- Email: [email protected]
[6] Neoglycoprotein-liposome and lectin-liposome conjugates as tools for carbohydrate recognition research
56
NEOGLYCOPROTEINS
[6]
the most critical peptide region of IL-2 required for p70 binding appears to be between amino acid residues 10 and 27 and this region is devoid of any lysines. On the other hand, the peptide epitope of IL-2 required for binding to the p56 receptor, which together with p70 mediates T cell activation, has a number of lysines that can be expected to become glycosylated under the chemical conditions used here. Consequently, the basic e-amino groups of lysines in the key peptide region can be acylated. This could result in a decreased ability to bind to the critical p56 receptor or to the receptor complex and consequently the diminution of T cell activation as measured by proliferation. It has been proposed that the toxic effects of IL-2 treatment may arise from the activation of helper T cells and the associated triggering of a cascade of other lymphokines, 6 and so it may be desirable to have IL-2 be capable of more selective NK and LAK cell activation, ultimately with a view to immunotherapy of some malignancies. Chemical glycosylation ofrIL-2 provides such an opportunity, and these results could be expanded to the exploration of other recombinant proteins.
[6] N e o g l y c o p r o t e i n - L i p o s o m e a n d L e c t i n - L i p o s o m e C o n j u g a t e s as T o o l s for C a r b o h y d r a t e R e c o g n i t i o n R e s e a r c h By NOBORU YAMAZAKI, MAKOTO KODAMA, and HANS-JOACHIM GABIUS
Introduction Specific interactions between carbohydrate ligands and lectin receptors on membrane surfaces play an important role in various biological processes such as cell-to-cell recognition, adhesion, and communication. For carbohydrate recognition research as well as for applied areas such as cell type-specific targeting, it is essential to provide a basic understanding of underlying mechanisms of the multivalent carbohydrate-protein interactions on membrane surfaces. To evaluate these interactions in in vitro and in vivo systems, model systems such as liposomes whose surfaces are modified by chemical conjugation of neoglycoproteins or lectins can provide appropriate tools. 1-4 1 N. Yamazaki, in " A d v a n c e s in C h r o m a t o g r a p h y 1986, Part II" (A. Zlatkis, ed.), p. 371. Elsevier, A m s t e r d a m , 1987. 2 N. Yamazaki, J. Membr. Sci. 41, 249 0989). 3 N. Yamazaki, S. Kojima, S. Gabius, and H.-J. Gabius, Int. J. Biochem. 24, 99 (1992).
METHODS IN ENZYMOLOGY, VOL. 242
Copyright © 1994by AcademicPress. Inc. All rights of reproduction in any form reserved.
[6l
NEOGLYCOPROTEIN-, LECTIN--LIPOSOME CONJUGATES
57
CHzOH HO" ~ i ~ ' ~
~
~
,o
O~NHz
,o
coo,
........ -((.,oo;; I~ ~
HNkc
NAN02 HCI
6,
,
<
CH~OH
HO.__..ww--
~,,~0~ N m N + CI-
0
HNAc
Nal04
COOH
OH
-
\
%
/
/
CHzOH
"o~"~, °\ HO
I '~"-"-NaBHJCN CHzOH ,o-..-
..... eo
HNXc
~H
-
F1G. 1. Outline of the reaction sequence for neoglycoprotein-liposome conjugation. Preparation of mannosylated bovine serum albumin (BSA)-coupled liposomes (Lipo) was chosen as an example.
This chapter describes procedures for the preparation and characterization of one type of versatile and stable liposomes, as well as procedures for covalent conjugation of neoglycoproteins and lectins to the liposomes and characterization of the conjugates. A scheme outlining the procedure described in this chapter for neoglycoprotein-liposome conjugation is represented in Fig. 1, and the procedure for lectin-liposome conjugation 4 N. Yamazaki, S. Gabius, S. Kojima, and H.-J. Gabius, in "Lectins and Glycobiology" (H.-J. Gabius and S. Gabius, eds.), p. 319. Springer-Verlag, Heidelberg, 1993.
