Volume 21, number 1

MOLECULAR • CELLULARBIOCHEMISTRY

October 13, 1979

BINDING INTERACTIONS OF GLYCOPROTEINS WITH LECTINS John T. DULANEY,

Biochemical Investigator, John F. Kennedy Institute, Baltimore, Maryland and Assistant Professor, Department of Pediatrics, Johns Hopkins University School of Medicine (Received June 5, 1978)

Snmmary Since plant lectins were used to help define differences between normal and transformed cell surfaces (reviewed in References1-4), they have been employed in many other situations where their sugar-recognition specificities could be used to advantage. One of these applications has been the purification and characterization of enzymes and other proteins; this work is reviewed here in order to define some of the variables that affect binding of glycoproteins to lectins, as well as to demonstrate how this technique has been profitably exploited for isolation of purified glycoproteins, and for their better understanding.

Introduction Three methods have been used to evaluate interactions of glycoproteins with lectins. One method is the study of the capacity of the glycoprotein to inhibit lectin-induced agglutination of erythrocytes bearing oligosaccharides of various blood group specificities; the inhibition occurs because the glycose components of the glycoprotein compete, with those borne by the Abbreviations: ConA, concanavalin A; WGA, wheat germ agglutinin; RCA6o (RCAn, mol. wt. 60,000) and RCA12o (RCAI, mol. wt. 120,000), Ricinus communis agglutinin; LCA, Lens culinaris agglutinin;LTA, Lotos tetragonolobus agglutinin; SBA, soybeanagglutinin;PNA, peanut agglutinin; Me-a-Glc, rnethyl-a-glucoside;Me-a-Man, methyl-amannoside; Gal, galactose;GlcNac, N-acetylglucosamine.

erythrocyte membrane, for reactive sites on lectins. The second technique is to examine the capability of the glycoprotein to form insoluble precipitin-like complexes (as suspensions in the test tube or as precipitates in immunodiffusion plates) with lectins specific for sugars which may be components of the glycoprotein. The third method is the determination of the ability of the glycoprotein to bind to lectin which has been attached covalently to an insoluble support such as Sepharose or Agarose, which itself should have no specific affinity for the glycoprotein. The glycoprotein can be eluted from the complex by treatment with a solution of a glycoside for which the lectin is specific, and which removes the glycoprotein by competing for the lectin's binding sites.

Tabulation of Glycoproteins which Interact with Lectins The latter two methods, but predominantly the third, were used in the studies whose results are summarized in Table 1. The interactions of some of these proteins with lectins have been well characterized, as will be discussed later, while others have received comment only in passing. The large number of lysosomal enzymes (that is, hydrolases with acid pH optima) which appear in Table 1.A. may merely be coincidental, reflecting the likelihood that since for several years lysosomal enzymes have been believed to be glycoproteins, more of this group of enzymes than any other have been tested for their interaction with lectins. Not only soluble,

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands

43

4~ 4~

A. Interactions of Enzymes with Lectins

Enzyme, Source and Reference ConA Acetylcholinesterase (rat brain (5), human serum a f t e r neuram~nidase treatment (6)) ~-N-Acetylgalactosaminidase (porcine l i v e r ; human l i v e r , placenta) (7,8,9) + ~-~-Acetylglucosaminidase (human urine) (I0) High clearance form + Low clearance form + B-N-Acetylhexosaminidase (human l i v e r , urine; human, monkey, sheep brain) Hex A (11-21) + Hex B (12-15, 17-20) + Hex C (17) Hex S (17) + Acid phosphatase (sheep brain) (15,20) + Adenosine deaminase (human l i v e r , kidney, intestine) (22) a form + d ~ form + d2 form + Alkaline phosphatase (Dictyostelium discoideum (23), bovine brain (118)) + Arylsulfatase A (chicken, sheep brain; rabbit kidney) (15,20,24-26) Arylsulfatase B (sheep brain, rabbit kidney, ox l i v e r ) (25,27,28) BI~ form (28) BI~ form (28) Ascorbate sulfatase (bovine l i v e r ) (29) Ceramidetrihexosidase (human l i v e r , placenta) (7,8) 3':5'-Cyclic-nucleotide phosphodiesterase (D. discoideum) (23) Dopamine-B-hydroxylase (bovine adrenal) (30~ Endoglucuronidase (specific for heparan sulfate) (human placenta (30a) Exonuclease (snake venom) (31) B-Galactocerebrosidase (human brain) (32) m-Galactosidase (:ceramidetrihexosidase) (human placenta, l i v e r ) (7,8,33) A form (7,8) "A-like" a c t i v i t y from Fabry disease l i v e r (7) B form (=~-N-acetylgalactosaminidase?) {7~,8) B-Galactosidase (human l i v e r ) Acid form A (34-40) Acid form B (34-38,40) Acid form B (bovine testes) (41) Neutral form(s) (=soluble B-glucosidase?) (35,39,42) l - c e l l enzyme (38,40)

Table I.

:} 351

(25)

+(6)

Reactivity with Lectin WGA RCA120

: ,TA 13 I

+,LCA (5)

Other

4~

A. (.Continued)

Enzyme, Source and Reference Galactosyltransferase ~ t erythrocytes) 6-Glucocerebrosidase (human placenta) (33,44-46) Glucose oxidase (Aspergillis ~ (47,48) ~-Glucosidase (human placenta) (33) ~-Glucosidase (,human liver, placenta, spleen) Membrane bound (=~-glucocerebrosidase) ( 3 3 ~ ] ~ Soluble (=neutral ~-galactosidase?) (39,40,421 B-Glucuronidase (sheep brain, rat preputial) (15,18,49) y=Glutamyltransferase (rat l i v e r , kidney and small intestine) Adult form (50-52) Fetal form (51,52) Glycogen synthetase (human polymorphonuclear leucocytes) (53) D-form l-form Hyaluronidase (bovine testes) (54) Iduronate sulfatase (human liver) (5--5~ m-lduronidase (human urine) (56) Form I (corrective) Form II (non-corrective) l-cell enzyme Li'pase (porcine pancreas~ (57) ~-Mannosidase (human liver) Acid form (33,58) Neutral form (58) ~-Mannosidase (rat liver) (59) Lysosomal (most acidic optimum) Golqi Cytosolic (most nearly neutral optimu~ 5'-NucleotiCase (D. discoideum; venom; porcine lymphocytes) (23,31,60) Peroxidase (horseradish) (47,61) Ribonuclease B (bovine pancreas)~2,l~F9~ Sphingomyelinase (human brain) (120)

Table I. ConA

Reactivity with Lectin WGA RCA12o

+, LCA (60)

Other

4=, 0%

*After treatment of glycoprotein with neuraminidase.

IgM (6,62,67) Insulin receptor (rat fat cell (75) Interferon (human fibroblasts) (76,77) Rhodopsin (bovine retinas) (78,79) Transferrin (human serum) (6,66,67,72)

M y ~ E ~TOm-T~%)

Asialo form Asialoagalacto form Human f o l l i c l e - s t i m u l a t i n g hormone Human growth hormone Human l u t e i n i z i n g hormone HL-A Antigens (IMI malignancy tissue culture cells) (747 Immunoglobulins (human serum) IgA (6,62) IgG Normal serum (6,67)

Glutamate-binding protein ( r ~ ~ 7 1 ) Hemopexin (human serum) (6,72) Hormones (73) Human chorionic gonadotropin Native form

}(70)

Partial + (72)

+

+

+* (6)

+* (6) +

+* (6) (67)

-

Partial * + (62,67)

+* (6)

+* (6)

(72)

+ (72)

+

+* (6)

+, LCA

;}SBA,PNA (70)

Reactivity with Lectin Other WGA RCA120

+ (62)

+

;}(7o)

+

Partial +

+

ConA + (63)

8. Interactions of Serum and Membrane Glycoproteins with Lectins

Protein, Source and Reference m~-Antitrypsin Thuman s e r u m ~ Carcinoembryonic antigen (liver-metastasized colon carcinoma) (64,65) Ceruloplasmin (human and goat serum) In fresh human serum (66,67) Sialylated form (68) Asialo form (6,6~ ~-Fetoprotein (human fetal cord blood) (69) Fetuin (fetal c a l f serum) Sialylated (native form) (68,70) Asialo form (6,68,70)

Tabie I.

but also membrane-localized enzymes such as /3-glucosidase, lipase and 5'-nucleotidase, interact with lectins, and both mammalian enzymes and those from bacteria (D. discoideum, A. niger) and plants (horseradish peroxidase). The extensive use of ConA (the lectin from jack bean, Canavalia ensi[ormis) for isolation and characterization studies is probably due largely to its commercial availability and comparatively inexpensive cost. Another advantage may be its specificity for a grouping (discussed later) which is present in many glycoproteins the structures of whose glycose chains has been determined. It may be that this oligosaccharide sequence is a common component of a large class of glycoproteins, which hence bind to ConA; lectins which may be more specific in the sense that they seem to recognize a single terminal sugar, such as RCA120 which preferentially interacts with terminal/3-galactosides, may therefore be more selective than ConA and bind fewer glycoproteins. It is interesting that enzymes which exist in multiple forms often have varying lectin-binding properties; note for example that the Hex C form of/3-N-acetylhexosaminidase17, the neutral forms of/3-galactosidase35'39'42 and amannosidase58"59, and the soluble form of/3glucosidase39'4°'42 fail to bind to ConA, whereas the other forms of these enzymes bind well to this lectin. In Table 1.B., notice especially the differences in binding which desialylation causes in the well-studied cases of fetuin and ceruloplasmin. The fact that these two, as well as several other serum glycoproteins6 bind to RCA12o after removal of their terminal sialyl groups, argues that/3-galactoside is exposed by the desialylation.

