Proc. Nati. Acad. Sci. USA
`Vol. 77, No. 5, pp. 2510-2513, May 1980
`Biochemistry
`Functional incorporation of synthetic glycolipids into cells
`(concanavalin A/agglutination/erythrocytes/liposome exchange/hydrophilic spacer arm)
`R. R. RANDO, J. SLAMA*, AND F. W. BANGERTER
`Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
`Communicated by E. R. Blout, February 4,1980
`
`Synthetic glycolipids containing an a-man-
`ABSTRACT
`noside group linked by a hydrophilic spacer arm to cholesterol
`were incorporated into bovine erythrocytes by exchange from
`glycolipid-containing liposomes. When the distance between
`the sugar and the cholesterol moieties was approximately 26 A,
`functional incorporation of these glycolipids could be easily
`detected, as revealed by the concanavalin A-mediated aggluti-
`nation of these cells. Bovine erythrocytes are not themselves
`susceptible to concanavalin A-mediated agglutination. The
`minimal concentration of concanavalin A required for a lu-
`tination of modified erythrocytes, containing 9.15 X lO1gly-
`colipid molecules per cell, was 4 Ag/ml. Under these conditions,
`only approximately 4% of the membrane-bound cholesterol had
`been exchanged for the synthetic glycolipid. The observed
`aggregation was reversible in the presence of a-methyl man-
`noside and did not occur when ,-galactosyl-containing gly-
`colipids were used in place of their a-mannoside isomers. These
`studies demonstrate a technique of sugar incorporation into cell
`membranes which should be of great advantage in studies on
`the roles of cell surface sugars in biological recognition. Fur-
`thermore, they demonstrate that the sugars need only be a short
`distance (26 A) from the membrane in order to functionally bind
`concanavalin A.
`Cell surface sugars have been implicated in various cellular
`recognition phenomena. On the surface of mammalian cells
`they function as virus, bacterial, and toxin receptors and are
`implicated as mediators in cell-cell adhesion (1). An approach
`to understanding the roles that cell surface sugars play in these
`phenomena involves the incorporation of chemically defined
`sugars onto the cell surface. The reconstitution or modulation
`of a recognition response as a consequence of the incorporation
`of a specific sugar or sequence of sugars can be taken as evi-
`
`the liberated aldehyde groups (2, 3). This method can be less
`than optimal because of the required chemical alteration of the
`membrane and the fact that the sugars are introduced in a
`random, nonuniform array. A second possible method that
`could potentially circumvent these difficulties, and hence po-
`tentially generate more information, is one involving the non-
`covalent incorporation of amphipathic sugar-containing
`compounds into the cell membrane. Synthetic glycolipids
`containing cholesterol as an anchor, linked by a spacer group
`to a sugar (structure I), can be incorporated into small unila-
`mellar liposomes, and these derivatized liposomes are rendered
`susceptible to aggregation by the appropriate lectins (4). The
`cholesterol-containing synthetic glycolipids distribute evenly
`on both sides of the bilayer and exhibit a condensing effect
`above the phase transition of both lipids (5). By these and other
`criteria the cholesterol analogs appear to be bound in the
`membrane much like cholesterol itself, with the polar oxygen
`being near the phosphate head group (6).
`Synthetic glycolipids analogous to structure I cannot im-
`mediately be applied to cells because of the hydrophobic nature
`of the spacer groups. It could be anticipated that due to the cell
`surface glycoproteins, relatively large distances between the
`sugar residue and cholesterol anchor would have to be achieved
`before the sugar could functionally interact with a binding
`protein. Simply polymerizing hydrophobic spacers of the am-
`inocaproyl type would not be expected to yield fruitful results.
`The hydrophobic chains would either "ball up" and interact
`with themselves in the aqueous environment or they would
`dissolve in the lipid bilayer. Neither situation would be fruitful
`for functional incorporation studies because the sugar residue
`
`of-Mannoside
`
`I
`
`HO
`
`CH20H
`
`OH
`o0
`
`0
`0
`HO ~~ --(CH2)2-NH-(C--(CH2-0O-CH2)2-CH2-NH),,-C,-0
`II
`a-Mannoside
`would remain sequestered too close to the membrane to interact
`dence for the functional role(s) of these sugars in the response.