58
NEOGLYCOPROTEINS
[6]
is the same except that unmodified lectins, instead of neoglycoproteins, are coupled to oxidized liposomes. Materials
All chemicals mentioned here, obtained from the indicated sources, are of analytical reagent grade and are used without further purification: L-a-dipalmitoylphosphatidylcholine, cholesterol, dicetyl phosphate, gangliosides (type III from bovine brain), and sodium cholate (Sigma Chemical Co., St. Louis, MO), and lectins of the highest available level of purity (Honen Corporation, Tokyo, Japan). Neoglycoproteins are prepared as follows: bovine serum albumin (BSA) is modified with the diazo derivatives ofp-aminophenyl glycosides, which results in yields of 10 -- 2 carbohydrate moieties per carrier protein, as described in detail elsewhere. 5 Preparation of Liposomes The following procedure for preparing a type of liposomes is a modified version of the published method,i-4 which is an adaptation of the controlled cholate dialysis method of Zumbuehl and Weder. 6 The procedure consists of two steps, namely, preparation of lipid-detergent mixed micelles and flow-through dialysis of the mixed micelles. The lipid composition of the liposomes, namely, L-a-dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), dicetyl phosphate (DCP), and gangliosides at a molar ratio of 35:45:5: 15, has been designed for achieving covalent coupling of liposome-bound garlgliosides to proteins via Schiff base formation, and for yielding homogeneous and stable liposomes. The lipids DPPC (17 mg), Chol (12 mg), DCP (2 mg), and gangliosides (15 rag) are mixed with sodium cholate (48 mg) in a 10-ml round-bottomed flask, suitable for a standard rotary evaporator, to give a lipid to detergent molar ratio of 0.6, and the solids are dissolved in 3 ml of chloroform/ methanol (1 : l, v/v) to make a clear solution. The solvent is evaporated under a stream of nitrogen on a rotating evaporator at about 30° in a water bath. The flask with the deposited lipid-detergent mixture is kept for about 1 hr under vacuum in a desiccator equipped with a high vacuum pump and stored overnight in v a c u o to ensure complete removal of organic solvents and to give a completely dry lipid film on the walls of the flask. The lipid film is dissoFced in 3 ml of tris(hydroxymethyl)methylaminopropanesulfonic acid (TAPS)-buffered saline [10 mM TAPS buffer (pH 8.4) 5 C. R. McBroom, C. H. Samanen, and I. J. Goldstein, this series, Vol. 28, p. 212. 6 O. Zumbuehl and H. G. Weder, Biochim. Biophys. Acta 640, 252 (1981).
[6]
NEOGLYCOPROTEIN-, LECTIN-LIPOSOME CONJUGATES
59
containing 150 mM NaCI] with gentle stirring for 1 hr in the flask, during which time the air is replaced by N2. The homogeneous lipid-detergent suspension is sonicated in a bath-type ultrasonicator equipped with time regulator for 1 hr with an intermission of 3 sec every 2 rain until a homogeneous suspension of mixed micelles is formed. To prepare homogeneous unilamellar liposomes, the lipid-detergent mixed micelle suspension obtained should be as transparent as possible before starting the following dialysis. For flow-through dialysis of the mixed micelles, a 10-ml ultrafiltration cell (Model 8010; Amicon Division, W. R. Grace & Co., Danvers, MA) is fitted with an Amicon Diaflo PMI0 membrane and equipped with a concentration/dialysis selector (Amicon Model CDS-10) connected to an 800-ml reservoir. Then 3 ml of the mixed micelle suspension is added and diafiltered with approximately 100 ml of TAPS-buffered saline (pH 8.4) for about 24 hr at room temperature, by applying about 2 atm N2 pressure. The volume of the prepared liposome suspension will increase after the diafiltration step, and it can be between 5 and 7 ml. The liposome suspension is filtered through a 0.45-/zm MillexHV membrane (Millipore, Bedford, MA) and stored in a glass tube with a Teflon-lined screw cap in a refrigerator at 40-7 °. Characterization of Liposomes To analyze the homogeneity and stability of liposomes, size distribution is an important characteristic among several physical properties. Gel-permeation chromatography (GPC), electron microscopy (EM), and dynamic light scattering (DLS) can be applied for size characterization of liposomes. In the following, a procedure for GPC analysis is described, and a typical GPC result is compared with analyses by EM and DLS. Analysis by Gel-Permeation Chromatography. A precise analysis can be performed by using a high-performance liquid chromatography (HPLC) system, which is equipped with an on-line degasser, a computer-controlled pump, a sample injector, and a data processor. A water-jacketed, thickwalled glass column (100 cm × 1 cm i.d.) is packed with Sephacryl S1000 Superfine (Pharmacia Fine Chemicals, Uppsala, Sweden), equilibrated, and eluted at 0.1 ml/min with phosphate-buffered saline [i.e., 10 mM phosphate buffer (pH 7.2) containing 150 mM NaCI]. The column is kept at 25° by circulating thermostatted water in the jacket. Column effluent is monitored by two detectors connected in series: a UV detector and a fluorometer for measuring absorbance at 280 nm and 90° light scattering at 633 nm, respectively. The measurement of light scattering permits estimation of the liposome content in the effluent with such high sensitivity that a 50-~1 injection of liposome suspension gives a well-defined elution
60
NEOGLYCOPROTEINS
320 nm
[6]
160 nm [ 70 nm
>,
(b) 2;o
'
600
800
Elution Time (rain) FIG. 2. Gel-permeationchromatography on a Sephacryl S 1000Superfine column. Elution
profiles of (a) one type of liposome whichis describedin this chapter and (b) another type of liposome which differs in lipid compositionare shown. The column was calibrated, as depicted by arrows, with three sizes of polystyrenelatex particles: 320, 160, and 70 nm. profile, as shown in Fig. 2. For calibration of the column with polystyrene latex particles (Polysciences, Warrington, PA), a Triton-containing buffer (phosphate-buffered saline, pH 7.2, containing 0.5% Triton X-100) is used to suspend the latex particles as well as to preequilibrate and elute the column. The use of Triton X-100 prevents aggregation of the latex particles. The result of GPC (Fig. 2a) is in good agreement with the results of EM (Fig. 3) and DLS (Fig. 4), and this agreement clearly supports the accuracy of the method described above. As shown in Fig. 2, two types of liposome preparations can be distinguished from one another by using the GPC analysis, which is convenient for routine size characterization of liposomes. Conjugation of Neoglycoproteins or Lectins to Liposomes The following procedure is a modified version of the published method 1-4 for conjugation of proteins to liposomes, which is an adaptation of the covalent coupling method of Heath et al. 7 As outlined in Fig. 1, the vT. D. Heath, B. A. Macher, and D. Papahadjopoulos,Biochirn. Biophys. Acta 640, 66 (1981).
