ANALYTICAL
BIOCHEMISTRY
189,
91-9-t
(19%))
Influence of Type of Linkage and Spacer on the Interaction of ,&Galactoside-Binding Proteins with Immobilized Affinity Ligands Hans-Joachim Max-Plan&-In&it& D-3400 Giittingen,
Received
February
Gabius fiir experimentelle Federal
Republic
of
Medizin, Germany
Abteilung
Chemie, Hermann-Rein-Str.
14, 1990
Affinity chromatography provides a powerful tool for isolation of carbohydrate-binding proteins. However, the choice of the ligand and spacer has an important impact on effectiveness. The influence of several different ligands on qualitative and quantitative aspects of the purification of two @-galactoside-specific lectins has been evaluated. Sepharose was modified by coupling four types of neoglycoproteins (galactosylated or lactosylated bovine serum albumin with increasing sugar content) and two naturally occurring asialoglycoproteins at similar densities. Carbohydrate ligands at essentially equal density were made accessible to the lectins by seven commonly used methods. The yield of mistletoe lectin was high when lactosylated neoglycoproteins were used for separation. For these resins the sugar incorporation exceeded 10 sugar groups per protein carrier molecule. The yield was similarly high with the asialoglycoproteins and with lactose; the sugar was coupled to the resin as ap-aminophenyl derivative or by means of divinyl sulfone activation. An epoxy group in linkages of galactose or lactose decreased the binding capacity. A quantitatively similar degree of protein yields was obtained for the /3-galactoside-binding protein of bovine heart, although different proteins were obtained when neoglycoproteins were used as ligand. The nature of the affinity ligand in lectin purification can increase the yield and may also influence the profile of the carbohydrate-binding proteins. o I990 Academic Press,
Inc.
The interest in mammalian lectins has increased in recent years (1). Several methods have proven very use-
1 Abbreviations used: carbodiimide hydrochloride; 0003-2697/90
Copyright All
rights
$3.00 1990 by of reproduction
0
3,
EDC,
1-ethyl-3-(3-dimethylaminopropyl)BSA, bovine serum albumin.
ful for lectin isolation (2). However, the use of different affinity ligands for purification of a certain class of lectins has shown that such changes can yield qualitative and quantitative differences (3-6). The linkage type and the structure of the aglycon can affect the interaction of lectins with neoglycoproteins in solution (7,8). Thus, rational selection of the affinity ligand is a prerequisite for optimal lectin recovery. When dealing with small amounts of starting material, e.g., cultured cells, or with pharmacologically active lectins from a source of limited availability, optimized yields are especially desirable. To establish rules for a rational synthesis of affinity matrices, 13 different combinations of linkers and spacers were prepared and the various affinity matrices were compared in their ability to efficiently purify ,&galactoside-binding proteins. The ligand selection includes (neo)glycoproteins and carbohydrates. Bovine heart and mistletoe, which is known to contain a lectin, (9,lO) are chosen as starting material. EXPERIMENTAL
Materials Sepharose 4B, AH-Sepharose 4B, CH-Sepharose 4B and Epoxy-activated Sepharose 6B were obtained from Pharmacia (Freiburg, FRG), p-nitrophenyl$-D-lactoside, l-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride (EDC), fetuin, and cy,-acid glycoprotein (orosomucoid) were purchased from Sigma (Munich, FRG), and cyanogen bromide and divinyl sulfone were from Merck (Darmstadt, FRG). Bovine heart was provided from a local slaughterhouse and dried mistletoe leaves were commercially available in a local pharmacy. Preparation of (neo)Glycoprotein.s Removal of sialic acid from sialoglycoproteins was achieved by acid treatment (pH 2 at 8O”C), yielding asialofetuin and asialoorosomucoid, as described (11). 91
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HANS-JOACHIM
GABIUS
Ho
4
a
14.3
C
b
FIG. 2. Polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate of fl-galactoside-binding proteins from fresh bovine heart, isolated on lactosylated BSA,, (a) and on immobilized lactose after divinyl sulfone activation (b), and from dried mistletoe leaves, representatively showing the lectin with its different A subunits and the B subunit after purification on immobilized p-aminophenyl lactoside (c). Positions of commercially available standard proteins for molecular weight designation are indicated.
FIG. 1. Representation of the structures of the neoglycoproteins, illustrating the product of coupling of the diazo derivative of p-aminophenyl lactoside to tyrosine groups of BSA (BSA,,, top), the product of coupling of p-isothiocyanato lactoside to t-amino groups of lysine residues of BSA (BSA&, the product of coupling of the galactose derivative of (2,3-epoxypropane)-4-oxybutyric acid to r-amino groups of lysine residues of BSA (BSA,,), and the product of reductive amination of lactose to c-amino groups of lysine residues of BSA after reduction by NaCNBH, (BSA,,, bottom).
Chemical glycosylation of carrier proteins allowed the use of additional proteins. Neoglycoproteins containing lactose or galactose groups were synthesized by four different procedures. Coupling reactions using p-aminophenyl-/3-D-lactoside as the diazonium salt or the pisothiocyanato derivatives (12,13), lactose, by reductive amination (14), and D-galactose, suitably modified by a spacer, were performed (15). The structures of the products are given in Fig. 1. The carrier molecule bovine serum albumin (BSA) had previously been treated with periodate to destroy any sugar contaminations (16). Protein content was assessed by a colloidal gold-binding assay (17). The sugar content was determined by a resorcinol-sulfuric acid micromethod (18), and was found to average 8-12, 28-32, 16-19, and 20-24 carbohydrate groups per carrier molecule for the four products. Preparation of Affinity Column Supports All (neo)glycoproteins were coupled to Sepharose 4B, which had been activated by cyanogen bromide (19).
Chemically deglycosylated asialoglycoproteins or the unmodified carrier protein were also coupled to the activated support and used in control experiments. The amount of protein coupled to the gel was estimated by determining the amount of protein in the supernatant and the washing solution after performing the coupling reaction, but before blocking with ethanolamine and further washing. The amino group of p-aminophenyl-0-D lactopyranoside was covalently linked to cyanogen bromide-activated Sepharose 4B and to CH-Sepharose 4B,
TABLE Effect
1
of the Type of Immobilized Yield of p-Galactoside-Binding
(neo)Glycoprotein Proteins
on the
Ligand Tissue
BSA,,”
Heart Leaves
32 248
BSAWb 62 505
BSA,, 42 440
BSAZZd 29 192
ASP’
ASOR’
71 536
74 550
Note. Yields are given in pg per 1.4 g fresh heart tissue or 6 g dried mistletoe leaves. Lactosylated BSA, derived from ‘diazotation, *thionylation, and ‘reductive amination; ‘galactosylated BSA, derived from coupling of galactose via an aliphatic linker;