Developmental Change and Genetic Defect in the Carbohydrate Structure of Band 3 Glycoprotein of Human Erythrocyte Membrane*

The chemical structure of Band 3 glycopeptide prepared from erythrocytes of normal adult (blood group OI), umbilical cord vessels (Oi), and an i adult variant who fails to develop I antigen (Oi), has been compared. Band 3 glycopeptide of cord erythrocytes gave, on permethylation analysis, predominantly 2,4,6-tri-O-methylgalactose and 3,6-di-O-methyl-2-N-methylacetamido-2-deoxyglucose, whereas the same glycopeptide of normal adult erythrocytes gave much higher amounts of 2,3,4,6-tetra-O-methylgalactose and 2,4-di-O-methylgalactose as compared with that of cord erythrocytes. Band 3 glycopeptide from i adult showed the same methylation pattern as cord-Band 3 glycopeptide. In accordance with these results, Band 3 glycopeptide of cord and i adult erythrocytes were hydrolyzed to mostly small oligosaccharides by endo-beta-galactosidase from Escherichia freundii, whereas that of normal adult produced a number of oligosaccharides with various sizes which was caused by branched structures. Based on these results and structures of released oligosaccharides, the major developmental change of carbohydrate structure in the erythrocyte membrane is the conversion of linear repeating Galbeta1 leads to 4GlcNAcbeta1 leads to 3Gal to a branched Galbeta 1 leads to 4GlcNAcbeta 1 leads to 3 (R leads to 6) Gal structure. i individual may result from the lack of the branching enzyme.


SUMMARY
The chemical structure of Band 3 glycopeptide prepared from erythrocytes of normal adult (blood group 01), umbilical cord vessels (Oi), and an i adult variant who fails to develop I antigen (Oi), has been compared. Band 3 glycopeptide of cord erythrocytes gave, on permethylation analysis, predominantly 2,4,6-tri-o-methylgalactose and 3,6-di-0-methyl-2-N-methylacetamido-L-deoxyglucose, whereas the same glycopeptide of normal adult erythrocytes gave much higher amounts of 2,3,4,6-tetra-O-methylgalactose and 2,4-di-o-methylgalactose as compared with that of cord erythrocytes. Band 3 glycopeptide from i adult showed the same methylation pattern as cord-Band 3 glycopeptide. In accordance with these results, Band 3 glycopeptide of cord and i adult erythrocytes were hydrolyzed to mostly small oligosaccharides by endo-fi-galactosidase from Escherichiu fmundii, whereas that of normal adult produced a number of oligosaccharides with various sizes which was caused by branched structures. Based on these results and structures of released oligosaccharides, the major developmental change of carbohydrate structure in the erythrocyte membrane is the conversion of linear repeating Gal/U -+ 4GlcNAc/31+ 3Gal to a branched Gal/31 + 4GlcNAcfil + 3 (R + 6)Gal structure.
i individual may result from the lack of the branching enzyme.
Membrane changes associated with the process of ontogenesis have been clearly demonstrated through the orderly appearance or disappearance of antigen markers such as "Fs" (l), blood group ABH (2), Forssman (3), and Ii antigens (4). The antigen i is converted to I during the development of fetal to adult erythrocytes (4) although rare individuals with a genetic defect cannot develop I antigen (adult i) (5). The Ii antigens have been regarded as the precursors of blood group ABH antigens (6), and a linear repeating Gal,!31 + 4GlcNAcPl + 3Gal structure for i (7)  (8) have been assigned recently. A similar progressive branching process in the carrier carbohydrate chains for glycoplipid A and H determinants has been implicated as being associated with the developmental process (9). Recently, Band 3, the major intrinsic membrane glycoprotein of human erythrocyte (for review, see Refs. 10 and ll), has been assigned by us as I antigen carrier (12). We have found also that endo+galactosidase from Escherichia freundii (13) abolished Ii antigenic activity of human erythrocytes resulting from shortening the carbohydrate chains of Band 3 as one of the most significant modifications (14,15). These observations prompted us to compare the carbohydrate structure of Band 3 from normal adult, fetus (umbilical cord), and adult i variant (5)  cording to the procedure of Laemmli (17). The glycopeptide from Band 3 protein was prepared by pronase digestion as described (18).
A portion of the glycopeptide was labeled by galactose oxidase/ NaB["HII as described (15). With this procedure, only the nonreducing terminal Gal or GalNAc was labeled (19). Endo-P-galactosidase was purified from the culture filtrate of E. freundii and was free from proteases and other glycosidases (13). The glycopeptide was digested with endo+galactosidase (125 munit/ml, final concentration) in 0.2 M sodium acetate buffer, pH 5.8, at 37'C for 18 h as described (15). Glycopeptides and oligosaccharides were permethylated by the method of Hakomori (20) and permethylated products were hydrolyzed and analyzed by the modified method (21) of Stellner et al. (22). Partially 0-methylated sugars were analyzed by Finnigan 3300-6110 gas chromatograph-mass spectrometer under the described condition (21). Paper chromatography was performed in a solvent system of ethyl acetate/pyridine/water (12:5:4, v/v) and radioactivity of chromatogram was determined as described (13).