[6]
NEOGLYCOPROTEIN-, LECTIN-LIPOSOME CONJUGATES
61
FIG. 3. Scanning electron micrograph of the same liposome preparation as used in Fig. 2a. Magnification: x 19,000. Bar: 1 /xm.
procedure for neoglycoprotein-liposome conjugation can be performed in a simple two-step reaction involving periodate oxidation of gangliosides in the liposome membrane and coupling of neoglycoproteins to oxidized liposomes by reductive amination. The periodate treatment at pH 8.4 oxidizes a large portion of the external gangliosides of the liposomes without oxidizing the internal gangliosides. This method provides a useful basis for covalent coupling of biologically active proteins to the outer surface of membranes without destroying the integrity of the liposomes. Periodate Oxidation. The liposome suspension described above, which contains approximately 46 mg of total lipid in 5-7 ml of TAPS-buffered saline (pH 8.4) in a glass tube, is mixed with 0.5-0.7 ml of 0.2 M sodium periodate in TAPS-buffered saline (pH 8.4) to obtain a final periodate concentration of 20 mM in the reaction mixture. The mixture is incubated at room temperature in the dark for 2 hr with gentle stirring. To separate residual periodate from the liposomes and to change the buffer, a 10ml ultrafiltration cell is fitted with a 0.03-/~m polycarbonate membrane (Nuclepore, Pleasanton, CA) and equipped with a concentration/dialysis
62
NEOGLYCOPROTEINS
A
E
6
(g Q °m
28.6 33.6 39.3 46.1 54.0 63.3
[6]
i~i~iii!i!~i!7ii7i7i~ii~i~77~7i7i7i7i7~7~ii!i~i~i~i~7~7ii~ ..................................................... .........................................
74.2
87.0 102 120 140 164 192 226 264 310 363
]
i Weight Distribution
Fic. 4. Bar histogram of dynamic light scattering of the same liposome preparation as shown in Figs. 2a and 3. selector connected to an 800-ml reservoir which contains phosphate-buffered saline [i.e., 10 mM phosphate buffer (pH 8.0) and 150 mM NaC1]. Then the reaction mixture is added and diafiltered overnight with approximately 100 ml of phosphate-buffered saline (pH 8.0) at about 10° by applying 1 atm N2 pressure. The oxidized liposomes obtained, the volume of the suspension being between 5 and 7 ml, can be stored at about 4 ° for at least several months under a nitrogen atmosphere in a tightly closed glass tube with Teflon-lined screw cap. Reductive Amination. The second step for coupling proteins to liposomes is usually performed by using one-tenth volume of the suspension of oxidized liposomes described above. The following procedure is applied to coupling of neoglycoproteins as well as lectins. In a 2-ml glass tube with a Teflon-coated micro stirring bar, 1-2 mg of solid neoglycoproteins or 2-3 mg of solid lectins are mixed with 0.5-0.7 ml of the oxidized liposome suspension, containing about 4.5 mg of total lipid in phosphatebuffered saline (pH 8.0). The mixture is incubated for 2 hr at room temperature with gentle stirring, and 5-7/~1 of 2 M sodium cyanoborohydride in phosphate-buffered saline (pH 8.0) is added to reach a final concentration of 20 mM in cyanoborohydride. The mixture is further incubated overnight at about l0 ° with gentle stirring. To separate unbound neoglycoproteins or lectins from the liposomes and to change the buffer and pH environment, a 10-ml ultrafiltration cell is fitted with a 0.03-/~m polycarbonate membrane and equipped with a concentration/dialysis selector connected to an 800-
[6]
NEOGLYCOPROTEIN-, LECTIN--LIPOSOME CONJUGATES
63
ml reservoir, containing phosphate-buffered saline [i.e., 10 mM phosphate buffer (pH 7.2) and 150 mM NaCI]. The reaction mixture is adjusted with phosphate-buffered saline (pH 8.0) until the total volume reaches 2 ml, then transferred to the cell and diafiltered overnight with approximately 100 ml of phosphate-buffered saline (pH 7.2) at about 10° by applying 1 atm N2 pressure. The suspension of neoglycoprotein-liposome conjugates or lectin-liposome conjugates obtained is filtered through a 0.2-/zm MillexHV membrane, and stored under a nitrogen atmosphere in a tightly closed glass tube with Teflon-lined screw cap in a refrigerator at 4°-7 °. Characterization of Neoglycoprotein-Liposome and Lectin-Liposome Conjugates
Analysis by Gel-Permeation Chromatography. To analyze the purity and stability of neoglycoprotein-coupled liposomes or lectin-coupled liposomes, GPC analysis is routinely carried out by using the same HPLC system as described above. Figure 5a shows the purity of a preparation of mannosylated BSA-coupled liposomes. The size distribution of this type of neoglycoprotein-liposome conjugate is identical with that of non-
320 nm
160 nm I 70 nm
t'-
-o
(a)
==
=~
0 09
(b) '
(C) 2;o
'
4;o
'
'
8;o
Elution Time (min)
FIc. 5. Gel-permeation chromatography on a Sephacryl S1000Superfinecolumn. Elution profiles of (a) mannosylated BSA-coupledliposomes, (b) N-acetylgalactosaminylatedBSAcoupled liposomes, which were stored for about 2 years at 4°, and (c) mannosylated BSA are shown. The column was calibrated as in Fig. 2.
