H-2 antigens (and some other alloantigens) that have molecular weights in the region of 50,000. Wheras a figure of 53,000 was allotted to the molecule carrying H-2-5 specificity [1] some others deviate sufficiently from this figure to allow separation by gel filtration, e.g. H-2.5 and H-2.2 have been separated in this way [2]. By ion exchange chromatography many H-2 antigens can be separated from each other, showing that a family of molecules exists where one molecule carries just one or only a few of the m a n y specificities determined by a particular genotype [3]. These studies have been carried out with products of enzymic degradation of m e m b r a n e preparations and they are possibly not naturally occurring m e m b r a n e units, although they must reflect organization of m e m b r a n e architecture since it could hardly be coincidence that such clearly definable specific molecules could arise by r a n d o m proteolysis. By use of detergents, crude m e m b r a n e derived material, grossly lipoprotein in nature, can be dispersed to provide a suspension from which H-2 activity is not sedimented by high speed centrifugation[4]. Generally these materials reaggregate on removal of the detergent and it has not been adequately demonstrated that a truly soluble H-2 antigen can be obtained in this way. In so far as some soluble activity has been f o u n d it might well be accounted for by autolytic degradation into molecules in the 50,000 mol. wt. region as already referred to. *This work was initiated while U.H. was working at the Sloan-Kettering Institute for Cancer Research, New York. 7

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U. HAMMERLING, D. A. L. DAVIES and A. J. MANSTONE

For these latter preparations, it has been shown not only that different specificities may be physically separated from one another, but, a fortiori, that the products of non H-2 histocompatibility loci are removed as 'impurities'. Such a separation of different gene products has not been shown for detergent prepared antigens, nor for preparations obtained by other physical methods such as ultrasonic treatment [5]. In this paper we give an account of H-2 antigens of relatively large molecular size obtained by the combined action of sodium dodecylsulphate (SDS) and starch stearate (SST). The basis of the method is that from membrane derived material dispersed with these agents, the bulk of crude lipoprotein can be sedimented by high speed centrifugation, leaving H-2 activity in the supernatants. SDS can then be dialysed away and the antigenic material fractionated chromatographically. H-2 active molecules subsequently remain in a water soluble form after removal of SST. The information provided in this, and the following paper, is largely a serological study comparing the high molecular weight material with alloantigenic products of smaller size that have previously been described. This preliminary work will be followed by more adequate chemical studies. MATERIALS AND METHODS Extracts Spleens were obtained from 320 (C57BL/10 X BALB/c)F1 hybrid mice and crude membrane fragment preparations made by elution of spleen cell suspensions with hypotonic salt as described previously[6]. The product of the 320 spleens was suspended in 27 ml water and amounted to 1.42 g (dry wt.). Antisera Monospecific antisera defining most of the known H-2 alloantigenic specificities and used for monitoring specific activity in chromatographic column fractions were described in detail in a recent publication [3]. Serological methods Specific H-2 activity was assessed by inhibition of immune cytotoxicity using ~lCr labelled lymph node target cells and guinea pig serum as source of complement. Inhibition tests were carried out by diluting antigen in a 75 per cent cytolytic dose of antiserum. Column fractions were generally tested as single point assays after preliminary tests had indicated appropriate doses of antigen for each specificity. Starch stearate This was synthesized as described by H~immerling and Westphal [7], and the preparation used contained 1.5 per cent stearic acid ester. Chromatographic methods Gel filtration on Sephadex G200 and ion-exchange chromatography on DEAE-Sephadex A50 has already been described in detail for mouse H-2 antigens [3] and human HL-A transplantation antigens [8]. Sepharose 4B was used

Mouse H-2 Transplantation Antigens

9

in columns 60 cnt × 1.5 cm, packed and eluted with 0.01 M Tris buffer p H 8-0.

