Cell surface modification by endo-beta-galactosidase. Change of blood group activities and release of oligosaccharides from glycoproteins and glycosphingolipids of human erythrocytes.

Endo-fi-galactosidase from Escherichia freundii has been shown to directly modify specific cell surface glycoconjugates. Antigenic changes caused by the enzyme treatment of human 01 adult, Oi-variant, and umbilical cord erythrocytes have been correlated with the altered cell surface glycoconjugate pattern and with the oligosaccharide pattern released from the cell surfaces. The following four kinds of antigenic changes were particularly remarkable, 1) abolishment of Ii-antigenic activity; 2) decrease of the reactivity with anti-ABH blood group and anti-paragloboside antibodies; 3) a slight, but distinct increase of the reactivity with antilacto-N-triosylceramide antibodies; and 4) no change of the reactivity with anti-globoside antibodies. Accompanying these antigenic changes, surface-labeled carbohydrates showed the following changes through endo+galactosidase treatment: 1) about 40% of the labeled galactose was released whereas only 5% of the labeled sialic acid was released. 2) Carbohydrate moiety of Band 3 and Band 4.5 glycoproteins were hydrolyzed by endo+galactosidase. In contrast, sialoglycoproteins (PAS-l, -2, and -3) were not affected. 3) The change in glycolipids occurred mainly with long chain carbohydrates such as Hz-, HZ-, and H1-glycolipids and in higher gangliosides. The oligosaccharide released from adult 01 active erythrocytes contained more heterogeneous higher molecular weight components than that released from adult Oi, or umbilical cord Oi erythrocytes which contained relatively homogeneous lower molecular weight components. The result is therefore consistent with I-active erythrocytes containing carbohydrate chains with more branching than i-active erythrocytes.

Endo-fi-galactosidase from Escherichia freundii has been shown to directly modify specific cell surface glycoconjugates.
Antigenic changes caused by the enzyme treatment of human 01 adult, Oi-variant, and umbilical cord erythrocytes have been correlated with the altered cell surface glycoconjugate pattern and with the oligosaccharide pattern released from the cell surfaces. The following four kinds of antigenic changes were particularly remarkable, 1) abolishment of Ii-antigenic activity; 2) decrease of the reactivity with anti-ABH blood group and anti-paragloboside antibodies; 3) a slight, but distinct increase of the reactivity with antilacto-N-triosylceramide antibodies; and 4) no change of the reactivity with anti-globoside antibodies. Accompanying these antigenic changes, surface-labeled carbohydrates showed the following changes through endo+galactosidase treatment: 1) about 40% of the labeled galactose was released whereas only 5% of the labeled sialic acid was released.
3) The change in glycolipids occurred mainly with long chain carbohydrates such as Hz-, HZ-, and H1-glycolipids and in higher gangliosides. The oligosaccharide released from adult 01 active erythrocytes contained more heterogeneous higher molecular weight components than that released from adult Oi, or umbilical cord Oi erythrocytes which contained relatively homogeneous lower molecular weight components. The result is therefore consistent with I-active erythrocytes containing carbohydrate chains with more branching than i-active erythrocytes.
Endo-P-galactosidase from Escherichia freundii has been shown to hydrolyze various substrates such as keratan sulfate (l), mutinous glycoproteins, milk oligosaccharides (2, 3), and glycosphingolipids (4). This enzyme has been found to hydrolyze specifically the ,&galactosidic bond of lacto-N-glycosyl series which has the common structure R + GlcNAcPl ---f 3GalPI + 4Glc (or GlcNAc) (2-4). This class of oligosaccharide chain is present as antigenic determinants for blood group ABH, Lewis (5, 6), Ii (7-g), P, (lo), and p (11) and possibly for tumor-associated antigens (12)(13)(14)(15). Thus, this enzyme is ex-* This investigation has been supported by Grants CA19224 and CA20026 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
petted to be useful to study the function and structure of these antigens.
Particularly, it is interesting to know whether endo-p-galactosidase can directly modify cell surface antigen in situ, since the enzyme is capable of hydrolyzing the internal ,&galactosyl linkage without removal of nonreducing sialosyl termini (4). Furthermore, the recent development of cell surface-labeling techniques (16)(17)(18)(19) enables us to identify the cell surface glycolipids and glycoproteins which could be modified by the enzyme treatment.