64
NEOGLYCOPROTEINS
320 nm
[6]
160 nm ~ 70 nm
C Q
"o o
O
if)
2;0
' 4 ; 0 ' 8;0 ' Elution Time (min)
8;o
FIG. 6. Gel-permeation chromatography on a Sephacryl S1000 Superfine column. An elution profile of a mistletoe lectin-coupled liposome preparation, which had been stored for 3 weeks at 4 °, is shown. The column was calibrated as in Fig. 2.
coupled liposomes, as compared with Fig. 2a, and the conjugate suspension does not contain free neoglycoproteins, as illustrated in Fig. 5a, c. This type of neoglycoprotein-liposome conjugate is stable and does not undergo changes in physical properties after storage at about 4° for several months, and it is noteworthy that one preparation of N-acetylgalactosaminylated BSA-liposome conjugates has been kept in a refrigerator for about 2 years without substantial property changes, as shown in Fig. 5b. Figure 6 shows the size distribution of mistletoe lectin-coupled liposomes. Some lectin-liposome conjugates tend to form liposome aggregates, which can be removed by filtration through a 0.22-~m Millex-HV membrane prior to use. Composition Analysis. To analyze the yield and quality of neoglycoprotein-coupled liposomes or lectin-coupled liposomes, protein and lipid compositions in each preparation are routinely determined, and coupled protein/total lipid ratios are calculated. The protein content of neoglycoprotein-liposome and lectin-liposome conjugates is estimated either by employing the modified Lowry method in the presence of 1% sodium dodecyl sulfate 8 or by using a commercial micro-BSA protein assay kit (Pierce Chemical Co., Rockford, IL) in the presence of 1% sodum dodecyl sulfate. For determination of the lipid concentration in the preparation, the cholesterol content of the liposome suspension is first analyzed by 8 M. A. K. Markwell, S. M. Haas, L. L. Bieber, and N. E. Tolbert, Anal. Biochem. 87, 206 (1978).
[7]
M O D I F I C A T I O N OF PROTEINS W I T H P E G
65
using a commercial kit for total-cholesterol determination, Determiner TC"555" (Kyowa Medex, Tokyo, Japan), in the presence of 0.5% Triton X-100. The amount of lipids in the liposome preparation is then estimated using the molar ratio oflipids, namely, DPPC, Chol, DCP, and gangliosides (35 : 45 : 5 : 15). Results by this method are consistent with results by microanalysis of phosphorus using the Bartlett assay. Coupled protein/total lipid ratios in the final products are usually between 0.1 and 0.2 g/g for neoglycoprotein-liposome conjugates, and between 0.1 and 0.4 g/g for lectin-liposome conjugates.
[7] M o d i f i c a t i o n of P r o t e i n s w i t h P o l y e t h y l e n e Glycol D e r i v a t i v e s By YuJI INADA, AYAKO MATSUSHIMA, MISAO HIROTO, HIROYUKI NISHIMURA, and YOH KODERA
Introduction Chemical modification of proteins became commonplace in the late 1950s: the techniques were developed to aid in the structural analysis of protein molecules. The intention of such a modification was to develop reagents which would specifically react with amino acid side chains in the protein molecule to discriminate the state of amino acid residues and to identify the amino acid involved in particular protein functions. From the 1970s, chemical modification of proteins by conjugation with synthetic or natural macromolecules has been performed. The purposes of these modifications include alteration of immunoreactivity, immunogenicity, and suppression of immunoglobulin E production, or making enzymes soluble and active in organic s o l v e n t s . 1,2'2a This chapter deals with the chemical modification of proteins with synthetic macromolecules, polyethylene glycol derivatives. Since polyethylene glycol [PEG; general formula HO(CHECHEO)nH] has been synthesized, many industrial and biochemical applications in the areas of pharmaceutics, cosmetics, and textiles have been developed to t y. Inada, A. Matsushima, Y. Kodera, and H. Nishimura, J. Bioact. Compat. Polym. 5, 343 (1990). 2 y. Inada, Y. Kodera, A. Matsushima, and H. Nishimura, in "Synthesis of Biocomposite Materials" (Y. Imanishi, ed.), p. 85. CRC Press, Boca Raton, Florida, 1992. 2a y. Inada, M. Furukawa, H. Sasaki, Y. Kodera, M. Hiroto, H. Nishimura, and A. Matsushima, Trends Biotechnol., in press.