Purification of Enzymes on Con-A Sepharose Columns The tight binding of glycoproteins to insolubilized lectins, such as those linked covalently to Sepharose or Agarose, has obvious implications for enzyme purification. Table 2 presents examples of the use of this technique to purify enzymes up to several hundred fold. These examples were chosen to illustrate aspects of lectin binding relevant to their practical use in enzyme purification (although many other cases

of purification by use of lectin adsorbents are represented in Table 1). Data are either taken directly from the papers cited, or if not given there in the form desired, calculated from other data such as column dimensions (to determine amounts of adsorbent used) or specific activities and total activities (to determine amounts of protein); such calculations (calc.) are indicated in the Table. The ratio of protein applied to the lectin-Sepharose column, to the amount of lectin protein comprising the column, varies from 0.2 to 113; there is apparently no correlation between this ratio and the fold-purification, or the yield of enzyme, achieved during this step. In fact, in the case of placental agalactosidase8 the amount of protein applied was 113 times as much as the lectin protein available for binding, yet the purification was 166-fold with a 150% yield (due, it is suggested, to removal of an inhibitor). At the opposite extreme, in the case of Vglutamyltransferase52 almost twice as much ConA-Sepharose protein was available as was present in the sample of enzyme, yet the purification was slightly less (104x) than the above example. In the cases where the purification was least (a-mannosidase, 7× 58) and greatest (glycogen synthetase, 811 x53), the ratios of applied protein/lectin protein were similar, 2.0 and 3.7 respectively. The enzyme activity recovered in each case was also similar, 87% and 70%, which are values in the middle of the overall range of recovered activities shown in Table 2. It is likely that several factors participate in determining the effectiveness of the lectin columns, for example: (a) the glycose content of the applied sample rather than, or as well as, the protein content; (b) the tissue source of the sample being purified; and (c) the degree of purification achieved up to the point of using the lectin column. The lectin used for all the isolations in Table 2 was ConA, covalently attached to Sepharose. The elution conditions varied considerably; citrate, phosphate, acetate and Tris buffers have been used, from pH 6.0 to 7.0, occasionally in presence of as much as 1 M Sodium chloride, and sometimes included the transition metal cation (Mn 2÷) required for Ca 2÷ binding, as well as the Ca 2+ required for glycose binding to concanavalin A al. Both Me-a-Glc and Me-aMan have been used for enzyme elution, at 47

OO

Arylsulfatase B

Arylsulfatase A

14.5 ml; 232 mg (calc.) 3.5 ml, 28.1 mg (calc.) I0 ml, 160 mg

Sheep brain; ethanol and salt fractionation; 905 ml, 1640 mg Rabbit kidney; DEAESephadex chromatography; 20 ml, 30.5 mg Sheep brain; ethanol, s a l t and pH 5 f r a c t i o n a t i o n ; 465 ml, 651 mg

20 mM Acetate, pH 6-I M NACI520 mM Me-a-Glc, 28°C

20 mM T r i s - C l , pH 7.4-150 mM NaCi-I mM MnCI=, CaCI=-600 mM Me-a-Man, 20-25°C

20 mM T r i s - a c e t a t e , pH 7.4520 mM Me-a-Glc, 28°C

50 mM Phosphate, pH 7.0-100 mM NaCI-520 mM Me-e-Glc, 20°C

50 ml, 400 mg

Human l i v e r ; centrifuged homogenate; l l , l l l mg (calc.)

I0 mM Phosphate, pH 7.0-500 mM Me-a-Man, 25°C

I00 mM Me-a-Man (probably in column-equilibrating buffer: 20 mM phosphate, pH 7.0-2 mM MgCI2, MnCI=-I M NaCI; see Reference (2]))

120 ml, 960 mg (calc.)

Human placenta; centrifuged homogenate; 280,000 mg, by repeated applications and desorptions

50 mM Phosphate, pH 7.0-500 mM Me-a-Glc, room temperature

50 mM C i t r a t e , pH 6.0-500 mM Me-a-Man

EluentL Temperature

Human kidney; heat-treated 44.2 ml, 380 mg and centrifuged homogen(calc.) ate; 10,375 ml, 35,275 mg ( c a l c . ) by 5 repeated a p p l i cations and desorptions

5 ml, 35 mg (calc.)

Human urine; DEAESephadex chromatography; 31 ml, 71 mg

B-N-Acetylhexosaminidase TA and B)

lO ml, 28 mg

a-N-Acetylgalactosaminidase

Bed Volume, Amount of Lectin

Porcine l i v e r ; s a l t f r a c t i o n a t i o n and DEAEc e l l u l o s e chromatography; 24 ml, 349 mg

Enzyme

Enzyme P u r i f i c a t i o n Using Concanavalin A-Sepharose Column Chromatography

Tissue Source; Pretreatment; ml and mg Applied

Table 2.

e/e x

42. 16.1

x

63.

x

9.2

4.65 mg 62. % 67.2 x

mg

5~.0

%

x

I00. ,

mg %

8.7

59.

347.5 mg ( c a l c . ) 73.7 % 24.1 x

1597. mg ( c a l c . ) 60. e,/e 13.3 x

%

57.

mg

x

13.7 2650.

%

62.5

3.15 mg

mg

9

Protein Eluted, ing; Activity Recovered,% ; Purification, Fold

(24)

(25)

(24)

(14)

(13); See also (21)

(12)

(ll)

(9)

Reference

Human l i v e r ; centrifuged homogenate; 3190 ml, 39~237 mg Human placenta; salt fractionation; 300 ml, 900 mg (calc.) Rat kidney cortex; acetonep r e c i p i t a t e d and heattreated; I0 mg Human polymorphonuclear leukocytes; centrifuged homogenate; 20 ml, 818.7 mg (calc.) Human l i v e r ; salt fractionation; 2495 mg (calc.)

B-Glucocerebrosidase (B-G1ucosidase)

y-Glutamyltransferase

Glycogen synthetase (D form)

~-Mannosidase

157 ml, 1256 mg (calc.)

28 ml, 224 mg

1.9 ml, !5.4 mg

50 ml, 400 mg

353 ml, 2826 mg

800 ml, 6400 mg

Human placenta; centrifuged, homogenate; ~2000 ml, 720,000 mg

B-Galactosidase A and B

10 ml, 80 mg

Human l i v e r ; centrifuged homogenate; -1650 ml, -5000 mg ( c a l c . )

3.5 ml, 28.1 mg (calc.)

Bed Volume, Amount o f Lectin

~-Galactosidase A and B (Ceramidetrihexosidase) {B form =~-N-Acetylgalactosaminidase?)

Tissue Source; Pretreatment; ml and m~ A p p l i e d Rabbit kidney; DEAEc e l l u l o s e and CMSephadex chromatography; 5.4 mg

(Continued)

Arylsulfatase B ( c o n t ' d . )

Enzyme

Table 2.

I0 mM Phosphate, pH 6.8-500 mM NaC1-O.l mM MgCI2, CaCI2, MnCI2-500 mM Me-e-Man, 18°C

50 mM T r i s - C l , pH 7.4-I mM DTT-500 mM NaCI-I mM CaCI~, MgCI2, MnCI2-100 mM Me-m-Man

M Me-~-.Man (elution using 1 M Man was only " v a r i a b l y successful")

50 mM C i t r a t e , pH 6.0-50 mM NACI0.02% azide-O.5% taurocholate5 mM MnCIT-5 mM CaCI2-100 mM Me-m-Man, room temperature

lO mM Phosphate, pH 7.0-500 mM NaCI-750 mM Me-m-Man, room temperature

25 mM Phosphate, pH 6.5-I M NaCI-200 mM Me-m-Man, 22°C

50 mM Phosphate, pH 7.0-500 mM NaCI-I M Me-m-Glc

20 mM Tris-Cl, pH 7.4-150 mM NaCl-I mM MnCl2, CaCl2-500 mM Me-s-Man, 20-25°C

E1uent, Temperature

mg

mg % x

mg % x (calc.)