Thus, it is possible to find out which glycoconjugates are responsible for those antigenicities on cell surface.
As the first step for applying the enzymatic modification of the intact cell surface, we treated the human erythrocytes by endo-/?-galactosidase. This paper describes the antigenic change of human erythrocytes by endo-P-galactosidase, the characterization of an altered pattern of cell surface glycoproteins and glycolipids associated with antigenic changes, and structural characterization of released oligosaccharides from cell surface caused by the enzyme treatment. It is also found in this study that released oligosaccharide pattern from cord (new born) and Oi variant cells (20)  Enzymes-Endo-P-galactosidase was purified from a culture filtrate of E. freundii as described previously (3). Purified endo-Pgalactosidase is free from protease and other glycosidases. P-Galactosidase was prepared from Jack bean meal according to Li and Li (30 GNeu, N-glycolylneuraminic acid; AcNeu, N-acetylneuraminic acid. All sugars mentioned in this paper have I) configuration except fucose, which has an L configuration. raphy of thin layer chromatogram was taken after treated with diethyl ether/2,5-diphenyloxazole (37 After removal of insoluble material by centrifugation, 10 Kabi units of galactose oxidase were added, and incubated at 37" C for 3 h. After incubation, the reaction mixture was titrated to pH 9.0, and 2.5 mCi of NaB[:'H], dissolved in 50 ~1 of 0.01 N NaOH was added. After standing for 2 h at room temperature, 2 mg of NaBHl was added and reduction was continued another 30 min. Reduction was stopped by adding a few drops of glacial acetic acid, and the solution was evaporated under nitrogen with repeated addition of methanol. Water-insoluble material which did not contain carbohydrate was removed by centrifugation, and water-soluble material was desalted by a Sephadex G-10 column eluted with water. Acetylation of Oligosaccharide Alcohols-HZ-Glycolipid, paragloboside, a-galactosylparagloboside, and ,&galactosylparagloboside were labeled bv aalactose oxidase/NaBI"Hl, according to Suzuki and Suzuki (40). Each glycolipid was hydrolized by endo-iii-galactosidase, and oligosaccharide was isolated and reduced to the alcohol by NaBH, as described previously (4). Oligosaccharide alcohols were dried over P205, and acetylated in pyridine/acetic anhydride (2:1, v/v) at room temperature for 20 h. Oligosaccharides released from surface-labeled cells were reduced to oligosaccharide alcohols and acetylated in the same manner as described above. Acetylated oligosaccharide alcohols were analyzed by thin layer chromatography in a solvent system: butylacetate/acetone/water (80:20:0.1, v/v). Fluorography of thin layer chromatography was taken after treated with diethyl ether/2,5diphenyloxazole (37).

Change of Erythrocyte
Antigenicity b-y Endo-P-galactosidase-Changes of various antigenic activities detectable by hemagglutination with various antisera have been observed as shown in Fig. 1. While hemagglutinability by anti-A, -B, and -H was generally decreased after treatment with endo-,8galactosidase,'J the loss of the reactivities with anti-1 and anti-i antisera were most remarkable.
A high agglutinability of OIadult cells with anti-1 Ma was completely abolished after the enzyme treatment.
Similarly, the agglutinability of Oi adult and Oi-cord erythrocytes with anti-i Hog and anti-i Dench was completely abolished after the enzyme treatment. i\n enhanced agglutinability of trypsin-treated adult erythrocytes by anti-1 (Ma or Step) was not abolished, but greatly reduced. However, the enhanced agglutinability of trypsin-treated cord erythrocytes to anti-i antisera (Dench and Hog) was completely abolished by endo-/?-galactosidase treatment. The reactivities of normal 01-adult, Oi-adult, and Oi-cord erythrocytes by anti-paragloboside antisera behaved in the same way as the reactivity to anti-1 antisera, namely, the agglutinability of intact cells was abolished and the enhanced agglutinability by trypsin treatment was decreased by the enzyme. The agglutinability by anti-globoside antibody was unchanged as expected since globoside was not susceptible to endo-,& galactosidase (4). In a striking contrast to the reactivities to anti-A, -B, -H, -1, -i, and paragloboside, agglutinability of trypsin-treated erythrocytes to anti-lacto-N-triosylceramide (LNTri) antibodies was distinctly greater after the endo-,&galactosidase treatment. This phenomenon was consistently observed irrespective of normal adult, Oi-adult, or umbilical cord Oi-erythrocytes. The result is compatible with the known specificity of the enzyme, i.e. a repeating Gal/31 + 4GlcNAcbl + 3Gal  The values of radioactivity are expressed as total counts per min when 0.5 ml of packed cell was used.