`with binding proteins. In order to circumvent this problem we
`A crucial step here is to devise specific ways to introduce sugars
`have developed a hydrophilic spacer group that combines the
`onto the cellular membrane. A chemical method has been
`water solubility of ethers with the ease of polymerization of
`formulated which involves the periodate oxidation of the sialic
`amino acids. In this communication we show that synthetic
`acid side chain of membrane-bound glycoproteins, followed
`glycolipids containing 3,6-dioxa-8-aminooctanoic acid-based
`by the condensation of sugar-containing acyl hydrazides with
`spacer groups can be functionally incorporated into cells.
`Abbreviation: Con A, concanavalin A.
`* Present address: Department of Chemistry, University of Chicago,
`Chicago, IL 60637.
`
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be hereby marked "ad-
`vertisement" in accordance with 18 U. S. C. §1734 solely to indicate
`this fact.
`
`2510
`
`MPI EXHIBIT 1043 PAGE 1
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`MPI EXHIBIT 1043 PAGE 1
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`

`

`Biochemistry: Rando et al.
`
`Proc. Natl. Acad. Sci. USA 77 (1980)
`
`2511
`
`glycolipids and the hydrophilic spacer groups will be published
`in detail dsewhere. The [6-3H]galactosyl-containing glycolipid
`used here was synthesized by treating the galactosyl-containing
`glycolipid with galactose oxidase followed by NaB3H4 reduction
`according to a published procedure (5).
`The concentrations of the sugar cholesterol compounds were
`determined by enzymatic assay of the free mannose after
`mercuric ion-catalyzed hydrolysis of the 1-thioglycoside linkage
`(9). To a solution of the cholesterol-mannose derivative (0.1-2
`jumol) in ethanol (1 ml) was added 0.2 M mercuric acetate in
`0.1 M acetic acid (0.1 ml). After 2 hr at 600C, the incubation
`mixture was treated with 2-mercaptoethanol (10,Ml) and the
`solvent was removed under reduced pressure. The residue was
`suspended in H20 (0.5 ml), shaken vigorously, and centrifuged
`in a clinical centrifuge. The liberated mannose was assayed by
`coupling the ADP produced by the hexokinase/ATP-catalyzed
`phosphorylation of the hexose to the enzyme system of pyruvate
`kinase/phosphoenolpyruvate and lactate dehydrogenase/
`NADH (4).
`The liposomes containing the synthetic glycolipids were
`prepared as described (4, 10). After sonication, the glycoli-
`pid-bearing liposomes were centrifuged at 12,000 X g to re-
`move insoluble material. The clear supernatant containing the
`unilamellar and multilamellar liposomes was then used in the
`exchange reactions with bovine erythrocytes. The erythrocytes
`were collected in heparin/phosphate-buffered saline (pH 7.4)
`from a local slaughterhouse on the day that they were to be
`used. The erythrocytes were repeatedly washed with ice-cold
`phosphate-buffered saline (pH 7.4) and centrifuged (2000 X
`g) three or more times. The buffy coat was removed by aspi-
`ration. The cells were continuously washed until no more white
`layer appeared. The cells were then incubated with the gly-
`colipid-containing liposomes at 370C for varying periods of
`time. The cells were centrifuged at 5000 X g and washed three
`times with phosphate-buffered saline. These cells were then
`used in the aggregation studies.
`
`3000
`
`V2000
`(D
`0.'
`
`1000 _
`
`00C E o
`
`20
`
`0
`
`5
`
`10
`15
`% glycolipid
`FIG. 2.
`Incorporation of glycolipids at varying concentrations
`into bovine erythrocytes. Liposomes were prepared containing 2, 5,
`8, and 15% of the [6-3H]galactosyl-containing glycolipid. The con-
`centration of phospholipid was adjusted to 10 ,umol/ml in each case.
`One milliliter of washed erythrocytes (6 X 108 cells) in phosphate-
`buffered saline was incubated with 40 M1 of each of the above stock
`solutions. After a 2-hr incubation, the samples were washed and lysed
`and radioactivity was measured.