Solubilization About half o f the eluate (680 mg) was used for SDS/SST solubilization. T h e suspension was diluted to 50 ml and SDS a d d e d to 0.3 per cent (w/v) to effect the dispersion; SST was then a d d e d to 1 mg/ml (0-1 per cent w/v) and the solution chilled. This was centrifuged at 105,000 g for 60 rain and the sedimented material put aside. T h e s u p e r n a t a n t was dialysed against water to remove SDS and centrifuged again to remove a little material separating f r o m solution on dialysis. T h e p r o d u c t weighed 153 mg and the solution was concentrated in a rotary e v a p o r a t o r at 4 ° to 6 ml in 0"01 M Tris p H 7.3 for application to a G-200 S e p h a d e x column. A similar a m o u n t o f c r u d e extract was solubilized autolytically by methods previously described for H-219] and HL-A[10] to give material o f molecular weight in the region o f 50,000 that has already been studied in detail. This preparation (270 rag) was also concentrated to 6 ml and studied at each chromatographic stage for comparison with the SDS/SST solubilized material. RESULTS T h e SDS/SST extract, before application to columns, was first tested for specificity in a cytotoxic inhibition test and the result is shown in Fig. 1. G o o d reactivity can be seen in a polyspecific system H-2-3,4,8,10,13,31,34 but there is no activity in a system H-2-1,11,23,25,32 where n o n e was to be expected. By c o m p a r i s o n with the starting material (also illustrated) it is clear that the yield was small (about 2 per cent). T h e yield f r o m autolytic solubilization was not assessed but generally falls between 10 and 50 per cent d e p e n d i n g on the partivular specificities sought [11]. 6o o

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Fig. 1. Specificity test of SDS/SST derived H-2 antigen as shown by inhibition of immune cytolysis. Starting material membrane derived crude lipoprotein) ~ A. SST soluble antigen shows inhibition in the system H-2 b anti-H-2 a, O------O but not in the system H-2 a anti-H-2 k, O C).

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U. H)~MMERLING, D. A. L. DAVIES and A. J. MANSTONE

Gelfiltration The crude soluble SDS/SST derived material was examined on a G-200 Sephadex column with the result shown in Fig. 2(B); this should be compared with the picture in Fig. 2(A) that is the result of running the same column with autolytically derived antigen. The antigen A/T (autolytically derived by incubation in Tris buffer) was almost twice the weight (270 mg) as the SST antigen (detergent solubilized) that weighed 153 rag. The additional 280 m/z absorbence of A/T antigen is mainly seen in the low molecular weight region of the column eluate; SST antigen showed relatively high absorbence in the excluded fraction as might have been expected. The H-2 activity measurements to locate the antigenically active material were made in a polyspecific system, H-2.3,4,8,10,13,31, 34; A/T antigen is substantially included with very small reactive peaks of higher and lower molecular weight. The SST antigen is substantially excluded with a small component corresponding to the A/T location, due, no doubt, to a small

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Fig. 2. Sephadex G-200 gel filtration. 280 mtz absorbence shown by broken lines; exclusion point indicated by DB and arrow. H-2 antigenic activity measured by inhibition of immune cytolysis in single point assays, 0 - - - - 0 . Measurements were made in a polyspecific system H-2 b anti-H-2 d. A, Antigen solubilized by gentle proteolysis. B, Antigen solubilized by SDS/SST.

Mouse H-2 Transplantation Antigens

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degree o f autolytic degradation that occurred during the course of SDS/SST processing. The material remaining after serological monitoring of the columns was pooled over the major activity region in each case (SST tubes 12 to 20 and A/T tubes 20 to 30), concentrated in the cold under reduced pressure, and dialysed against starting buffer for DEAE-Sephadex chromatography (0.05M Tris buffer pH 8"0). DEAE-chromatography

The two H-2 active preparations recovered from G-200 Sephadex, i.e. the high molecular weight fraction of the SST column, and the intermediate molecular weight fraction of the A/T column were applied to two DEAE columns which were eluted with a linear molarity gradient up to 0-35M (0.05M Tris pH 9"0 plus 0"3 M NaC1) over a volume of 1000 ml. The elution pattern for the A/T column is shown semidiagrammatically in Fig. 3, after monitoring with several monospecific H-2 alloantisera. A description of the separations of different H-2 specificities will be found in detail elsewhere [3]. By comparison of this with the picture shown in Fig. 4, of SST derived antigen, it will be seen that all specificities occur in a region centred on about tube 100, but that in addition each specificity occurs in a position expected of A/T antigen, where such positions deviate sufficiently from the tube 100 position to reveal different peak positions• This suggests that SST antigen has depolymerised in part into A/T type antigen during the processing. In order to illuminate this point, pools were made over the ranges of antigenic reactivity of these two columns (tubes 40 to 80 of the A/T column and tubes 60 to 120 of the SST column) and each pool concentrated to 4 ml for reexamination on G-200 Sephadex. LReexamination on G-200 Sephadex

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Fig. 3. DEAE ion exchange chromatogram of autolytically derived antigen showing positions of several H-2 antigens of the H-2 a component of the F1 hybrid phenotype, measured in monospecific systems. 280 m/x absorbence indicated by dotted line. Column eluted with a linear molarity gradient.