Step as seen in lacto-N-norhexaosylceramide or its analogue were hydrolyzed to yield lacto-N-triosylceramide and various oligosaccharides with this enzyme (4). However, the structure created by the enzyme was not readily accessible to antibody, therefore only trypsin-treated and endo-P-galactosidase-treated cells showed an enhanced reactivity with antilacto-N-triosylceramide antibodies.
The time course of the decrease of I, i, and H showed the rapid decrease of hemagglutinability and reached a plateau after incubation at 37" C for 1 h with 125 milliunits of the enzyme/ml (data not shown). This rate was very similar to the hydrolysis rate of the isolated sialosyl paragloboside or Hz-glycolipid with the same enzyme concentration (4).
Endo-P-galactosidase Treatment of Surface-labeled Cells-The cell surface galactose residues or sialic acid residues were labeled by galactose oxidase-NaB["H& method or periodate oxidation-NaB["H14 method, respectively. Labeled cells were treated by endo-P-galactosidase as described under "Materials and Methods," and cells and supernatant were fractionated as summarized in Fig. 2. The yield of radioactivity in each step of adult I cells is shown in Table I.
With endo-fi-galactosidase treatment, the surface galactoselabeled cell released 40% of the radioactivity (see Table I, Steps 1,2, and 3), and the radioactivity of both protein fraction (Step 6) and glycolipid fraction (Step 5) was decreased compared to controls. Based on these data (Table I, Steps 2, 5, and 6), it could be calculated that approximately 70% of released oligosaccharides were derived from glycoproteins whereas 30% of those were derived from glycolipids.
In contrast, sialic acid labeled cells released less than 5% of total radioactivity after enzyme treatment (Table I) (Gels 1 and 3). Gels 1 and 2 are Coomassie blue-stained gels and Gels 3 and 4 are fluorographs of Gels 1 and 2, respectively. B, labeled i adult cells after incubation with endo+-galactosidase (Gels 6 and 8) or without the enzyme (Gels 5 and 7). Gels 5 and 6 are Coomassie blue-stained gels and Gels 7 and 8 are fluorographs of Gels 5 and 6, respectively. C, labeled cord cells after incubation with endo-/3-galactosidase (Gels 10 and 12) or without the enzyme (Gels 9 and II). Gels 9 and 10 are Coomassie blue-stained gels and Gels 11 and 12 are fluorographs of Gels 9 and 10, respectively. teins were essentially derived from Band 3 and Band 4.5 carbohydrate moiety. In addition, PAS-l and PAS-2 appear to be affected slightly to lose the radioactivity, suggesting the presence of a small population of the susceptible carbohydrate chain in PAS-l and PAS-2.
Similar results were obtained in the case of adult i and cord cells (Fig. 3, Gels 5 to 12). Interestingly, endo+-galactosidasedigested Band 3 migrated slightly faster than nondigested sample in SDS-polyacrylamide gel electrophoresis and showed a less broad band (Fig. 3, Gels 6 and 10).