`
`600_
`
`0)
`
`o
`
`400
`
`200
`
`0
`
`1
`
`2
`
`3
`
`4
`
`Time, hr
`Incorporation of glycolipids into bovine erythrocytes.
`FIG. 1.
`Liposomes were prepared containing 5 mol % [6-3H]galactosyl-con-
`taining glycolipid (M, = 796), #B-[6_3HjGal-S-(CH2)2-NH-C0-
`(CH2-0CH2)2-CH2-NH-CO2Cho1 (specific activity, 2.35 X 105
`cpm/,umnol). The stock solution of the liposomes was 10,umiol of
`phospholipid per ml as determined by phosphate assay. To test tubes
`containing 6.9 X 108 cells in 1 ml of phosphate-buffered saline was
`added 40 ul (4770 cpm) of the transparent liposomal preparation. The
`erythrocytes were incubated for various periods of time at 37°C with
`shaking in a Dubinoff shaking bath. At the indicated times the cells
`were centrifuged at 3000 X g and washed three times with 10 ml of
`phosphate-buffered saline. The final pellet was lysed in 10 ml of 10
`mM potassium phosphate (pHI 7.5). The membrane pellet was cen-
`trifuged at 27,000 X g for 10 min and washed twice with 10 ml of the
`phosphate buffer. The final pellet was dissolved in Aquasol (New
`England Nuclear) and radioactivity was measured.
`Specifically, we show that bovine erythrocytes, which cannot
`be agglutinated by the c-mannosyl binding lectin concanavalin
`A (Con A), can be rendered so as a consequence of the incor-
`poration of synthetic glycolipids of the type shown in structure
`II. Furthermore, we show that the distance requirement from
`the cholesterol anchor to the sugar in order for Con A-mediated
`aggregation to occur is relatively modest (n = 2).
`MATERIALS AND METHODS
`Con A (three times recrystallized) was purchased from Miles.
`Solutions of Con A in 50 mM Tris-HCl/140 mM CaC12 were
`centrifuged at 5000 x g for 5 min before use. The concentra-
`tions of Con A solutions were determined by absorbance
`readings at 280 nm (Alcmg/ml = 1.3). Egg phosphatidylcholine
`was prepared and purified by the method of Litman (7). The
`phospholipid concentrations were determined as inorganic
`phosphate after ashing and acid hydrolysis (8). The phospho-
`lipid was dissolved in benzene at a concentration of 40 ,umol/ml
`and stored at -70°C under an atmosphere of nitrogen. The
`purity of the preparation was checked routinely by thin-layer
`chromatography (silicic acid; CHC13/CE[30H/H20, 65:25:4).
`The glycolipids were prepared by a procedure similar to that
`already published (4, 5). The main differences were that the
`thiosugar was alkylated with N-trifluoroacetamido iodo-
`ethane instead of with N-trifluoroacetamido iodohexane and
`the coupling reaction was done with N-trifluoro-31,6-dioxaoc-
`tanoic acid instead of with N-trifluoroacetyl aminocaproic acid.
`These changes eliminated the hydrophobicity of the earlier
`spacer groups. All of the compounds used here gave satisfactory
`elemental analyses, and their NMR and infrared spectra were
`entirely consistent with the assigned structures. Full details of
`the synthesis of a-D-mannosyl- and fl-Dgalactosyl-containing
`
`MPI EXHIBIT 1043 PAGE 2
`
`MPI EXHIBIT 1043 PAGE 2
`
`

`

`ezj0512
`
`Biochemistry: Rando et al.
`
`Proc. Natl. Acad. Sci. USA 77 (1980)
`
`shown. Rough linearity was observed in both cases at low
`phospholipid concentrations.