Isolation of Glycopeptides
from Band 3-Band 3 protein purified from erythrocytes of each source showed essentially a single band with a similar mobility upon examination by SDS-polyacrylamide gel electrophoresis (data not shown). When the pronase digest of Band 3 glycoprotein labeled with galactose oxidase/NaB[3H]4 was subjected to gel filtration, two major radioactive peaks were observed (Fig. 1). Chemical analysis, however, showed that only the first peak contained detectable amounts of carbohydrate, which is consistent with the result reported previously (23). The major peak (indicated by horizontal arrow in Fig. 1) was, therefore, used for further studies. The chemical quantity of the glycopeptide eluted at Fractions 55 to 65 was small and variable, and the glycopeptide ' The abbreviations used are: I-Band 3, i-Band 3, and cord-Band 3,  was not degraded by endo+-galactosidase. Therefore, this glycopeptide was not subjected to further analysis in the present study. Whether this glycopeptide is related to the major glycopeptide is under investigation. The elution pattern of the major peak was not changed after mild alkaline treat-Endo-/?-galactosidase Digestion of the Glycopeptide-Galactose oxidase/NaB[3H]4-labeled Band 3 glycopeptide was digested by endo-P-galactosidase and subjected to gel filtration on Sephadex G-50. As shown in Fig. 3, A, B, and C, the digest from i-or cord-Band 3 showed mostly the smallest component while that from I-Band 3 showed a number of oligosaccharides with various sizes. Since the above technique can show only the oligosaccharides derived from the nonreducing termini of glycopeptides, the oligosaccharide profile of the enzyme digest was separately analyzed on Bio-Gel P-4 after being labeled at the reducing termini of the oligosaccharides by reduction with NaB[3H]4 followed by NaBH*.' As shown in Fig. 3, E and F, the digest from i-or cord-Band 3 glycopeptide predominantly produced disaccharide as well as small amounts of tri-and tetrasaccharide.
The oligosaccharide eluted at Fractions 28 to 36 was found to be a sialic acidcontaining tetrasaccharide, which is known to behave as a high molecular weight substance under this condition (21,25). More than 75% of galactose present in the original glycopeptide was recovered in the di-, tri-, and tetrasaccharide fractions based on neutral sugar quantitation using the unreduced digest.
The early eluted oligosaccharide fraction (Fractions 48 to 55 in Fig. 3D), which is characteristic of I-Band 3, was found to be a branched oligosaccharide by permethylation analysis. Fractions 28 to 37 in Fig. 30 were found to be a mixture of sialic acid-containing oligosaccharides and higher neutral oligosaccharides. ment (21). The chemical composition of the major glycopeptide is shown in Table I. The comnosition is exnressed as I mannose being 3.0 since the Band 3 molecule appears to have a sinale carbohvdrate attachment site (23). which seems to " ., contain 3 mannose residues (16). Accordingly, the molecular weights of the glycopeptides were calculated to be about 7500 for I-Band 3 and 5500 for i-or cord-Band 3 from the chemical composition shown in Table I. The calculated sizes of the glycopeptides were basically consistent with those based on gel filtration (23, 24) (see also Fig. 1) although the accurate determination of the molecular weights of the glycopeptides requires further structural analysis.