In contrast, sialoglycoproteins which had been labeled by periodate-NaB[3H]4 method did not show obvious changes by the enzyme treatment (Fig. 4), except that two bands between PAS-l and PAS-2 in cord cells were lost (Fig. 4, Gel 6). The short time exposed film also showed no indication of change after enzyme treatment except as described above. Glycolipids of Surface-labeled Cells-Membrane glycolipids were extracted and fractionated as summarized in Fig. 2 and Table I. Fig. 5A shows the fluorography pattern of the thin layer chromatogram of neutral glycolipid (Step 9 in Fig.  2 and Table I) from galactose oxidase/NaB[3H]4-labeled adult I cells. It is apparent that endo-fi-galactosidase hydrolyzes the carbohydrate portion of HZ-and H4-glycolipid completely, and hydrolyzes some part of Hs-glycolipid. Fig. 5B shows the fluorography of the thin layer chromatogram of ganglioside (Step 10 in Fig. 2 and Table I)  lightly PAS-l, and -2 (Fig. 3, Gels 1 and 3). Significantly, most of the radioactivity located in Band 3 and Band 4.5 disappeared after endo+-galactosidase treatment (Fig. 3, Gel 4) Table I) was applied on a column of Sephadex G-50. As shown in Fig. 6, A and B, oligosaccharides of various molecular size were observed from adult I cells. The gel chromatography profiles of the endo-P-galactosidase digest of Band 3 glycopeptides or glycolipids are shown in Figs. 7 and 8, respectively. The elution position of oligosaccharide P-l from galactose oxidase-labeled cells (Fig. 6) is identical to the smallest size oligosaccharide peak from Band 3 glycopeptide (Fig. 7) or from glycolipids (Fig. 8). These results indicate that most of the higher oligosaccharides originated from the Band 3 carbohydrate moiety, but small size oligosaccharides (P-l and P-2 in Fig. 6A) could have originated from HZ-, H3-, and Hqglycolipid as well.
The gel chromatography profiie of the sialic acid-labeled oligosaccharides is similar to that of the galactose-labeled oligosaccharide, although total radioactivity of the former is 10 times less than that of the latter (see Fig. 6, A and B). It is possible that a small amount of sialic acid in Band 3 (38) and Band 4.5 has been labeled by tritium and hydrolyzed to oligosaccharides.
Small size oligosaccharides also may have originated from higher gangliosides.
Comparison of Oligosaccharides Released from Adult I, Adult i, and Cord Cells-Although oligosaccharides released from adult I cells showed various molecular sizes, oligosaccharides were released almost exclusively as smallest size from i and cord cells (Fig. 6, C to F). It appeared that carbohydrate chains of i cells could be more extensively degraded and only short oligosaccharides were produced. It is likely therefore that i cells have a different structure of oligosaccharide chains, which is more susceptible to endo-/?-galactosidase than that of normal adult I cells.
Structural Characterization of Oligosaccharides-Since the amount of oligosaccharide released from cells was too little to be analyzed by chemical means, the oligosaccharides were characterized as follows. The smallest size oligosaccharide (Fig. 6, P-1) from galactose oxidase labeled adult I, adult i, and cord cells was subjected to paper chromatography.
In all cases, two components, P-la and P-lb were observed and showed the same mobility as authentic Gal/?1 + 4GlcNAcbl --, 3Gal and Fuc~vl + 2Galbl+ 4GlcNAcPl+ 3Gal, respectively (Fig. 9). P-la and P-lb were eluted from filter paper after paper chromatography as described above and subjected to thin layer chromatography after derivatization to oligosaccharide alcohol acetates. As seen from Fig. 10, oligosaccharide alcohol acetates of P-la and P-lb migrated to the positions corresponding to the acetates of Galpl + 4GlcNAcPl ---* 3Galol and Fuccul + 2Galpl + 3GlcNAc/?l+ 3Galo1, respectively.  Suzuki and Suzuki (40). Each labeled glycolipid fraction (2 x lo5 cpm) was incubated with 12.5 milliunits of endo-/?-galactosidase in 100 ~1 of 0.2 M sodium acetate buffer, pH 5.8, containing 100 pg of sodium deoxytaurocholate at 37" C for 2 h. After incubation, 600 ~1 of chloroform/ methanol (2:1, v/v) was added and upper water phase was taken as oligosaccharide fraction.
Each oligosaccharide fraction was applied on Sephadex G-50 column as the same manner as in Fig. 6. A, oligosaccharides from Hz-glycolipid fraction; B, oligosaccharides from Ha-glycolipid fraction; C, oligosaccharides from HI-glycolipid fraction.
The release of radioactive galactose residue from each oligosaccharide by exo-glycosidase was examined by paper chromatography.