`The a-D-mannosyl-containing synthetic glycolipids of the
`type shown in Structure II were prepared, incorporated into
`liposomes, and exchanged into the bovine erythrocytes. The
`modified erythrocytes were then treated with varying con-
`centrations of Con A and the extent of agglutination was as-
`sessed with microtiter plates (2). The results of these experi-
`ments are shown in Table 1. These experiments show that
`functional incorporation of the synthetic glycolipids into the
`bovine erythrocytes was achieved. The effect was specific for
`the a-mannosyl residue because f3-galactosyl-containing gly-
`colipids were not agglutinable and the agglutination response
`in the presence of II was specifically blocked by added
`a-mannosides but not by f3-galactosides. In addition, cells
`treated with pure phosphatidylcholine-based liposomes were
`also not rendered agglutinable by Con A. Furthermore, the
`incorporation was reversible. Bovine erythrocytes containing
`II (n = 2 or 4) were rendered nonagglutinable by Con A by
`simply incubating them with cholesterol-containing liposomes.
`The synthetic glycolipids exchanged out of the cells as the
`cholesterol exchanged in. By microscopic reversibility, then,
`the synthetic glycolipid must have been incorporated into the
`erythrocyte by an exchange process rather than by another
`process such as fusion. The synthetic glycolipids are, however,
`bound in the erythrocyte membrane in a stable form in the
`absence of exchange-mediating liposomes. It is of interest to
`compare the effects of the spacer group length on the suscep-
`tibility of the modified erythrocytes to aggregate. When n =
`0 or 1 (II), aggregation either did not proceed or it proceeded
`poorly, requiring relatively large amounts of Con A. When n
`= 2 or 4 (II), aggregation proceeded rapidly at 4 jig of Con A
`per ml. The distance from the sulfur group to the carbamate
`carboxyl group is approximately 16 A in II (n = 1) and 26 A in
`II (n = 2). We have shown that within experimental error gly-
`colipids with one or two spacer groups are incorporated to the
`
`Table 1.
`
`Glycolipid
`
`Lowest lectin concentration that causes agglutination of
`native and modified bovine erythrocytes
`Concen-
`a-Methyl
`mannoside,
`tration,
`mM
`%
`
`ConA,
`Ug ml-l*
`>500
`>500
`>500
`>500
`50
`4
`>500
`4
`>500
`
`t
`10
`10
`5
`15
`5
`5
`5
`5
`
`40
`-
`40
`
`I
`II (n =0)
`II (monomeric spacer, n = 1)
`II (monomeric spacer, n = 1)
`II (dimeric spacer, n = 2)
`II (dimeric spacer, n = 2)
`II (tetrameric spacer, n = 4)
`II (tetrameric spacer, n = 4)
`III (fl-Gal-dimeric spacer,
`n = 2)
`>500
`10
`To 1 ml of washed erythrocytes in phosphate-buffered saline (5 X
`108 cells) was added 0.1 ml of liposome (10 ,umol/phospholipid mol-
`ecule per ml of phosphate-buffered saline) containing the synthetic
`glycolipid at the indicated concentrations. The samples were incu-
`bated with shaking at 37°C for 2 hr; the cells were then thoroughly
`washed and resuspended in 1 ml of phosphate-buffered saline. Fifty
`microliters of the prepared cells was placed in microtiter wells along
`with 50,ul of various concentrations of Con A in phosphate-buffered
`saline. The cells were allowed to remain at room temperature for 2 hr
`and then agglutination was scored. In the absence of liposomes, no
`agglutination occurred. In the presence of a-methyl mannoside at the
`indicated concentration, agglutination did not occur. When aggluti-
`nation was positive by this assay, the results were checked by micro-
`scopic examination. In all instances, the results from both determi-
`nations concurred.
`* Lowest concentration to cause agglutination.
`t Liposomes were used without glycolipid in this experiment.
`
`~0
`
`00 1000A
`
`0.0
`
`C E
`
`0
`
`2
`
`6
`8
`10
`1 2
`4
`Phospholipid, uM X 10-2
`
`1 4
`
`16
`
`FIG. 3.
`Incorporation of glycolipids at varying liposome con-
`centrations into bovine erythrocytes. Liposomes containing 10%o of
`the [3Hlgalactosyl-containing glycolipid at a final phospholipid
`concentration of 10 imol/ml were made. To 1 ml of 7 X 108 cells in
`phosphate-buffered saline was added 10, 20, 40, 80, and 160 ,1 of the
`liposomal preparation. The total volumes were kept constant by ad-
`dition of the appropriate amount of buffer to each sample. The cells
`were incubated at 370C with shaking for 2 hr and then washed and
`lysed. Radioactivity was then measured.