METHODS 1N ENZYMOLOGY, VOL. 242
Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.
NEOGLYCOPROTEINS
[6]
the most critical peptide region of IL-2 required for p70 binding appears to be between amino acid residues 10 and 27 and this region is devoid of any lysines. On the other hand, the peptide epitope of IL-2 required for binding to the p56 receptor, which together with p70 mediates T cell activation, has a number of lysines that can be expected to become glycosylated under the chemical conditions used here. Consequently, the basic e-amino groups of lysines in the key peptide region can be acylated. This could result in a decreased ability to bind to the critical p56 receptor or to the receptor complex and consequently the diminution of T cell activation as measured by proliferation. It has been proposed that the toxic effects of IL-2 treatment may arise from the activation of helper T cells and the associated triggering of a cascade of other lymphokines, 6 and so it may be desirable to have IL-2 be capable of more selective NK and LAK cell activation, ultimately with a view to immunotherapy of some malignancies. Chemical glycosylation ofrIL-2 provides such an opportunity, and these results could be expanded to the exploration of other recombinant proteins.
[6] N e o g l y c o p r o t e i n - L i p o s o m e a n d L e c t i n - L i p o s o m e C o n j u g a t e s as T o o l s for C a r b o h y d r a t e R e c o g n i t i o n R e s e a r c h By NOBORU YAMAZAKI, MAKOTO KODAMA, and HANS-JOACHIM GABIUS
Introduction Specific interactions between carbohydrate ligands and lectin receptors on membrane surfaces play an important role in various biological processes such as cell-to-cell recognition, adhesion, and communication. For carbohydrate recognition research as well as for applied areas such as cell type-specific targeting, it is essential to provide a basic understanding of underlying mechanisms of the multivalent carbohydrate-protein interactions on membrane surfaces. To evaluate these interactions in in vitro and in vivo systems, model systems such as liposomes whose surfaces are modified by chemical conjugation of neoglycoproteins or lectins can provide appropriate tools. 1-4 1 N. Yamazaki, in " A d v a n c e s in C h r o m a t o g r a p h y 1986, Part II" (A. Zlatkis, ed.), p. 371. Elsevier, A m s t e r d a m , 1987. 2 N. Yamazaki, J. Membr. Sci. 41, 249 0989). 3 N. Yamazaki, S. Kojima, S. Gabius, and H.-J. Gabius, Int. J. Biochem. 24, 99 (1992).
METHODS IN ENZYMOLOGY, VOL. 242
Copyright © 1994by AcademicPress. Inc. All rights of reproduction in any form reserved.
[6l
NEOGLYCOPROTEIN-, LECTIN--LIPOSOME CONJUGATES
57
CHzOH HO" ~ i ~ ' ~
~
~
,o
O~NHz
,o
coo,
........ -((.,oo;; I~ ~
HNkc
NAN02 HCI
6,
,
<
CH~OH
HO.__..ww--
~,,~0~ N m N + CI-
0
HNAc
Nal04
COOH
OH
-
\
%
/
/
CHzOH
"o~"~, °\ HO
I '~"-"-NaBHJCN CHzOH ,o-..-
..... eo
HNXc
~H
-
F1G. 1. Outline of the reaction sequence for neoglycoprotein-liposome conjugation. Preparation of mannosylated bovine serum albumin (BSA)-coupled liposomes (Lipo) was chosen as an example.
This chapter describes procedures for the preparation and characterization of one type of versatile and stable liposomes, as well as procedures for covalent conjugation of neoglycoproteins and lectins to the liposomes and characterization of the conjugates. A scheme outlining the procedure described in this chapter for neoglycoprotein-liposome conjugation is represented in Fig. 1, and the procedure for lectin-liposome conjugation 4 N. Yamazaki, S. Gabius, S. Kojima, and H.-J. Gabius, in "Lectins and Glycobiology" (H.-J. Gabius and S. Gabius, eds.), p. 319. Springer-Verlag, Heidelberg, 1993.