From P-la, radioactive galactose was liberated by fi-galactosidase but not by cY-galactosidase. From P-lb, neither /3-galactosidase nor a-galactosidase could release radioactive galactose. However, /3-galactosidase together with a+fucosidase could release radioactive galactose from P-lb. Based on these results, the structures of P-la and P-lb were tentatively estimated to be GalPl + 4GlcNAc/31+ 3Gal and Fucal --, 2G@l---, 4GlcNA$l+ 3Gal, respectively. Oligosaccharide P-2 ( Fig. 6A) showed five components by paper chromatography and has not been characterized further. However, these oligosaccharides might be branched oligosaccharides since P-2 was produced only from branched HZor HI-glycolipid but not from straight chain Hz-glycolipid (8) (see Fig. 8). The smallest oligosaccharide (Fig. 6, S-l) from sialic acid-labeled adult I, adult i, and cord cells were applied to paper chromatography and showed the same mobility as authentic AcNeucw2 ---, 3Galbl + 4GlcNAcbl --) 3Gal (data not shown). a, G@l + 4GlcNAcbl+ 3Gal prepared from paragloboadult I cells (Fig. 6A, p-1 4GlcNAc Bl -+ 3Galol: 3. GalBl + 4GlcNAcBl G 3Gkol; 4, L-F&~ + 2GalBlb 4GlcNA& > 3dalol; 5, P-la frdm adult I cell; 6, P-lb from adult I cell. One to four are acetates of authentic oligosaccharides prepared from corresponding glycolipids as described in the legend of Fig. 9. Experimental details are described under "Materials and Methods." DISCUSSION Enzymatic modification of the cell surface, particularly surface antigenicity and immunogenicity, has been a major topic in transplantation immunology. Enhanced immunogenicity of tumor cells through sialidase treatment has been reported by a number of investigators although some controversy had also been reported (41)(42)(43)(44). Conversion of blood group B erythrocytes to 0 erythrocytes by coffee bean (Ygalactosidase was reported to be useful for practical transfusion purpose (45), however, the reactivities of various sugar residues on cell surfaces to various exo-glycosidase are generally limited except to sialidase and probably to a few exceptional enzymes.
Modification of cell surface carbohydrate by endo-glycosidases could be an exciting approach if a suitable enzyme can be applied. However, no suitable endo-glycosidase has so far been reported to degrade a specific cell surface glycoprotein or glycolipid. Endo-P-galactosidase of E. freundii has an obvious merit in that the enzyme can hydrolyze a specific ,i3galactosyl linkage without modification of the peripheral carbohydrate chain and its substrate specificity has been well established (4). Thus, the enzyme is ideal to modify a specific carbohydrate chain at the cell surface. This study is aimed at the antigenic modification of erythrocytes as a model with the endo+-galactosidase and further to correlate with the changes in a specific cell surface carbohydrate to the antigenicity change caused by the enzyme treatment.
Four kinds of antigenicity changes of erythrocytes have been observed: 1) abolished Ii-antigenic activity, 2) decrease of the reactivities with antibodies to ABH and paragloboside, 3) a slight, but distinctive increase to antibodies directed to lacto-N-triosylceramide, and 4) no change in the reactivity with anti-globoside antibodies. Through endo+?-galactosidase treatment of surface-labeled cells; 1) about 40% of the labeled galactose was released while only 5% of the labeled sialic acid was released (Table I) and 2) the change in glycoproteins occurred mainly in Band 3 glycoprotein, the major intrinsic membrane protein.
3) The change in glycolipids occurred in those with long chain carbohydrate such as HZ-, H3-, and H4-glycolipids and in higher gangliosides (Fig. 5). 4) Oligosaccharides were released from the cell surface which showed characteristic patterns according to I-active adult or i-active fetal erythrocytes (Fig. 6). From these results, it is apparent that endo+galactosidase modified carbohydrate chains of Band 3, Band 4.5 and long chain glycolipids resulting in the abolishment of Ii antigenic activity and the decrease of ABH and paragloboside antigenic activity. As a consequence, a new structure having N-acetylglucosamine as nonreducing terminus was created which may show the reactivity with antibodies directed to lacto-N-triosylceramide.