319.9 mg 87. % 6.9 x

0.71 mg 70. % 811. x

0.08 mg 83. 104. x

97.5 mg (calc.) 80. % 7.7 x

522. mg 56. % 41.7 x

7100. 150. 166.

35. 75. 12.

71, % 36.6 x

0.I

Protein Eluted, mg; Activity Recovered, %; P u r i f i c a t i o n , Fold

(58); See also (33)

(53)

(52)

(44); See also (33,45,80)

(34)

(8)

(7); See also (33)

(25)

Reference

concentrations from 100 rr~ to 1 M. The membrane-bound enzyme, /3glucocerebrosidase, was purified in presence of the detergent, 0.5% taurocholate. Most of the columns were run at room temperature or somewhat lower, although in a few cases the temperature was not specified. These parameters will be discussed in detail later.

Survey o[ Additional Lectin-Binding Studies Table 3 lists proteins which have been submitted to lectin-binding studies, the primary purpose of most of which was to examine the characteristics of the binding reaction itself, or the use of it to help characterize or to differentiate among various proteins or their different forms. The results are briefly described under the heading "Remarks"; in cases where they contribute to a better understanding of the characteristics of the binding reaction, they are discussed more fully in later sections of the text.

Glycoside Specificities of Lectins The specificity of lectins for the sugars which they bind is not absolute; lectins associate preferentially with certain glycose ligands however, in the same way that enzymes act best on preferred substrates. The most quantitative understanding of these associations has resulted from equilibrium dialysis experiments, and from studies of hemagglutination inhibition, using simple glycoses or methyl glycosides, or glycoprotein oligosaccharides of known structure. It is beyond the scope of this Review to discuss all the evidence bearing on the specificities of the commercially available lectins; instead, the resuits of a few relevant studies will be summarized below. Certain more complex oligosaccharides have a far greater ability to inhibit ConA-iriduced hemagglutination, or to bind to the lectin, than the simple ligand Me-~t-ManS2-86., For example, ovalbumin glycopeptide with an apparent association constant for ConA of 2.5 × 10 6 is 178× more inhibitory in a hemagglutination system than Me-a-Man (association constant 1.4 × 104)83. These and other findings support 50

the idea that ConA recognizes a "mannobiosylN-acetylglucosamine" structure. It is not necessary that mannose be terminal since the addition of N-acetylglucosamine, galactose and Nacetylneuraminic acid distal to mannose do not destroy the binding activity of immunoglobulin glycopeptides to ConA s4 (see also82 and aT). In fact, "the presence of terminal Nacetylglucosamine residues linked/31, 2 to the underlying mannose contributes greatly to the ability of these glycopeptides to interact with concanavalin A ''s4. The position of substitution of the mannose residues is important, as shown by the fact that "the presence of at least two t~-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be retained by a concanavalin A-Sepharose column ''s5 and that "neutral mannose-rich Nglycosidic glycopeptides containing several terminal and (2-O)-substituted a-mannose residues are strongly bound to the lectin ''s6"88. It may be noted that such glycopeptides seem to contain the common inner core of those carbohydrate chains bound to protein via asparagine linkage (reviewed inS9'9°), which may thus be the common factor relating those glycoproteins which bind to ConA. The fact that Me-t~-Glc is, like Me-t~-Man, an effective ligand for displacing bound glycoproteins from ConA is presumably related to similar conformational features in both and has "led to the hypothesis that it is the hydroxyl groups at C-3, C-4 and C-6 of the a-Dglucopyranosyl and ,~-D-mannopyranosyl ring forms which bind" to the ConA 91. WGA, like ConA, interacts more strongly with macromolecular glycosides than with its preferred monosaccharide ligand,/3-Nacetylglucosaminide92"93. The ability of a series of per N-acetylated chitodextrins to inhibit WGA-induced hemagglutination indicated that "the wheat germ agglutinin binding site [for optimum binding] is complementary to a sequence of three/3-(1--~ 4)-linked N-acetyl-Dglucosamine units (N,N',N"-triacetyl chitotriose ''93. However, results of equilibrium dialysis experiments with WGA, Nacetylneuraminic acid and N-acetylglucosamine led to the proposal "that the initial step in the agglutination of cells by WGA is the nonspecific binding of the lectin to sialic acid residues on the cell surface," possibly orienting the lectin

Source Human brain e x t r a c t , and other tissues

Sheep b r a i n , soluble f r a c t i o n from lysosomes

Human l i v e r , kidney and i n t e s t i n e extracts

Enzyme

~-N-Acetylhexosaminidases A,B,C, and S

Acid phosphatase

Adenosine deaminase

Table 3.

During a p p l i c a t i o n of sample, 500 mM NaCI was included "to minimize e l e c t r o s t a t i c i n t e r a c t i o n s between ConA and other p r o t e i n s . " Optimum pH f o r desorption from column was pH B.O (42% release) using 580 mM Me-~-Glc or 130 mM Me-a-Man. Recovery was greater when "the sugars were kept in contact with the column loaded with the enzymes f o r a long time." When column e f f i c i e n c y dropped, i t could be a c t i v a t e d by passing over i t a s o l u t i o n of 1 mM CaCI= and 1 mM MnCI: and washing out excess metal ions.

Varied, but included 50 mM b u f f e r , 4°C

Forms a and d 2 adsorbed to both ConA and WGA, d~ form to ConA only. Neuraminidase does not a f f e c t binding of d2 to WGA,

(17)

(zz)

(zo)

Ref. were eluted at about w/v) Me-a-Man or about w/v) Me-~-Glc. Hex S but not Hex C.

Rcmarks Hex A and B 206 mM (4%, 412 mM (8~, also bound,

I 0 0 mM Acetate, pH 6 . 0 - I M NaCI-IO mM MnCl:-lO mM MgCl~-lO mM CaCl~-gradient of 0 to 520 mM ( I 0 ~ , w/v) Me-~-Man or Me-a-Glc, 25°C

Dissociating Conditions

ConA-Sepharose, WGAFor WGA, 50 mM phosphate, Sepharose; up to 0.5 pH 7.0-200 mM NACI-452 ml of a l : l (w/v) mM (10%, w/v) GIcNAc e x t r a c t applied w i t h out overloading to column containing 15 mg WGA

ConA-Sepharose, I ml ( c a l e . ) containing 6 mg ConA; 1.8 mg protein applied

ConA-Sepharose

Adsorbent

A. Studies of the Binding of Enzymes to Lectins

Source Dictyostelium --d~coid-euTplasma membranes solubilized by D0C

Sheep brain, soluble fraction from lysosomes Ox l i v e r extract, after several purification steps

Snake venom

Alkaline phosphatase

Arylsulf~tase A

Arylsulfatase B (BI~ and BIB) form

Exonuclease (Pnosphodiesterase I)

A. (continued)

Enzyme

Table 3. Dissociatin 9 Conditions

ConA-Sepharose

ConA-Sepharose

ConA-Sepharose, I ml (calc.) containing 6 mg ConA; 1.8 mg protein applied

200 mMAcetate, pH 6-I00 mM Me-o-Man

Linear gradient from 20 mM Tris, pH 7.4-I00 mM NaClI mM MnCIz, CaCl2 ( s t a r t ing buffer), to starting buffer plus 400 mMMe-B(~?)Man, at room temperature

Varied, but contained 50 mM buffer, 4°C

lO mM Tris-Cl, pH 7.5-0.2% ConA-Sepharose (a 15fold excess of lectin DOC-IO0 mM Me-e-Glc, 4°C was used over amount of protein applied)

Adsorbent

(28)

(31)

Step gave lO-fold increase in specific a c t i v i t y , 80% recovery. BI~ and BI~ forms s l i g h t l y resolved.

About 80% of the exonuclease could not be eluted by up to 500 mM Me-o-Man, once bound. (Venom 5'nucleotidase was also irreversibly bound). Immobilized exonuclease was stable for at least a month at room temperature.

(20) Desorption was pH-dependent, optimal at pH 8.0. 85% of enzyme was released using 500 mM Me-e-Glc at this pH, none was eluted at pH 5.0.

Ref, (23)

Remarks

Enzyme from log Rhase cells was t o t a l l y bound, enzyme from mature cells was not, The enzymes3':5 'cyclic-nucleotide phosphodiesterase and 5'-nucleotidase behaved similarly.

Ln

~-Galactosidase A and B bound to ConA (35) Sepharose, p a r t i a l l y bound to WGAand negligibly bound to LTA-Agarose; neutral B-galactosidase not bound to ConA-Sepharose. B-Galactosidase A and B not eluted by Me-a-Man at 2°C.

lO mM Phosphate, pH 7.0-I0 mM NaCl, plus (a) 750 mM Me-a-Man or (b) 750 mM DGlcNAc or (c) 750 mM LFucose, at 22°C

Bound enzyme was not eluted by l M (46) I0 mM Acetate, pH 5.0-500 NaCI or by increasing the pH to 7.5, mM NaCl-500 mM Me-:-Man, or by decreasing the pH to 5.0 and 50% ( v / v ) ethylene g l y c o l adding 500 mMMe-a-Man to the buffer, s t a b i l i z e r s (lO mM mercaptobut specifically required the ethanol-5 mM EDTA-O.2% (w/v) presence of ethylene glycol for Cutscum) elution. Enzymerecovery 85%, 30x purification.