`
`RESULTS AND DISCUSSION
`The synthetic glycolipids were incorporated into the bovine
`erythrocytes by a liposome-exchange technique used for cho-
`lesterol exchange (11). Briefly, egg yolk phosphatidylcholine-
`based liposomes were prepared containing the synthetic gly-
`colipids. Simply incubating these liposomes with bovine
`erythrocytes allows an exchange process to occur between the
`synthetic glycolipids in the liposomes and the cholesterol in the
`erythrocytes. In Fig. 1 a time course for the incorporation of
`radioactive glycolipids into erythrocytes is shown under defined
`conditions. After about 1-2 hr, the exchange appeared to reach
`equilibrium. Under these conditions, the erythrocytes contained
`3.05 X 106 synthetic glycolipid molecules per cell. Given the
`average surface area of a bovine erythrocyte to be 109 ,um2, the
`average density of sites can be calculated to be 2.79 X 104
`sites/jm2, assuming that the synthetic glycolipids do not
`transverse the bilayer. The glycolipids used here cannot be
`incorporated into the erythrocytes by direct addition. Incu-
`bation of the erythrocytes with a dispersion of glycolipid gives
`approximately 5% of the amount bound to cells relative to the
`exchange technique. Under the conditions of the liposomal
`exchange technique, 17.2% of the total glycolipid incorporated
`in the liposomes exchanges with the erythrocytes.
`If a one-to-one exchange process is assumed, only approxi-
`mately 1-2% of the total cholesterol in the bovine erythrocyte
`has been exchanged with the synthetic glycolipids. In Fig. 2,
`the relationship between the percentage of glycolipid in the
`liposomes and the amount incorporated in the erythrocytes is
`shown. The total phospholipid concentration and time of in-
`cubation (2 hr) were kept constant in these experiments. In Fig.
`3, the relationship between the amount of phospholipid added
`(at a fixed percentage of glycolipid) and the amount incorpo-
`rated in the erythrocytes after a fixed incubation time (2 hr) is
`
`MPI EXHIBIT 1043 PAGE 3
`
`MPI EXHIBIT 1043 PAGE 3
`
`

`

`Biochemistry: Rando et al.
`
`Proc. Natl. Acad. Sci. USA 77 (1980)
`
`2513
`
`porated into the bovine erythrocytes by the periodate/
`a-mannosyl hydrazide method, the cells were agglutinated at
`a minimum concentration of 7.5 tug of Con A per ml (2).
`Therefore, both techniques give roughly equivalent results.
`In order to be certain that the aggregates formed upon the
`Con A-mediated agglutination of the modified erythrocytes
`were typical of agglutinated cells, photomicrographs were taken
`of control cells, modified and agglutinated cells, and modified
`and agglutinated cells with a-methyl mannoside. As can be
`judged from Fig. 4, the agglutinated cells look typical of ag-
`gregating cells and can be easily dispersed by added a-methyl
`mannoside. The latter results show that the aggregates were not
`the result of an irreversible process such as cell-cell fusion.
`When the sugar was added before the lectin, agglutination was
`prevented; when the sugar was added after, it was reversed.
`In this communication we have shown that synthetic cho-
`lesterol-containing glycolipids can be exchanged from liposomes
`into bovine erythrocytes. These cells, which normally cannot
`be agglutinated by Con A, can be rendered so by incorporating
`a-thiomannosyl-containing glycolipids into their outer mem-
`brane. One of the more surprising aspects of this work was the
`relatively modest distance requirement between the cholesterol
`anchor and the sugar needed for agglutination. This distance
`requirement is not dissimilar to that seen with these synthetic
`glycolipids in small unilamellar liposomes (4). Assuming that
`the cholesterol anchor is bound in the erythrocyte membrane
`in a way similar to the way it is bound in liposomes, this suggests
`that the glycoprotein coat of the erythrocyte does not prevent
`access of so large a protein as Con A close to the bilayer. This
`technique of sugar incorporation is, of course, not limited to
`erythrocytes. We have recently found that synthetic glycolipids
`of the kind discussed here can be functionally incorporated into
`such diverse cells as embryonic skeletal muscle cells and liver
`hepatocytes. Furthermore, the use of water-soluble spacer
`groups should allow the determination of the minimal distance
`a sugar (or other receptor) must be from the membrane before
`it becomes a functional receptor. This dimension of the mem-
`brane is not obtainable by electron microscopy, and its deter'
`mination should be of interest for a molecular understanding
`of what terms such as crypticity mean when applied to recep-
`tors.