Permethylation of the Major Glycopeptide of Band 3-The
Overall Structure of I-, Cord-, and i-Band 3 Glycopeptide-Based on the permethylation of the glycopeptide (Fig. 2) and structural characterization of oligosaccharides (Fig. 3), it can be concluded that the carbohydrate side chains of i-and cord-Band 3 are mainly made up of the straight chain structures, R + GalPl -+ 4GlcNAcbl + 3GalpI + 4, whereas that of I-Band 3 contains the branched structure, R 4 Gal/?1 + 3GlcNAc/31+ 3 (R' -+ 6)Gal (Fig. 4). The carbohydrate chain of i-Band 3 was extensively digested by endo+galactosidase to produce mostly di-to tetrasaccharides, whereas that of I-Band 3 produced oligosaccharides of various sizes (Fig. 3) because a linear chain can be degraded easily by this enzyme but branched galactose is hardly hydrolyzed (26). The greater amount of nonreducing terminal galactose in I-Band 3 glycopeptide shown by permethylation (Fig. 2) is consistent with the fact that I-Band 3 carbohydrate chain has a branched structure.
major glycopeptide from each Band 3 prenaration was permethylated and partially 0-methylated sugars were analyzed as alditol acetate derivatives. As shown in Fig. 2, I-Band 3 and i-Band 3 showed distinctly different gas chromatography patterns. Thus, i-Band 3 produced mainly 3-O-substituted galactose and 4-O-substituted N-acetylglucosamine, whereas I-Band 3 produced substantial amounts of 3,6-di-O-substituted Since a negligible amount of nonreducing terminal N-acetylglucosamine was present in the starting glycopeptide (see ' Since glycoprotein fraction often contains borohydride-reducible components (Schiff base iminium group, prophyrin pyridinium group), it was essential to pretreat the fraction with NaBH4 in order to eliminate any nonspecific label (19). The glycopeptide was first treated with NaBH+ desalted on Sephadex G-10 column, and then digested by endo-,&galactosidase. were obtained by monitoring the total ion current. The following peaks were identified aa alditol acetates oE 1, 2,3,4-tri-O-methyl Fuc; 2, 2,3,4,6-t&a-O-methyl Gal; 3,3,4,6-t&Omethyl Man; 4,2,4,6-t&O-methyl Gal; 5, 3,4.6-t&O-methyl Gal; 6, 2,3,4&i-Omethyl Gal; 7, 3.  Fig. 1) was incubated with or without endo. &galactosidase and then applied to a Sephadex G-50 column as described in the legend to Fig. 1. The enzmetreated C---j and the no,,treated-contml t---j. A, I-Band 3: B, cord-Band 3; C, i-Band 3. D, E, and F, the oligosaccharide profile after being labeled at the reducing terminus. Nanlabeled glycopeptide corresponding to Fractions 33 to 53 in Fig. 1 (-50l to 3OOpg) was digested with endo-P-galactosidwe, reduced by NL~B [~H], and followed by NaBH,, desalted by a Sephadex G-10 column, and applied on a column (0.9 x 150 cm) of Bio-Gel P-4 WI to 400 mesh) equtibrated and &ted with water. D. I-Band 3; E, cord-Band 3; F, i-Band 3. results of permethylation), G@l + 4GlcNAcBl + 3&l, of this chain was degraded by endo-fi-galactosidase and the Fucol + ZGa@l -t 4GlcNAcfil + 3&d, and sialic acidside chain is essentially linear in this molecule. However, the containing tetrasaccharides produced by endo-&galactosidase g?ycopeptide isolated from i-Band 3 contains about 12 residues are derived from the nonreducing terminal portion of carbo-of Gal (Table I). Therefore, the overall structure of i-Band 3 hydrate chains while the disaccharide (GlcNAcjl + 3Gal) glycopeptide is envisioned as follows. Two or three carbohymust be produced from the internal part of the chain (see Fig. drate chains, each being a linear chain and assigned as a "side 4). The molar ratio of disaccharide to tri-plus tetrasaccharide chain," me linked to a %xe structure" which provides the produced from i-Band 3 was found to be 32~1.0 based on paper branching point for the linear side chains, probably through chromatography. This indicates that the number of the mannose. In fact, di-O-or t&O-substituted mannose was GlcNAcfil -3Gal repeating units per one carbohydrate side detected in methyl&ion analysis of the whole glycopeptide chain may not exceed 4 to 5 (see Fig. 4), since more than 75% (Fig. 2). 3)". Although the residue which links to C-6 of galactose is not characterized yet, the structure shown here is likely based on the established structure for I antigenicity (8). Two or three chains shown here are linked to the same core portion (R2). See text also.

DISCUSSION
This paper clearly demonstrates the developmental change and genetic defect in the carbohydrate structure of Band 3. As shown in Fig. 4, the most critical change from fetus (i) to adult (I) cells is branching at position C-6 of galactose. To our knowledge, this is the first demonstration on a clear chemical basis of the structural change of cell surface carbohydrate during development.
The same change might occur in cell surface glycoconjugates of human erythrocytes in general, since oligosaccharides released from surface-labeled cells by endo+?-galactosidase gave a similar characteristic pattern according to I-or i-cells as described (15).
The structure of the Band 3 carbohydrate has a few interesting features. Particularly, it has H antigenic determinants indicating that the molecule might serve as a carrier for ABH antigens as well as Ii antigens. Recently, glycopeptides with Gal/?1 + 4GlcNAcbl + 3 repeating structure were isolated from pronase digest of human erythrocyte membrane and found to be ABH-active (27) and digestable by endo-P-galactosidase (28). Thus, one of the origins of these glycopeptides appears to be Band 3.
Another interesting feature is that blood group-active side chains are linked to the core portion, which appears to be composed of mannose and N-acetylglucosamine, having an alkali-stable linkage to the peptide. Thus, the carbohydrate moiety of Band 3 shows a unique hybrid structure, consisting of blood group carrier chains and a core portion of serum-type glycoprotein.
The scheme for the structure of this molecule which consists of unique side chains, an inner core, and the linking portion between the side chains and the core, provide us with a new system for biosynthesis and structural change during ontogenesis.