ConA: lO mM Phosphate, pH 6.0-I M NaCI-500 mM Me-a-Man RCA: I0 mM Phosphate, pH 6.0-150 mM NaCllO mM Gal

lO0 mM T r i s - C l , pH 8.1-10 mM CaCl2-1 mM Mg(OAc)2520 mM (lO%w/v) Me-a-Man

(a) ConA-Sepharose; (b) WGA-Agarose; (c) LTA-Agarose

ConA-Sepharose

ConA-Sepharose

ConA-Sepharose and RCA~20-Sepharose

ConA-Sepharose

Human l i v e r e x t r a c t

Human l i v e r enzyme, highly p u r i f i e d

Rat l i v e r and small intestine extracts

Human urine concentrate, and secretions from I - c e l l disease fibroblasts

Porcine pancreas, a f t e r gel f i l t r a tion and ion exchange chromgraphy

~-Galactosidase A and B

~-Glucocerebrosidase (B-Glucosidase)

y-Glutamyltransferase

a-lduronidase

Lipase

(56)

(57)

Both the l - c e l l c o r r e c t i v e and nonc o r r e c t i v e forms o f the u r i n a r y enzyme bound to ConA. The corr e c t i v e form bound well tb RCA, but the noncorrective form bound only s l i g h t l y , a-lduronidase isolated from I - c e l l fibroblast secretions bound almost quantitatively to RCA. Both Me-a-Glc and sucrose were less effective at displacing lipase from ConA, even at 40~ concentration ('2 M for Me-a-Glc).

50); ee also (51,52)

(39); See ~ so (42)

Neutral B-galactosidase, which has B-glucosidase a c t i v i t y (and may be equivalent to ~-xylosidase?), does not bind to ConA-Sepharose.

750 mM Me-a-Man, room temperature

ConA-Sepharose

Human l i v e r extract

B-Galactosidase (acid form)

50 mMTris-Cl, pH 7.5-500 mM The adult form of the enzyme bound to ConA-Sepharose, but the fetal form NaCl-l mMMgCI2-MnCI2-CaCI2did not, until treated with neura4 mMazide-0.5~ (w/v) T r i minidase. ton x-lO0-200 mMMe-a-Man, 4°C

(40); See also (38)

Adsorption and elution profiles similar to those for the enzyme from normal tissue, although enzyme from one I-cell case showed almost a 40%-decreased binding.

lO mM Phosphate, pH 7.0500 mM NaCI-700 mM Me-~-Man, 22°C

Ref.

Remarks

Dissociating Conditions

Adsorbent ConA-Sepharose; 47193 mg protein in centrifuged homogenate applied to 2 ml adsorbent containing 16 mg ConA

Source Extract from (human) I - c e l l disease liver

....

A. (continued)

B-Galactosidase A and B (I-cell enzyme)

Enzyme

Table 3.

Human serum, after salt fractionation and ion-exchange chromatography Liver-metastasizing colon carcinoma

Ceruloplasmin from goat serum, fetuin from fetal c a l f serum Human fetal (cord) blood, after ionexchange chromatography and gel filtration; 290 mg applied Rat brain synaptic membrane solubilized in lO mM phosphate~ pH 7.45-I Triton X-lO0 Human serum

Carcinoembryonic antigen

Ceruloplasmin and Fetuin, enzymatically desialylated

~-Fetoprotein

Glutamate-binding protein

Heinepexi n (heine-binding proteinl WGA-Sepharose, 5 mg lectin per mI Sepharose

ConA-Sepharose

ConA-Sepharose; 80 ml, 640 mg ConA

RCA~20-Sepharose, I0 mg lectin per ml Sepharose

ConA-Sepharose

C~nA-Sepharose

Adsorbent

2.8 mg Protein eluted containing 650 ~g ~-fetoprotein. Purification I06x, 59% recovery.

The f u l l y sialylated proteins do.not bind; 180 ug asialoceruloplasmin binds per mg of RCA-Sepharose, 82 ug asialofetuin per mg.

89% Recovery from ConA-Sepharose column with 8.4x purification. After i n i t i a l elution and a 20hour wait, additional a c t i v i t y was recovered from the column.

80% Recovery from ConA-Sepharose column, 4.3x purification.

Remarks

50 mM Phosphate, pH 7.0-200 mM NaCI-O.02% azide-452 mM GIcNAc

223 mg Protein passed over 9 ml column (two applications and desQrptions were necessary); 33 mg eluted with 91% recovery and 6.3x purification (capacity of lectin-gel about 2 mg hemopexin per 5 mg lectin). Transferrin did n~t hind_

lO mM Phosphate, pH 7.45-0.5% Recovery from the column was 92% Triton X-100-0.5 mM CaCI2with 1.6x purification. 139 mM D-Man-130 mMMe-~-Man, o-a':c

104 mM Me-e-Glc

50 mM Phosphate, pH 6.8-200 mMNaCl-100 ~M lactose, 25°C

200.mM Citrate, pH 6.5-I00 mMMe-o-Man

50 mM Phosphate, pH 7.6-100 mMMe-e-Glc, 4~C

Dissociatin 9 Conditions

B. Studies of the Binding of Serum and Membrane GIycoproteins to Lectins

~1-Antitrypsin

Table 3. Source

Protein

(72)

(71)

(69)

(68); See also (70)

(65); See also (64)

(6s)

Ref.

IM] (malignancy) tissue culture cell line membranes, solubilized with DOC Rat fat cell membranes, solubilized with Triton X-lOg

Human fibroblasts

Bovine retinas, rod outer segments

HL-A Antigens HL-A 3, 12 and W27

Insulin receptor

Interferon

Rhodopsin

Human chorionic gonadotropin Human f o l l i c l e stimulating hormone Human lutenizing hormone Human growth hormone

Source Human fluids

continued)

Protein

B.

Hormones

Table 3.

Ref. (73)

(74)

(75)

(76); See also (77)

(78); See also (79)

Remarks All hormones except growth hormone bound to ConA-Sepharose and were eluted with 200 mM Me-~-Glc. Asialo-HCG required 1M Me-~-GIc for elution, but asialoagalactoHCG could not be eluted at a l l , even with 1 M EDTA. HCG could be purified 3.2x with 85-99% recovery. The antigens could be eluted from LCA with at least an 8x increase in specific a c t i v i t y . Antigens could not be eluted from ConAcolumns "even after extensive elution with glucopyranosidecontaining buffers." Binding protein purified 300 x with 90% recovery.

The requirement of ethylene glycol is interpreted to mean that binding in this case is due not only to carbohydrate Recognition. but also to hydrophobic interaction. Interferon Gould be purified 750x. Other detergents are also satisfactory for eluent: 3% dodecyltrimethylammonium bromide, I% Triton X-lO0, I% lauryl dimethylamine oxide. Periodate-oxidized rhodopsin does not bind to ConA.

200 mM or I M Me-~-Glc

5 mMTris-Cl, pH 8:0-150 mM NaCl-O.05% DOC-520 mM Me-~-Glc

lO0 mM Bicarbonate, pH 8.4-0.1% Triton X-lO00.1% albumin containing either (for ConA) 300 mMMe-~-Glc or (for WGA) 300 mM GlcNAc, 24°C 20 mM Phosphate, pH 7.4-I M NaCl-lO0 mMMe-s-Man50% (v/v) ethylene glycol

50 mM Acetate, pH 5.0-I.4% cetyltrimethylammonium bromide-I ~ CaClv, MnClz-lO0 mM D-Glc, 23°C

ConA-Sepharose

ConA-Sepharose

(a} (b)