`
`This work was supported by Department of Energy Contract DE-
`AC02-79EV10268 and partially by National Science Foundation Grant
`PCM 77-23588. R.R.R. is the recipient of National Institutes of Health
`Research Career Development Award GM 00014; J.S. is the recipient
`of National Institutes of Health Postdoctoral Fellowship Al 05752.
`
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`
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`
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`9.
`
`10.
`
`11.
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`Hughes, R. C. (1976) Membrane Glycoproteins (Butterworth,
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`Orr, G. A. & Rando, R. R. (1978) Nature (London) 272, 722-
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`Rando, R. R., Orr, G. A. & Bangerter, F. W. (1979) J. Biol. Chem.
`254,8318-8323.
`Orr, G. A., Rando, R. R. & Bangerter, F. W. (1979) J. Biol. Chem.
`254,4721-4725.
`Rando, R. R. & Bangerter, F. W. (1979) J. Supramol. Struct. 11,
`295-309.
`Huang, C., Charlton, J. P., Shyr, C. I. & Thompson, T. E. (1970)
`Biochemistry 9, 3422-3426.
`Litman, B. J. (1973) Biochemistry 12, 2545-2554.
`Ames, B. M. & Dubin, D. T. (1960) J. Biol. Chem. 235, 769-
`775.
`Krantz, M. J. & Lee, Y. C. (1976) Anal. Biochem. 71, 318-
`321.
`Barenholz, Y., Gibbes, D., Litman, B. J., Goll, J., Thompson, T.
`E. & Carlson, F. D. (1977) Biochemistry 16,2806-2810.
`Bruckdorfer, K. R. & Graham, J. M. (1976) in Biological Mem-
`branes, eds. Chapman, D. & Wallach, D. F. H. (Academic, New
`York), Vol. 3, pp. 103-152.
`
`B
`
`**.
`
`gQv
`
`Aggregating cells: 1 ml of bovine erythrocytes (5 X 108
`FIG. 4.
`cells) in phosphate-buffered saline was modified with liposomes
`containing 5% II (n = 2) as in Table 1. Aliquots (0.1 ml) of the cells
`were placed in three test tubes: (A) control; (B) Con A (50,g/ml) was
`added; (C) 40 mM a-methyl mannoside was added followed by Con
`A (50,ug/ml). The cells were incubated for 10 min at room tempera-
`ture; aliquots were removed and examined by light microscopy with
`bright-field objectives at a magnification of X125. When a-methyl
`mannoside was added to agglutinated cells, the aggregates were dis-
`persed (not shown).
`
`same extent into the erythrocyte. Therefore, the differences
`observed here cannot be due to differential incorporation of the
`glycolipid. Further increases beyond 26 A do not appear to
`enhance the aggregation response. When the hydrophobic
`spacer groups were used (I), no aggregation of the cells occurred
`in the presence of Con A, supporting our contention that hy-
`drophilic spacer groups are- required. Under conditions where
`cells modified with II (n = 2) were aggregated with 4 ,ug of Con
`A per ml, 9.15 X 106 sites per cell were introduced and the av-
`erage density of sites equaled 8.4 X 104 sites per 'm2. Com-
`paring this value with that obtained by the chemical method
`for the incorporation of sugar residues onto the cell surface, we
`found that when 7.1 X 106 a-mannosyl residues were incor-
`
`MPI EXHIBIT 1043 PAGE 4
`
`MPI EXHIBIT 1043 PAGE 4
`
`