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Fig. 4. DEAE ion exchange chromatogram of SDS/SST derived antigen run as for that shown in Fig. 3. This shows positions for some of the H-2 d and the H-2 b antigens of the hybrid cells used for extraction. By comparison with Fig. 3, all specificities react in one position (centred on tube 100) and, in addition, react in positions indicated from Fig. 3. c o l u m n is shown in Fig. 5. T h e results are entirely as expected, all H-2 reactivity being r e t a r d e d (partially included) as it a p p e a r e d originally (Fig. 2A); however in the r e - r u n several monospecific systems were sought, showing, incidentally, some size differences a m o n g H-2.4, H-2.31 a n d H-2.37. W h e n SST antigen was r e e x a m i n e d , (Fig. 6), some changes had o c c u r r e d f r o m the original picture (Fig. 2B) for wheras some specificities h a d largely m a i n t a i n e d their large m o l e c u l a r 0

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Fig. 5. Examination on G-200 Sephadex of material pooled from the DEAE run using A/T antigen illustrated in Fig. 3. 280 m/* absorbence shown as dotted line. Systems monitored are H-2.4, O-----O; H-2-31, O Q; H-2.37, A A. These are all well included by reference to the exclusion point (BD and arrow) and are in the molecular weight range of 30,000 to 60,000.

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Fig. 6. Examination on G-200 Sephadex of material pooled from the DEAE run using SST antigen illustrated in Fig. 4. The specificities shown are in two pictures to provide clarity; these may be superimposed being derived from a single column run. For all specificities, some activity is excluded and some included. size (e.g. H-2"29 and H-2-37) others were mainly d e g r a d e d into the size range o f A / T antigen (H-2-4 and H-2-31). T h u s the SST antigen appears to be somewhat unstable u n d e r the conditions in which it had been handled. A pool was m a d e f r o m the material remaining in tubes 20 to 50 o f the SST second G-200 S e p h a d e x c o l u m n and this was concentrated to 4 ml for examination on Sepharose 4B.

Sepharose 4B gelfiltration T h e c o l u m n shown in Fig. 7 was loaded with blue dextran, which showed the exclusion point (BD1 at tube 11) and m a r k e d at approximately a level o f 2 million tool. wt. (BD2 at tube 37); e x t r e m e inclusion is m a r k e d by CuSO4 (tube 54). Many specificities were m o n i t o r e d on this c o l u m n but for clarity, only two are shown in Fig. 7. As might have been expected f r o m the results shown in Fig. 6B, H-2"4 gives two peaks, that to the right is taken to be the G-200 r e t a r d e d fraction

14

U. HAMMERLING, D. A. L. DAVIES and A. J. MANSTONE

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Fig. 7. Examination on Sepharose 4B of material pooled from the column shown in Fig. 6. Markers are BD1 (about 20 million mol. wt. for proteins), BD2 (about 2 million mol. wt.), Cu 2+ inclusion point. 280 m/x absorbence shown by dotted line. H-2'4 (of H-2 d) and H-2"5 (of H-2 b) both distribute in two peaks, that on the left representing true SDS/SST soluble antigen. and that to the left marking the position o f the high molecular size SST antigen. Specificity H-2"5 also shows two peaks in the same positions as H-2"4. T h e two principal 280 m~t absorption peaks coincide with and are d u e to blue d e x t r a n itself. T h e distribution of non H-2 alloantigens on the Sepharose columns is described in the following paper. T h e possibility that the effects described might be attributable to adsorption o f soluble H-2 molecules ( - 5 0 , 0 0 0 mol. wt. version) to starch stearate, with subsequent partial reversion is r e n d e r e d unlikely by the n o n H-2 data given in the following paper. It was also tested directly as follows. Soluble H-2 antigen (H-2") proteolytically derived (5 rag) was dissolved in 5 ml 0"02 M Tris buffer p H 8.0, dialysed overnight against distilled water and applied to a G-200 Sephadex column; serological analysis o f this r u n in shown in Fig. 8. A similar sample was a d d e d to 0.5 ml o f starch stearate solution (10 rag/ ml) and then processed in the same way; the serological analysis o f the G-200 S e p h a d e x column fractions is also shown in the figure. H-2 activity is confined in both instances to the r e t a r d e d fraction and no activity could be detected in the excluded region o f the second r u n where starch stearate was located (by its iodine reaction) as shown also in Fig. 8. DISCUSSION Chemical data on transplantation antigens are mainly confined to studies o f m e m b r a n e fragments derived by limited proteolysis. Since these are enzymic artefacts little can be d e d u c e d from them about m e m b r a n e structure. Nevertheless the production o f discrete molecules having the properties o f globular glycoproteins and each carrying only one or a small n u m b e r o f the H-2 specific