ConA-Sepharose WGA-Sepharose

LCA-Sepharose

ConA-Sepharose

Dissociatin 9 Conditions

Adsorbent

"to bring the N-acetylglucosamine binding site into close proximity to molecules on the cell membrane that contain N-acetylglucosamine ''94. In summary, it appears that W G A interacts with N-acetylglucosamine specifically and with Nacetylneuraminic acid nonspecifically2z. R C A has been isolated in two forms separable on the basis of their molecular weights, which are approximately 60,000 and 120,00095. RCA6o hemagglutination is inhibited slightly better by N-acetylgalactosamine than by /3(1--* 4)-linked galactose 95; RCAlz0, on the other hand, appears to be considerably more specific for/3 (1--~ 4)-linked galactosides than for any other glycosides tested 95-9a. N-Acetylneuraminic acid linked distal to/3-galactose in oligosaccharides diminished their ability to inhibit RCA-induced hemagglutination 97'99. The best inhibitors of LTA-induced hemagglutination were found to be methyl-a-Lfucopyranoside and a difucosyl oligosaccharide bearing the H blood grouping, which contained fucose linked a (1--~ 2) to/3-galactoside and o~(1---~3) to/3-N-acetylglucosaminide 1°°. LCA-induced hemagglutination was inhibited most potently by "glycopeptides having a branched oligosaccharide structure with Nacetylglucosamine residues in the outer branches and mannose residues in the core"l°1; treatment with/3-N-acetylglucosaminidase markedly diminished the inhibitory capactity 87"1°°. If galactose was linked/3(1-->4) to the Nacetylglucosamine, the inhibitory activity was also impaired, but not if the linkage was /3(1 --* 6) TM. Once again, oligosaccharides associate more strongly than do the simple glycose ligands; the apparent association constant for LCA with IgM glycopeptide was 9.2 × 104, whereas that for LCA with mannose was 2.3 × 102 83, and these authors remark on the apparent requirement of an "Nacetylglucosaminylmannobiose" structure for binding to LCA in contrast to the "mannobiosyl-N-acetylglucosamine" requirement of ConA. Other lectins now also commercially available, and the glycosides which appear to associate preferentially with them, are: soybean agglutinin, terminal or- or/3-Nacetylgalactosaminidesl°2'l°3; peanut agglutinin, terminal/3-galactosidesl°4; Dolichos biflorus agglutinin, terminal a-N56

acetylgalactosaminidesl°5; and Bandeirea simplicifolia agglutinin, terminal a-galactoside 1°6"1°7. A better understanding of the binding preferences of these lectins, as well as an increasing number of those less well characterized, should assist greatly in selecting from among them lectins for a particular purpose such as glycoprotein purification, especially if something is known of the structure of its oligosaccharide moiety. It is also clear that interactions (or lack of them) between the glycoprotein under study, and a given lectin, may allow inferences to be drawn about the composition or structure of the glycose portion of the glycoprotein, if enough is known about the specificity of the lectin.

Factors Affecting Giycoprotein Binding by Lectins Several variables appear to be important in the binding of glycoproteins to lectins and in their elution by glycosides: temperature; time of contact between the lectin and the glycoprotein on the one hand, and between the complex and the eluting glycoside on the other; pH; ionic strength; and concentration of competing glycoside. In addition, there is the question of what stabilizers of the lectin or glycoprotein, or glycoprotein solubilizers, may or should be present.

1. Capacity of adsorbent Use of adsorption techniques in isolation studies also raises the question of the capacity of the adsorbent. In the case of insolubilized lectins, this aspect has not been extensively studied per se, although the data summarized in Table 2 is relevant; only in a few cases is it known how many mg of the purified glycoprotein will bind, under defined conditions, to known amounts of the lectin. For example, 180/~g of asialoceruloplasmin or 82/~g of asialofetuin bind to 1 mg of RCA12o linked to Sepharose 68, and 400 t~g of hemopexin (serum heme-binding protein) bind to a volume of WGA-Sepharose which contains I mg of W G A 72. Two groups have estimated t h a t 7% 23 o r 2 0 % 27 of their tissue extract would be glycoprotein, and calculated the amount of lectin to be used in the column accordingly. The best method would clearly be to examine the degree of binding between

preparation and lectin in a small scale experiment before running the full scale isolation, and in cases where the binding is important to structural arguments, to re-apply any nonadsorbed material to a second identical column to make sure the binding capacity of the first was not exceeded.

2. Temperature dependence of binding It is said that the rate of dissociation of e n z y m e - C o n A complexes is slow at low temperatures 2°. This may account for the fact that /3-galactosidase was not eluted from its complex with ConA-Sepharose by 750 mM M e - a - M a n at 2 °C, but was eluted quantitatively at 22 ° 35; and other workers 2° have stated that "recovery of the [lysosomal] enzymes from the columns was greater when the ConA-specific sugars were kept in contact with the column loaded with the enzymes for a long time" (in this case, one hour at 4°C). In a study of the temperaturedependence of the rates of association and dissociation of/3-N-ace0ylhexosaminidase with ConA-Sepharose, it was shown that in 100 mM phosphate, p H 7.0, about 7 hours was required for complete binding of the enzyme at 4°C, whereas at 25 ° and 37 ° enzyme was bound more rapidly; at 25 ° binding was nearly complete between 2 and 3 hours 18. Furthermore, it was reported that after it had been bound, elution of the enzyme from lectin in presence of 500 mM M e - a - M a n was even more temperaturedependent than the binding step was. After 2 hours at 4°C only 20% of the enzyme had been released, and this amount was not increased after 5 more hours. On the other hand, after 4 hours at 37 ° almost 90% of the enzyme was eluted. At 25 ° the elution rate and eventual limit of elution were intermediate between those at 37 ° and 4 ° . Another group found that when [3H]fetuin or asialo[3H]fetuin was added to a series of lectin-Sepharose columns at different temperatures, binding to SBA, P N A and W G A over a 10-minute period was greater at 4 ° than at 23 °, whereas in the case of C o n A the greater binding occurred at 23°; for RCA120, binding was similar at both temperatures 7°. From a practical point of view, since recoveries from lectin columns have generally been fairly good (Tables 2 and 3), it seems likely that slow, continuous sample addition to, and elution from, columns will give satisfactory

yields over the conventional temperature range (2°C to room temperature). It is important, in any event, not to disregard time and temperature in experiments involving lectin-glycoprotein interactions.

3. Effect of pH The p H dependence of binding between lysosomal enzymes and ConA, as well as other factors influencing the association, have been studied in several papers by Bishayee, Bachhawat and their co-workers 15"2°'48. These workers measured the amount of enzyme precipitated by C o n A under different conditions, and showed that the optimum p H for precipitation of all enzymes tested (acid phosphatase, arylsulfatase A, /3-glucuronidase, /3-galactosidase and /3-N-acetylhexosaminidase) was p H 5.0 in 50 mM acetate-500 m ~ NaC115. In a study of the elution of ConA-Sepharose-bound hydrolases, in the presence of 50 mM buffer-500 mM M e - a Glc, it was found that none of the bound form of arylsulfatase A was released at p H 5.0 whereas 85% was released at p H 8.0. Values for other enzymes were: acid phosphatase, 34% at p H 5.0, 42% at p H 8.0;/3-glucuronidase, 0% at p H 5.0, 4 0 - 4 5 % at p H 8 . 0 ; / 3 - N acetylhexosaminidase, 13% at p H 5.0, 32% at p H 8.02°. The authors speculate that this indicates the presence of at least two types of isoenzymes of acid phosphatase a n d / 3 - N acetylhexosaminidase, differing in their susceptibility to elution at this p H 48. H e r e again, judging from the acceptable recoveries observed in most cases where purifications were run at around neutral pH, slow continuous elution between p H 6 and p H 8 appears likely to give satisfactory results.

4. Ionic strength effects Elevated ionic strength (usually in the form of high concentrations of NaC1) has often been utilized in purification steps involving formation and dissolution of lectin-glycoprotein complexes. One group of workers has stated that "during the application of sample to the columns, 0.5 M NaC1 was included in order to minimize the electrostatic interactions between C o n A and other proteins ''2°. In other cases as well, high NaC1 concentrations were present during application of the sample to the column and during washing prior to elution with 57

glycoside: /3-N-acetylhexosaminidase12"13'17"19; /3-galactosidaseT'35;/3-galactosidase A36;/3glucocerebrosidase46; ~/-glutamyltransferaseS°; glycogen synthetaseS3; ~t-mannosidaseSS; and a group of other lysosomal hydrolases2°. The presence of NaC1 may actually enhance the binding of some glycoproteins; it has been noted that the presence of 200 mM NaCI caused a 3-fold increase in the formation rate constant of a complex between ConA and Ricinus communis agglutinin (itself a glycoprotein)~°8, and that the presence of NaCI in the buffer more than doubled the binding efficiency of fetuin to ConA- and WGA-Sepharose, and more than tripled its binding to SBA- and PNA-Sepharose 7°. These findings are consistent with the idea that the reaction between lectins and glycoproteins is partially ionic in character (as well as hydrophobic, as will be mentioned later), and that larger steric interactions occur during the association than merely recognition of a carbohydrate moiety. However, NaC1 also plays a role in the elution of glycoproteins from lectins. Examination of Tables 2 and 3 will show numerous instances in which 500 mM or 1 M NaCI was included in the medium used to elute enzymes and other proteins from lectin columns. In fact, addition of 1 MNaC1 to the solution of buffer and glycoside was required for elution of hyaluronidase from ConA-Sepharose54; and while in the absence of NaC1, 100 mM Me-aGlc released only 5% of the enzyme from an arylsulfatase A-ConA complex at pH 5, in the presence of 500 rnM NaC1 almost 90% of the enzyme was freed 15. On the other hand, in the same studya5 it was shown that the presence of NaC1 did not affect the already high rate of release of acid phosphatase at pH 5 from a complex with ConA. In summary, it therefore seems likely that inclusion of NaCI during adsorption and elution could be of general advantage in suppressing unwanted non-specific binding to lectins during isolation steps or glycoprotein binding studies, and may be a useful adjuvant during elution.