58
NEOGLYCOPROTEINS
[6]
is the same except that unmodified lectins, instead of neoglycoproteins, are coupled to oxidized liposomes. Materials
All chemicals mentioned here, obtained from the indicated sources, are of analytical reagent grade and are used without further purification: L-a-dipalmitoylphosphatidylcholine, cholesterol, dicetyl phosphate, gangliosides (type III from bovine brain), and sodium cholate (Sigma Chemical Co., St. Louis, MO), and lectins of the highest available level of purity (Honen Corporation, Tokyo, Japan). Neoglycoproteins are prepared as follows: bovine serum albumin (BSA) is modified with the diazo derivatives ofp-aminophenyl glycosides, which results in yields of 10 -- 2 carbohydrate moieties per carrier protein, as described in detail elsewhere. 5 Preparation of Liposomes The following procedure for preparing a type of liposomes is a modified version of the published method,i-4 which is an adaptation of the controlled cholate dialysis method of Zumbuehl and Weder. 6 The procedure consists of two steps, namely, preparation of lipid-detergent mixed micelles and flow-through dialysis of the mixed micelles. The lipid composition of the liposomes, namely, L-a-dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), dicetyl phosphate (DCP), and gangliosides at a molar ratio of 35:45:5: 15, has been designed for achieving covalent coupling of liposome-bound garlgliosides to proteins via Schiff base formation, and for yielding homogeneous and stable liposomes. The lipids DPPC (17 mg), Chol (12 mg), DCP (2 mg), and gangliosides (15 rag) are mixed with sodium cholate (48 mg) in a 10-ml round-bottomed flask, suitable for a standard rotary evaporator, to give a lipid to detergent molar ratio of 0.6, and the solids are dissolved in 3 ml of chloroform/ methanol (1 : l, v/v) to make a clear solution. The solvent is evaporated under a stream of nitrogen on a rotating evaporator at about 30° in a water bath. The flask with the deposited lipid-detergent mixture is kept for about 1 hr under vacuum in a desiccator equipped with a high vacuum pump and stored overnight in v a c u o to ensure complete removal of organic solvents and to give a completely dry lipid film on the walls of the flask. The lipid film is dissoFced in 3 ml of tris(hydroxymethyl)methylaminopropanesulfonic acid (TAPS)-buffered saline [10 mM TAPS buffer (pH 8.4) 5 C. R. McBroom, C. H. Samanen, and I. J. Goldstein, this series, Vol. 28, p. 212. 6 O. Zumbuehl and H. G. Weder, Biochim. Biophys. Acta 640, 252 (1981).
[6]
NEOGLYCOPROTEIN-, LECTIN-LIPOSOME CONJUGATES
59
containing 150 mM NaCI] with gentle stirring for 1 hr in the flask, during which time the air is replaced by N2. The homogeneous lipid-detergent suspension is sonicated in a bath-type ultrasonicator equipped with time regulator for 1 hr with an intermission of 3 sec every 2 rain until a homogeneous suspension of mixed micelles is formed. To prepare homogeneous unilamellar liposomes, the lipid-detergent mixed micelle suspension obtained should be as transparent as possible before starting the following dialysis. For flow-through dialysis of the mixed micelles, a 10-ml ultrafiltration cell (Model 8010; Amicon Division, W. R. Grace & Co., Danvers, MA) is fitted with an Amicon Diaflo PMI0 membrane and equipped with a concentration/dialysis selector (Amicon Model CDS-10) connected to an 800-ml reservoir. Then 3 ml of the mixed micelle suspension is added and diafiltered with approximately 100 ml of TAPS-buffered saline (pH 8.4) for about 24 hr at room temperature, by applying about 2 atm N2 pressure. The volume of the prepared liposome suspension will increase after the diafiltration step, and it can be between 5 and 7 ml. The liposome suspension is filtered through a 0.45-/zm MillexHV membrane (Millipore, Bedford, MA) and stored in a glass tube with a Teflon-lined screw cap in a refrigerator at 40-7 °. Characterization of Liposomes To analyze the homogeneity and stability of liposomes, size distribution is an important characteristic among several physical properties. Gel-permeation chromatography (GPC), electron microscopy (EM), and dynamic light scattering (DLS) can be applied for size characterization of liposomes. In the following, a procedure for GPC analysis is described, and a typical GPC result is compared with analyses by EM and DLS. Analysis by Gel-Permeation Chromatography. A precise analysis can be performed by using a high-performance liquid chromatography (HPLC) system, which is equipped with an on-line degasser, a computer-controlled pump, a sample injector, and a data processor. A water-jacketed, thickwalled glass column (100 cm × 1 cm i.d.) is packed with Sephacryl S1000 Superfine (Pharmacia Fine Chemicals, Uppsala, Sweden), equilibrated, and eluted at 0.1 ml/min with phosphate-buffered saline [i.e., 10 mM phosphate buffer (pH 7.2) containing 150 mM NaCI]. The column is kept at 25° by circulating thermostatted water in the jacket. Column effluent is monitored by two detectors connected in series: a UV detector and a fluorometer for measuring absorbance at 280 nm and 90° light scattering at 633 nm, respectively. The measurement of light scattering permits estimation of the liposome content in the effluent with such high sensitivity that a 50-~1 injection of liposome suspension gives a well-defined elution
60
NEOGLYCOPROTEINS
320 nm
[6]
160 nm [ 70 nm
>,
(b) 2;o
'
600
800
Elution Time (rain) FIG. 2. Gel-permeationchromatography on a Sephacryl S 1000Superfine column. Elution
profiles of (a) one type of liposome whichis describedin this chapter and (b) another type of liposome which differs in lipid compositionare shown. The column was calibrated, as depicted by arrows, with three sizes of polystyrenelatex particles: 320, 160, and 70 nm. profile, as shown in Fig. 2. For calibration of the column with polystyrene latex particles (Polysciences, Warrington, PA), a Triton-containing buffer (phosphate-buffered saline, pH 7.2, containing 0.5% Triton X-100) is used to suspend the latex particles as well as to preequilibrate and elute the column. The use of Triton X-100 prevents aggregation of the latex particles. The result of GPC (Fig. 2a) is in good agreement with the results of EM (Fig. 3) and DLS (Fig. 4), and this agreement clearly supports the accuracy of the method described above. As shown in Fig. 2, two types of liposome preparations can be distinguished from one another by using the GPC analysis, which is convenient for routine size characterization of liposomes. Conjugation of Neoglycoproteins or Lectins to Liposomes The following procedure is a modified version of the published method 1-4 for conjugation of proteins to liposomes, which is an adaptation of the covalent coupling method of Heath et al. 7 As outlined in Fig. 1, the vT. D. Heath, B. A. Macher, and D. Papahadjopoulos,Biochirn. Biophys. Acta 640, 66 (1981).