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Fig. 8. Sephadex G-200 gel filtration. Sample of H-2 a soluble (proteolytically derived) antigen (5 mg) monitored for H-2"1,5,11,23,25, C) C). In a subsequent run, a similar sample with starch stearate e~----O; position of starch stearate on the column G . . . . A. determinants suggests that they arise by n o n - r a n d o m proteolysis f r o m a nonr a n d o m l y assembled polymer. Soluble, as applied to SST antigen is purely operational. Generally, detergent-derived H-2 antigen preparations come out o f solution when the d e t e r g e n t is removed. SST antigen cannot be centrifuged down over 3 hr at 100,000 g and behaves as a soluble substance for effective c h r o m a t o g r a p h y and is not excluded by Sepharose 4B. Unlike the proteolytically derived preparations, all specificities o f the H-2 p h e n o t y p e used are expressed in the material, and to the extent that DEAE ion exchange c h r o m a t o g r a p h y effects some resolution this is due to degradation o f the p a r e n t substance to smaller units having the characteristics o f the lower molecular weight p r o d u c t whose DEAE column resolution characteristics have already been described in detail [3]. A t t a c h m e n t o f these smaller autolytically derived molecules to starch stearate to give activity in high molecular regions o f gel filtration columns, is not the explanation because they have been shown not to adsorb on to starch stearate. T h e p r e p a r a t i o n o f the high molecular weight SST antigen now described does not involve a r u p t u r e o f covalent bands and a cell m e m b r a n e is clearly not itself a macromolecule. I n d e e d many discrete molecular species are m e m b r a n e derived but n o n is generally accepted as a subunit o f m e m b r a n e structure. T h e SST antigen has been obtained, thus far, in too small a yield to justify discussion o f the substance as a m e m b r a n e unit hut its non H-2 alloantigen content permits some points to be m a d e [12]. T h e likelihood is that the e c o n o m y o f n a t u r e does not require a unit o f purely structural m e m b r a n e material to which functional entities are attached, but that the functional molecules wholly compose the structure; one might exclude red cells f r o m such a generalization. In any event there is a pressing need to associate functional entities o f m e m b r a n e s with antigenically defined units to hasten the establishment o f a structural model. dcknowledgements-We

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thank Miss B. J. Alkins and Mr. V. S. G. Baugh for technical assist-

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

U. HAMMERLING, D. A. L. DAVIES and A. J. M A N S T O N E REFERENCES SummerellJ. M. and Davies D. A. L., Transplantation Proc. 1,479 (1969). Shimada A. and Nathenson S. G., Biochem. biophys. Res. Commun. 29, 828 (1967). Davies D. A. L., Transplantation 8, 51 (1969). Hilgert I., Kandutsch A. A., Cherry M. and Snell G. D., Transplantation 8,451 (1969). Kahan B. D. and Reisfeld R. A., Tramplantation Proc. 1,483 (1969). Davies D. A. L., Immunology 11,115 (1966). H~immerling U. and Westphal O., Eur.J. Biochern. 1, 46 (1967). Davies D. A. L., Colombani J., Viza D. C. and Dausset J., Biochem. biophys. Res. Commun. 33, 88 (1968). Nathenson S. G. and Davies D. A. L., Ann. N.Y. Acad. Sci. 129, 6 (1966); Proc. natn. Acad. Sci. U.S.A. 56, 476 (1966); Davies D. A. L., Transplantation 5, 31 (1967). Davies D. A. L., Manstone A. J., Viza D. C., Colombani J. and Dausset J., Transplantation 6, .571 (1968). Davies D. A. L., Syrup. on Blood and Tissue Antigens, Ann Arbor, Michigan, 1969 (Edited by Aminoff D.). Academic Press (in press 1969). Davies D. A. L., H~immerling U. and Alkins B.J., Immunochemistr~, 8, 17 (1971).