5. Concentration requirements for glycoside There appears to be no definite rule as to the concentration of glycoside that will be effective in desorbing a glycoprotein from its complex with lectin; for example, the concentration of 58

Me-a-Man used to elute enzymes from ConASepharose columns has varied 20-fold, from 50 naM for galactosyl transferase43 to 1 ~ for ~/-glutamyltransferase52, a-galactosidase 33 and a-glucosidase33, although it is not clear that such a high concentration was actually necessary to elute the enzyme. Gradient elution of glycoproteins from lectin columns should be extremely helpful in determining affinities of the protein for the lectin, or for helping to distinguish among several forms of an enzyme; unfortunately, few such results have been reported. In the two cases in which enzymes were eluted using gradients,/3-N-acetylhexosaminidase A and B were eluted from ConA-Sepharose at about 206 mM Me-t~-Man (but required about twice this concentration of Me-a-Glc for elution) 17, and arylsulfatase Bl,v and B1/3 were eluted after 50 ml of a 150 ml linear gradient to 400 n ~ Me-/3(a?)-Man 2s, which should correspond to about 130 naM. A separation of rat brain glycopeptides was accomplished on a ConA-Sepharose column by using a concentration gradient of Me-a-Glc, eluting two glycopeptide fractions at about 15 n'~ and 100 naM Me-,~-Glc; the more tightly-bound fraction was richer in mannose content 1°9. Similar findings were reported for carcinoembryonic antigen64 which was eluted from ConASepharose in a Me-a-Man gradient in three peaks, suggested to be molecular variants, at about 60 rnu, 150 m~a and 300 naM Me-a-Man. Aside from these examples, only two other studies have been noted which report the minimum concentration of glycoside required under a given set of conditions for dissociation of glycoprotein-lectin complexes: /3-Nacetylhexosaminidase was said to be maximally released from ConA-Sepharose, at 25 °C, at a concentration of 300 mM Me-a-Mania; while in another study, this time at 4 °C and in the presence of 500 ~ NaC1,/3-Nacetylhexosaminidase as well as arylsulfatase A, acid phosphatase and/3-glucuronidase, were optimally eluted at 130 mM Me-a-Man or 500 InM Me-a-Glc 2°. Finally, it was found that Me-ot-Glc at a concentration of 30 rnM, in 500 rn~ NaC1 and at 37°, inhibited completely the formation of insoluble complexes between ConA and arylsulfatase A or/3-Nacetylhexosaminidase or acid phosphatase 15. In view of the fact that a number of these

studies 1sA7,xS'2°'2s, as well as still others also utilizing relatively low concentrations of glycosides'44"47, were performed near room temperature, and since glycoside-induced dissociation of lectin-enzyme complexes is sometimes slower at low temperatures ~s'2°,35, it can be speculated that at room temperature, as a general rule, elution could occur to a greater extent at lower glycoside concentrations than would be required at low temperatures. The reason why Me-a-Man is more effective than Me-a-Glc at dissociating enzyme-ConA complexes is doubtless due to the fact that the association constant of ConA for Me-a-Man is almost 5× that for Me-a-Glc ~°, hence Me-aMan will dissociate glycoprotein-ConA complexes at lower concentrations than Me-a-Glc. (Although it does not help to define the concentration of glycoside that will be required to dissociate lectin-complexed glycoproteins, it is nevertheless worth noting that the stability of arylsulfatase A- and acid phosphatase-ConA complexes is higher by 3 to 4 orders of magnitude than that of the Me-a-Glc-ConA complex 15.) The effect of pH on the concentration of glycoside required for elution of acid phosphatase and arylsulfatase A from their ConA complexes has been examined 15 and the results show a considerable pH dependence. For example, at pH 4.0 the acid phosphatase complex is 50% dissociated at about 20 mM Me-a-Glc, while at pH 6.0 almost 200 rnM Me-a-Glc is required to reach this extent of dissociation; arylsulfatase A is 50% dissociated at about 250 mM Me-a-Glc at pH 6.0, but at pH 9.0 is one-half dissociated at about 10 mM Me-a-Glc. Finally, it should be recalled that the concentrations of NaC1 and glycoside required for dissociation of arylsulfatase A-ConA complexes are also interdependent 15. It is clear from the results discussed above that the concentration of ligand required for dissociation of glycoprotein-lectin complexes is a function of a number of other variables, which must be taken into consideration when devising protocols for studying lectin binding.

6. Other modifiers of lectin binding Various substances have been present during lectin binding studies or enzyme purifications for a variety of reasons. The cations Ca 2+ and Mn 2+ have often been added as their chloride

salts, usually at a concentration of 1 to 2 raM, in order to preserve the glycoside-binding ability of ConA; Mg z+ has also been added, but presumably to no effect since it cannot substitute for the Ca 2÷ or the transition metal (Mn2÷) requirements of ConA sl. (SBA and LCA also require metals for activity~.) EDTA was present in two cases 33"46, and if it assisted in the dissociation of the glycoprotein-lectin complex, presumably did so by removing the required cations. The thiol-stabilizing agents dithiothreitol and mercaptoethanol were added to preparations of glycogen synthetase5s and/3glucocerebrosidase46, respectively, and an antibacterial agent (azide) was included in other cases 44"5°'72. Detergents were important components of the eluting media for a number of membrane-derived proteins, and included deoxycholate, taurocholate, Tritons X-100 and WR1339, Cutscum and cetyltrimethylammonium bromide (see Tables 2 and 3). It may be advisable, however, to keep detergent concentrations as low as possible; it has been reported that the binding of fetuin and asialofetuin is not substantially affected by nonionic detergents, but cationic and zwitterionic detergents cause significant inhibition of ConAand SBA-Sepharose activities7°. An interesting (and essential) component of the eluent for/3-glucocerebrosidase46 was ethylene glycol, at a concentration of 50% (v/v). This requirement was similar to the requirement for hydrophobic solutes such as dioxane or propanediol to dissociate interferon from Conm 76'77, which has been cited as evidence for hydrophobic interactions between ConA and some proteins. Others 44 have found that a detergent (taurocholate) could also be used successfully to elute/3-glucocerebrosidase from ConA-Sepharose.

Stabilization of Lectin-lmmobilized Enzymes Evidence has been presented in a number of studies that enzymes are not only catalytically active in the presence of or bound to lectins, but may actually be more stable than in a free state./3-N-Acetylhexosaminidaseis, acid phosphatase is, arylsulfatase A 26, snake venon exonuclease31, /3-galactosidase15"111, /3glucuronidase is, glucose oxidase4s, 3'glutamyltransferases2 and hyaluronidase4s are 59

all enzymatically active after binding to ConA, ind.icating that their ConA-binding sites are not the same as their catalytic sites. In fact, the Km of/3-N-acetylhexosaminidase ~5, acid phosphatase x5 and arylsulfatase m 26 increased from twoto three-fold. Stabilization of the enzyme was noted for glucose oxidase 48, exonuclease and arylsulfatase A; bound exonuclease was still active after a month at room temperature 31, and the half-life of arylsulfatase A at 55°C increased from 5 minutes in the soluble form to several hours when bound 26. A detailed study of arylsulfatase A bound to ConA 26 showed the same anomalous time-activity relationship and the same kinetics of inhibition by pyrophosphate as for the soluble enzyme; the pH optimum was slightly displaced, but the activation energy was about the same. Hyaluronidase lost some activity upon binding by ConA 48, but instead of being due to a change in Kin, it was found that this was due to a diminished Vmax; the authors suggest that this might have occurred because access to the large molecular weight substrate was limited by the enzyme's binding to ConA.

Glycoprotein Nature of Proteins which Bind to Lectins The question has not yet been raised here regarding the importance of confirming by independent means that proteins associating tightly with lectins are actually glycoproteins. In some of the papers cited in this Review, the proteins, purified to electrophoretic homogeneity, did contain sugars: arylsulfatase A24; a galactosidase A and Bs; /3-glucuronidase49; /3N-acetylhexosaminidase A 11'12'16 and B~2; and lipase 57. In other cases, electrophoretic bands containing enzyme or other biological activity also stained positively for carbohydrate: arylsulfatase B27; ascorbate sulfatase29; a mannosidase59; and glutamate-binding protein 7~. The glycoprotein nature of still other enzymes and serum proteins is well known and has been covered in numerous reviews (see for example112-~5). Finally, a number of the enzymes or proteins listed in Table 1 have been isolated in pure form in other studies, albeit sometimes from different tissues or organisms, and in those studies shown or suggested to be glycoproteins; such cases have not been reviewed for this paper. 60

It was stated earlier that the inert insoluble support to which lectins could be attached for glycoprotein binding studies should itself have no specific affinity for the glycoprotein. Yet it must be pointed out that agarose, or Sepharose which is a bead-gelled agarose, the most frequently used supports, are derived from agar which may contain D-glucuronic acid and pyruvic acid, and that agarose itself is composed of the cross-linked disaccharide, 4-O-/3-Dgalactopyranosyl-3,6-anhydro-L-galactose, linked 1 ~ 3 to form the polysaccharide (reviewed in 116). The presence of galactose, or impurities such as glucuronic acid, in agarose could mean that this substance under some conditions might bind galactosidase, or other glycosidases, by virtue of its resemblance to their substrate. It should also be recalled that electrostatic and hydrophobic associations have been invoked as components of the binding interaction between lectins and glycoproteins. These associations, to the extent that they are nonspecific, could occur as well between lectins and nonglycosylated proteins, resulting iJ1 nonspecific binding under some conditions if not corrected.