[6]
NEOGLYCOPROTEIN-, LECTIN-LIPOSOME CONJUGATES
61
FIG. 3. Scanning electron micrograph of the same liposome preparation as used in Fig. 2a. Magnification: x 19,000. Bar: 1 /xm.
procedure for neoglycoprotein-liposome conjugation can be performed in a simple two-step reaction involving periodate oxidation of gangliosides in the liposome membrane and coupling of neoglycoproteins to oxidized liposomes by reductive amination. The periodate treatment at pH 8.4 oxidizes a large portion of the external gangliosides of the liposomes without oxidizing the internal gangliosides. This method provides a useful basis for covalent coupling of biologically active proteins to the outer surface of membranes without destroying the integrity of the liposomes. Periodate Oxidation. The liposome suspension described above, which contains approximately 46 mg of total lipid in 5-7 ml of TAPS-buffered saline (pH 8.4) in a glass tube, is mixed with 0.5-0.7 ml of 0.2 M sodium periodate in TAPS-buffered saline (pH 8.4) to obtain a final periodate concentration of 20 mM in the reaction mixture. The mixture is incubated at room temperature in the dark for 2 hr with gentle stirring. To separate residual periodate from the liposomes and to change the buffer, a 10ml ultrafiltration cell is fitted with a 0.03-/~m polycarbonate membrane (Nuclepore, Pleasanton, CA) and equipped with a concentration/dialysis
62
NEOGLYCOPROTEINS
A
E
6
(g Q °m
28.6 33.6 39.3 46.1 54.0 63.3
[6]
i~i~iii!i!~i!7ii7i7i~ii~i~77~7i7i7i7i7~7~ii!i~i~i~i~7~7ii~ ..................................................... .........................................
74.2
87.0 102 120 140 164 192 226 264 310 363
]
i Weight Distribution
Fic. 4. Bar histogram of dynamic light scattering of the same liposome preparation as shown in Figs. 2a and 3. selector connected to an 800-ml reservoir which contains phosphate-buffered saline [i.e., 10 mM phosphate buffer (pH 8.0) and 150 mM NaC1]. Then the reaction mixture is added and diafiltered overnight with approximately 100 ml of phosphate-buffered saline (pH 8.0) at about 10° by applying 1 atm N2 pressure. The oxidized liposomes obtained, the volume of the suspension being between 5 and 7 ml, can be stored at about 4 ° for at least several months under a nitrogen atmosphere in a tightly closed glass tube with Teflon-lined screw cap. Reductive Amination. The second step for coupling proteins to liposomes is usually performed by using one-tenth volume of the suspension of oxidized liposomes described above. The following procedure is applied to coupling of neoglycoproteins as well as lectins. In a 2-ml glass tube with a Teflon-coated micro stirring bar, 1-2 mg of solid neoglycoproteins or 2-3 mg of solid lectins are mixed with 0.5-0.7 ml of the oxidized liposome suspension, containing about 4.5 mg of total lipid in phosphatebuffered saline (pH 8.0). The mixture is incubated for 2 hr at room temperature with gentle stirring, and 5-7/~1 of 2 M sodium cyanoborohydride in phosphate-buffered saline (pH 8.0) is added to reach a final concentration of 20 mM in cyanoborohydride. The mixture is further incubated overnight at about l0 ° with gentle stirring. To separate unbound neoglycoproteins or lectins from the liposomes and to change the buffer and pH environment, a 10-ml ultrafiltration cell is fitted with a 0.03-/~m polycarbonate membrane and equipped with a concentration/dialysis selector connected to an 800-
[6]
NEOGLYCOPROTEIN-, LECTIN--LIPOSOME CONJUGATES
63
ml reservoir, containing phosphate-buffered saline [i.e., 10 mM phosphate buffer (pH 7.2) and 150 mM NaCI]. The reaction mixture is adjusted with phosphate-buffered saline (pH 8.0) until the total volume reaches 2 ml, then transferred to the cell and diafiltered overnight with approximately 100 ml of phosphate-buffered saline (pH 7.2) at about 10° by applying 1 atm N2 pressure. The suspension of neoglycoprotein-liposome conjugates or lectin-liposome conjugates obtained is filtered through a 0.2-/zm MillexHV membrane, and stored under a nitrogen atmosphere in a tightly closed glass tube with Teflon-lined screw cap in a refrigerator at 4°-7 °. Characterization of Neoglycoprotein-Liposome and Lectin-Liposome Conjugates
Analysis by Gel-Permeation Chromatography. To analyze the purity and stability of neoglycoprotein-coupled liposomes or lectin-coupled liposomes, GPC analysis is routinely carried out by using the same HPLC system as described above. Figure 5a shows the purity of a preparation of mannosylated BSA-coupled liposomes. The size distribution of this type of neoglycoprotein-liposome conjugate is identical with that of non-
320 nm
160 nm I 70 nm
t'-
-o
(a)
==
=~
0 09
(b) '
(C) 2;o
'
4;o
'
'
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Elution Time (min)
FIc. 5. Gel-permeation chromatography on a Sephacryl S1000Superfinecolumn. Elution profiles of (a) mannosylated BSA-coupledliposomes, (b) N-acetylgalactosaminylatedBSAcoupled liposomes, which were stored for about 2 years at 4°, and (c) mannosylated BSA are shown. The column was calibrated as in Fig. 2.