Thus, it can be concluded that at least some portion of carbohydrate chain of Band 3 and Band 4.5 and long chain glycolipids are responsible to express Ii-activity. This result is consistent with the recent finding that long chain glycolipids such as lacto-N-norhexaosylceramide, or lacto-N-isooctaosylceramide (9, 46) and Band 3 protein are the antigenic carriers for Ii-determinants (47). Endo-P-galactosidase hydrolyzed galactose-labeled oligosaccharide chains about 10 times more than sialic acid-labeled oligosaccharide chains. The results indicated that carbohydrate chains labeled by galactose oxidase/NaB["H14 were much more susceptible than those labeled by periodate/ NaB[3H]4. The former was represented by the Band 3 molecule which has a susceptible carbohydrate chain composed of mainly galactose and N-acetylglucosamine (38, 48), whereas the latter was represented by the carbohydrate chains of glycophorin which are mainly composed of AcNeucu2 -+ 3Galpl + 3 (AcNeucu2 + 6)GalNAc (49). Thus, the combination of cell surface labeling and endo-,&galactosidase treatment can be a useful method to discriminate the structure of various oligosaccharide chains on cell surfaces. Recently, the glycopeptides which are mainly composed of galactose and Nacetylglucosamine were isolated from protease-released fraction of human erythrocytes and termed as erythroglycan (50). Since these glycopeptides were susceptible to endo+-galactosidase (50), the glycopeptides appear to originate from Band 3 and Band 4.5.
Analysis of a released oligosaccharide pattern gave us useful information about the structure of the carbohydrate chain of the original molecule. A large variety of oligosaccharides with different molecular sizes were released from adult I-active erythrocytes in a striking contrast to the homogeneous small size oligosaccharides released from i-active adult, or i-active umbilical cord erythrocytes. A linear carbohydrate chain such as R + GlcNAcPl + 3GalBl + 4GlcNAc -+ R can be hydrolyzed readily by endo+galactosidase whereas the galactosidic linkage at a branched structure such as R + GlcNAcPl -+ 6(R + GlcNAcPl --+ 3)Ga1/31-+ 4GlcNAc was hardly hydrolyzed by this enzyme (4). Therefore, the release of oligosaccharides with various molecular sizes from adult Ierythrocytes are probably derived from the heterogeneous branched structures.
In contrast, a homogeneous small size oligosaccharide can be released probably from a linear structure with little branching. This interpretation is supported by the recent structural studies on i-active and I-active glycolip-ids (9, 46), i.e. i-activity is determined by straight structure Galpl + 4GlcNAcbl + 3Gal/31 ---f 4GlcNAQl + 3Ga1, whereas I activity is determined by branched structure Gal/31 -+ 4GlcNAcPl+ 3(Gal/31 + 4GlcNAc ,81-+ 6)Gal. Furthermore, direct structural analysis of Band 3 carbohydrate chain from I-and i-erythrocytes confirmed the above conclusion (51).
Glycolipids with short carbohydrate chains such as sialosylparagloboside, paragloboside, and H1-glycolipids were hydrolyzed when isolated glycolipids were incubated with endo-,&galactogidase (4), but not on cell surface (Fig. 5). Only a long chain glycolipid with more than about 6 sugar residues were hydrolyzed on cell surface. This indicates that the susceptible galactosidic linkage close to glycosylceramide moiety is not accessible by endo+-galactosidase on cell surface. Although HZ-glycolipid has a susceptible linkage at the 4th sugar residue from ceramide as shown in in vitro analysis under a high concentration of the enzyme (4), this galactosyl residue is branched and therefore is unsusceptible on cell surface. Thus, HI-, Hn-glycolipid and their analogues are not highly susceptible to the enzyme treatment, therefore a remaining HI-, and H3-glycolipid may contribute H-antigenic activity after the endo+-galactosidase treatment. As a summary, combining cell surface labeling technique and endo-P-galactosidase digestion brought us extensive characterization of cell surface glycoproteins and glycolipids, especially those which are Ii antigenic carriers in human erythrocytes.
Human fetal and cord red blood cells have i antigenic activity but will develop to I active red blood cells in a normal adult (52). It also has been reported that the change of Ii blood group is associated with the tumorigenic change (15). Endo-P-galactosidase will, therefore, be an indispensable tool to analyze the developmental and oncogenic change of cell surface glycoproteins and glycolipids in these cells.