"Endogenons" Lectins Finally, it is worth noting that a number of poteins with carbohydrate-binding specificities have been reported recently in a variety of mammalian and other vertebrate tissues; they have been comprehensively reviewed 117 particularly with reference to their possible role in intracellular and intercellular recognition processes during development.

Conclusion It is clear from the work reviewed here that the reversible binding of glycoproteins to lectins can be an important adjunct to their better understanding. While each glycoprotein or its oligosaccharide may present a slightly different problem in terms of optimum conditions for binding and release, the important variables are amenable to experimental examination and have already been profitably employed for purposes of purification; moreover, as practice grows in

selection and usage of lectins, their abilities to distinguish among various forms of microheterogeneous glycoproteins may become not merely a useful but an essential tool.

Acknowledgement While this review was being prepared, the author was supported in part by NIH grants NS-13513 and HD-10981.

References 1. 2. 3. 4. 5. 6.

7.

8. 9.

10. 11. 12. 13. 14.

15. 16. 17. 18. 19.

20.

Sharon, N. and Lis, H., 1972. Science 177, 949-959. Burger, M., 1973. Fed. Proc. 32, 91-101. Nicolson, G. L., 1974. Intl. Rev. Cytol. 39, 89-190. Lis, H. and Sharon, N., 1973. Ann. Rev. Biochern. 42, 541-574. Wenthold, R. J., Mahler, H. R. and Moore, W. J,, 1974. J. Neurochem. 22, 945-949. Uhlenbruck, G., Baldo, B. A., Steinhausen, G., Schwick, H. G., Chatterjee, B. P., Horejsi, V., Krajhanzl, A. and Kocourek, J., 1978. J. Clin. Chem. Clin. Biochern. 16, 19-23. Schram, A. W., Hamers, M. N., Brouwer-Kelder, B., Donker-Koopman, W. E. and Tager, J. M., 1977. Biochim. Biophys. Acta 482, 125-137. Kusiak, J. W., Quirk, J. M. and Brady, R. O., 1978. J, Biol. Chem. 253, 184-190. Sung, S.-S. J. and Sweeley, C. C., 1975. Current Trends in Sphingolipidoses and Allied Disorders (Volk, B. W. and Schneck, L. eds.) pp. 323-337, Plenum Press, New York. von Figura, K., 1977. Eur. J. Biochem. 80, 535-542. Banerjee, D. K. and Basu, D., 1975. Biochem. J. 145, 113-118. Lee, J. E. S. and Yoshida, A., 1976. Biochem. J. 159, 535-539. Wiktorowicz, J. E., Awasthi, Y. C., Kurosky, A. and Srivastava, S. K., 1977. Biochem. J. 165, 49-53. Sandhoff, K., Conzelmann, E. and Nehrkorn, H., 1977. Hoppe-Seyler's Z. Physiol. Chem. 358, 779787. Bishayee, S. and Bachhawat, B. K., 1974. Biochim. Biophys. Acta 334, 378-388. Aruna, R. M. and Basu, D., 1975. J. Neurochem. 25, 611-617. Swallow, D. M., Evans, L , Saha, N. and Harris H., 1976. Ann. Hum. Genet., Lond. 40, 55-66. Brattain, M. G., Kimball, P. M., Pretlow, T. G. II and Marks, M. E., 1977. Biochem. J. 163, 247-251. Bearpark, T., Bouquelet, S., Fournet, B., Montreuil, J., Spik, G., Stirling, J. and Strecker, G., 1977. F.E.B.S. Lett. 84, 379-384. Bishayee, S. and Bachhawat, B. K., 1974. Neurobiol. 4, 48-56.

21. Cohen, C. M., Weissmann, G., Hoffstein, S., Awasthi, Y. C. and Srivastava, S. K., 1976, Biochemistry 15, 452-460. 22. Swallow, D. M., Evans, L. and Hopkinson, D, A., 1977. Nature 269, 261-262. 23. Crean, E. V. and Rossomundo, E. F., 1977. Biochem. Biophys. Res. Commun. 75, 488--495. 24. Balasubramanian, K. A. and Bachhawat, B. K., 1975. Biochim. Biophys. Acta 403, 113-121. 25. Helwig, J.-J., Farooqui, A. A., Bollack, C. and Mandel, P., 1977. Biochem. J. 165, 127-134. 26. Ahrnad, A., Bishayee, S. and Bachhawat, B. K., 1973. Biochem. Biophys, Res. Commun. 53, 730-736. 27. Balasubramanian, K. A. and Bachhawat, B. K., 1976. J. Neurochern. 27, 485-492. 28. Farooqui, A. A. and Roy, A. B., 1976. Biochim. Biophys. Acta 452, 431--439. 29. Carlson, R. W., Downing, M. and Tolbert, B., 1977. Biochemistry 16, 5221-5227. 30. Rush, R. A., Thomas, P. E., Kindler, S. H. and Udenfriend, S., 1974. Biochem. Biophys. Res. Cornmun. 57, 1301-1305. 30a. Klein, U. and von Figura, K., 1976. Biochern. Biophys. Res. Cornmun. 73, 569-576. 31. Sulkowski, E. and Laskowski, M., Sr., 1974. Biochem. Biophys. Res. Comrnun. 57, 463--468. 32. Wenger, D. A., Sattler, M. and Clark, C., 1975. Trans. Am. Soc. Neurochern. 6th Annual Meeting, p. 151. 33. Braidman, I. P. and Gregoriadis, G., 1977. Biochern. J. 164, 439-445. 34. Miller, A. L., Frost, R. G. and O'Brien, J. S., 1976. Anal. Biochern. 74, 537-545. 35. Norden, A. G. W. and O'Brien, J. S., 1974. Biochem. Biophys. Res_ Commun. 56, 193-198. 36. Norden, A. G. W., Tennant, L. L. and O'Brien, J. S., 1974. J. Biol. Chem. 249_ 7969-7976. 37. Miller, A. L., Frost, R. G. and O'Brien, J. S., 1977. Biochem. J. 165,591-594. 38. Holmes, E. W., Miller, A. L., Frost, R. G. and O'Brien, J. S., 1975. Am. J. Hum. Genet. 27, 719-727. 39. Shapira, E., David, A., DeGregorio, R. and Nadler, H. L., 1976. Enzyme 21,332-341. 40. Miller, A. L., 1978, Biochim. Biophys. Acta 522, 174-186. 41. Hieber, V., Distler, J., Myerowitz, R., Schrnickel, R. D. and Jourdian, G. W., 1976. Biochem. Biophys. Res. Cornmun. 73, 710-717, 42. Ben-Yoseph, Y., Shapira, E., Edelman, D., Burton, B. K. and Nadler, H. L., 1977. Arch. Biochem. Biophys. 184, 373-379. 43. Podolsky, D. K,, Weiser, M. M., Lamont, J. T. and Isselbacher, K., 1974. Proc. Natl. Acad. Sci. (USA) 71,904-908. 44. Dale, G. L. and Beutler, E., 1976. Proc. Natl. Acad. Sci. (USA) 73, 4672-4674. 45. Dale, G. L., Villacorte, D. G. and Beutler, E., 1976. Biochem. Biophys. Res. Commun. 71, 1048-1053. 46. Peters, S. P., Coyle, P., Coffee, C. J., Glew, R. H., Kuhlenschmidt, M. S., Rosenfeld, L. and Lee, Y. C., 1977. J. Biol. Chem. 252, 563-573. 61