64
NEOGLYCOPROTEINS
320 nm
[6]
160 nm ~ 70 nm
C Q
"o o
O
if)
2;0
' 4 ; 0 ' 8;0 ' Elution Time (min)
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FIG. 6. Gel-permeation chromatography on a Sephacryl S1000 Superfine column. An elution profile of a mistletoe lectin-coupled liposome preparation, which had been stored for 3 weeks at 4 °, is shown. The column was calibrated as in Fig. 2.
coupled liposomes, as compared with Fig. 2a, and the conjugate suspension does not contain free neoglycoproteins, as illustrated in Fig. 5a, c. This type of neoglycoprotein-liposome conjugate is stable and does not undergo changes in physical properties after storage at about 4° for several months, and it is noteworthy that one preparation of N-acetylgalactosaminylated BSA-liposome conjugates has been kept in a refrigerator for about 2 years without substantial property changes, as shown in Fig. 5b. Figure 6 shows the size distribution of mistletoe lectin-coupled liposomes. Some lectin-liposome conjugates tend to form liposome aggregates, which can be removed by filtration through a 0.22-~m Millex-HV membrane prior to use. Composition Analysis. To analyze the yield and quality of neoglycoprotein-coupled liposomes or lectin-coupled liposomes, protein and lipid compositions in each preparation are routinely determined, and coupled protein/total lipid ratios are calculated. The protein content of neoglycoprotein-liposome and lectin-liposome conjugates is estimated either by employing the modified Lowry method in the presence of 1% sodium dodecyl sulfate 8 or by using a commercial micro-BSA protein assay kit (Pierce Chemical Co., Rockford, IL) in the presence of 1% sodum dodecyl sulfate. For determination of the lipid concentration in the preparation, the cholesterol content of the liposome suspension is first analyzed by 8 M. A. K. Markwell, S. M. Haas, L. L. Bieber, and N. E. Tolbert, Anal. Biochem. 87, 206 (1978).
[7]
M O D I F I C A T I O N OF PROTEINS W I T H P E G
65
using a commercial kit for total-cholesterol determination, Determiner TC"555" (Kyowa Medex, Tokyo, Japan), in the presence of 0.5% Triton X-100. The amount of lipids in the liposome preparation is then estimated using the molar ratio oflipids, namely, DPPC, Chol, DCP, and gangliosides (35 : 45 : 5 : 15). Results by this method are consistent with results by microanalysis of phosphorus using the Bartlett assay. Coupled protein/total lipid ratios in the final products are usually between 0.1 and 0.2 g/g for neoglycoprotein-liposome conjugates, and between 0.1 and 0.4 g/g for lectin-liposome conjugates.
[7] M o d i f i c a t i o n of P r o t e i n s w i t h P o l y e t h y l e n e Glycol D e r i v a t i v e s By YuJI INADA, AYAKO MATSUSHIMA, MISAO HIROTO, HIROYUKI NISHIMURA, and YOH KODERA
Introduction Chemical modification of proteins became commonplace in the late 1950s: the techniques were developed to aid in the structural analysis of protein molecules. The intention of such a modification was to develop reagents which would specifically react with amino acid side chains in the protein molecule to discriminate the state of amino acid residues and to identify the amino acid involved in particular protein functions. From the 1970s, chemical modification of proteins by conjugation with synthetic or natural macromolecules has been performed. The purposes of these modifications include alteration of immunoreactivity, immunogenicity, and suppression of immunoglobulin E production, or making enzymes soluble and active in organic s o l v e n t s . 1,2'2a This chapter deals with the chemical modification of proteins with synthetic macromolecules, polyethylene glycol derivatives. Since polyethylene glycol [PEG; general formula HO(CHECHEO)nH] has been synthesized, many industrial and biochemical applications in the areas of pharmaceutics, cosmetics, and textiles have been developed to t y. Inada, A. Matsushima, Y. Kodera, and H. Nishimura, J. Bioact. Compat. Polym. 5, 343 (1990). 2 y. Inada, Y. Kodera, A. Matsushima, and H. Nishimura, in "Synthesis of Biocomposite Materials" (Y. Imanishi, ed.), p. 85. CRC Press, Boca Raton, Florida, 1992. 2a y. Inada, M. Furukawa, H. Sasaki, Y. Kodera, M. Hiroto, H. Nishimura, and A. Matsushima, Trends Biotechnol., in press.
METHODS 1N ENZYMOLOGY, VOL. 242
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