47. Avrameas, S. and Guilbert, B., 1971. Biochimie. 53, 603-614. 48. Surolia, A., Bishayee, S., Ahrnad, A., Balasubramanian, K. A., Thambi-Dorai, D., Podder, S. K. and Bachhawat, B. K., 1975. Concanavalin A (Chowdhury, T. K. and Weiss, A. K. eds.) pp. 95-115, Plenum Press, New York. 49. Stahl, P., Six, H., Rodman, J. S., Sehlesinger, P., Tulsiani, D. R. P. and Touster, O., 1976. Proc. Natl. Acad. Sci. (USA) 73, 4045-4049. 50. Kottgen, E., Reutter, W. and Gerok, W., 1976. Biochem. Biophys. Res. Commun. 72, 61-66. 51. Kottgen, E., Reutter, W. and Gerok, W., 1978. Eur. J. Biochem. 82, 279-284. 52. Takahashi, S., Pollack, J. and Seifter, S., 1974. Biochim. Biophys. Acta 371, 71-75. 53. Soiling, H. and Wang, P., 1973. Biochem. Biophys. Res. Commun. 53, 1234-1239. 54. Balasubramanian, K. A., Abraham, D. and Bachhawat, B. K., 1975. Ind. J. Biochem. Biophys. 12, 204-206. 55. Yutaka, T., Fluharty, A. L., Stevens, R. L. and Kihara, H., 1977. Fed. Proc. 36, 643. 56. Shapiro, L. J., Hall, C. W., Leder, I. G. and Neufeld, E. F., 1976. Arch. Biochem. Biophys. 172, 156-161. 57. Garner, C. W., Jr. and Smith, L. C., 1972. J. Biol. Chem. 247,561-565. 58. Phillips, N. C., Robinson, D. and Winchester, B. G., 1976. Biochem. J. 153, 579-587. 59. Opheim, D. J. and Touster, O., 1978, J. Biol. Chem. 253, 1017-1023. 60. Hayman, M. J. and Crumpton, M. J., 1972. Biochem. Biophys. Res. Commun. 47, 923-930. 61. Brattain, M. G., Marks, M. E. and Pretlow, T. G., II, 1976. Anal. Biochem. 72, 346-352. 62. Goldstein, I. J., So, L. L., Yang, Y. and Callies, Q. C., 1969. J. Immunol. 103, 695-698. 63. Liener, I. E., Garrison, O. R. and Pravda, Z., 1973. Biochem. Biophys. Res. Commun. 51,436-443. 64. Harvey, S. R. and Chu, T. M., 1975. Cancer Res. 35, 3001-3008. 65. Carrie.o, R. J, and Usategui-Gomez, M., 1975. Cancer Res. 35, 2928-2934. 66. Harris, H. and Robson, E. B., 1963. Vox Sang. 8, 348-355. 67. Leon, M. A., 1967. Science 158, 1325-1326. 68. Surolia, A., Ahmao, A. and Bachhawat, B. K., 1975. Bioehim. Biophys. Acta 404, 83-92. 69. Page, M., 1973. Can. J. Biochem. 51, 1213-1215. 70. Lotan, R., Beattie, G., Hubbel, W. and Nichoison, G. L., 1977. Biochemistry 16, 1787-1794. 71. Michaelis, E. K., 1975. Bioehem. Biophys. Res. Commun. 65, 1004-1012. 72. Vretblad, P. and Hjorth, R., 1977. Biochem. J. 167, 759-764. 73. Dufau, M. L., Tsuruhara, T. and Catt, K. J., 1972. Bioehim. Biophys. Acta 278,281-292. 74. Dawson, J. R., Silver, J., Sheppard, L. B. and Amos, D. B., 1974. J. Immunol. 112, 1190-1193. 75. Cuatrecasas, P. and Tell, G. P. E., 1973. Proc. Natl. Acad. Sci. (USA) 70, 485-489.

62

76. Davey, M. W., Huang, J. W., Sulkowski, E. and Carter, W. A., 1974. J. Biol. Chem. 249, 6354-6355. 77. Davey, M. W., Sulkowski, E. and Carter, W. A., 1976. Biochemistry 15,704-713. 78. Steinemann, A. and Stryer, L., 1973. Biochemistry 12, 1499-1502. 79. Renthal, R., Pober, J. S., Steinemann, A. and Stryer, L., 1976. Concanavalin A as a Tool (Bittiger, H. and Schnebli, H. P. eds.) pp. 427-434, John Wiley and Sons, New York. 80. Braidman, I. and Gregoriadis, G., 1976. Biochem. SOc. Trans. 4, 259-261. 81. Kalb, A. J. and Levitzki, A., 1968. Biochem. J. 109, 669-672. 82. Goldstein, I. J., Reichert, C. M. and Misaki, A., 1974. Ann. N.Y. Acad. Sci. 234, 283-296. 83. Young, N. M. and Leon, M. A., 1974. Biochim. Biophys. Aeta 365,418-424. 84. Kornfeld, R. and Fen-is, C., 1975. J. Biol. Chem. 250, 2614-2619. 85. Ogata, S.-I., Muramatsu, T. and Kobata, A., 1975. J. Biochem. (Tokyo) 78, 687-696. 86. Krusius, T., Finne, J. and Rauvala, H., 1976. F.E.B.S. Lett. 71, 117-120. 87. Toyoshima, S., Fukuda, M. and Osawa, T., 1972. Biochemistry 11, 4000-4005. 88. Krusius, T. and Finne, J., 1977. Eur. J. Biochem. 78, 369-379. 89. Kornfeld, R. and Kornfeld, S., 1976. Ann. Rev. Biochem. 45, 217-237. 90. Waechter, C. J. and Lennarz, W. J. (1976) Ann. Rev. Biochem. 45, 95-112. 91. Goldstein, I. J. and So, L. L., 1965. Arch. Biochem. Biophys. 111,407--414. 92. Smith, D. F., Neff, G. and Walborg, E. F., Jr., 1973. Biochemistry 12, 2111-2118. 93. Goldstein, I. J., Hammarstrom, S. and Sundblad, G., 1975. Biochim. Biophys. Acta 403, 53-61. 94. Greenaway, P. J. and LeVine, D., 1973. Nature New Biol. 241, 191-192. 95. Nicholson, G. L. and Blaustein, J., 1972. Biochim. Biophys. Acta 266, 543-547. 96. Van Wauwe, J. P., Loontiens, F. G, and De Bruyne, C. K., 1973. Biochim. Biophys. Acta 313, 99-105. 97. Irimura, T., Kawaguchi, T., Terao, T. and Osawa, T., 1975. Carbohydrate Res. 39, 317-327. 98. Etzler, M. E., 1974. Ann. N.Y. Acad. Sci. 234, 260-275. 99. Nieholson, G. L., 1973. J. Natl. Cancer Inst. 50, 1443-1451. 100. Pereira, M. E. A. and Kabat, E. A., 1974. Ann. N.Y. Acad. Sci. 234, 301-305. 101. Kornfeld, S., Rogers, J. and Gregory, W., 1971. J. Biol. Chem. 246, 6581-6586. 102. Lis, H., Sela, B.-A., Sachs, L. and Sharon, N., 1970. Biochim. Biophys. Acta 211,582-585. 103. Pereira, M. E. A. and Kabat, E. A., 1974. Carbohydrate Res. 37, 89-102. 104. Lotan, R., Skutelsky, E., Danon, D. and Sharon, N., 1975. J. Biol. Chem. 250, 8518-8523. 105. Etzler, M. E. and Kabat, E, A., 1970. Biochemistry 9, 869-877.

106. Hayes, C, E. and Goldstein, 1. J., 1974. J. Biol. Chem. 249, 1904--1914. 107. Murphy, L. A. and Goldstein, I. J., 1977. J. Biol. Chem. 252, 4739--4742. 108. Podder, S. K., Surolia, A. and Bachhawat, B. K., 1974. Eur. J. Biochem. 44, 151-160. 109. Krusius, T., 1976. F.E.B.S. Lett. 66, 86-89. 110. So, L. L. and Goldstein, I. J., 1968. Biochim. Biophys. Acta 165, 398-404. 111. Bourque, E. and Kanfer, J., 1972. Life Sci. 11 (Part II), 1119-1205. 112. Pazur, J. H. and Aronson, N. N., Jr., 1972. Adv. Carbohydrate Chem. and Biochem. 27, 301-341. 113. Spiro, R. G., 1973. Adv. Prot. Chem. 27, 349-467. 114. Bahl. O. P. and Shah, R. H., 1977. The Glycoconjugates (Horowitz, M. I. and Pigman, W. eds.) Vol. 1, pp. 385-421, Academic Press, New York. 115. Bacchus, H., 1977. Critical Reviews in Clinical Laboratory Sciences 8,333-362. 116. Araki, C., 1959. Carbohydrate Chemistry of Substances of Biological Interest (Fourth Int'l. Congress of Biochemistry, Vienna, 1958, Vol. I, Symposium 1), (Wolfrom, M. L. ed.) pp. 15-30, Pergamon Press, New York. 117. Simpson, D. L., Thorne, D. R. and I_oh, H., 1978. Life Sciences 22, 727-748. 118. Pitlick, F. A., 1976. Biochim. Biophys. Acta 428, 27-34. 119. Baynes, J. W. and Wold, F., 1976. J. Biol. Chem. 251, 6016-6024. 120. Yamaguchi, S. and Suzuki, K., 1977. J. Biol. Chem. 252, 3805-3813.

63