Primary Structure of the Oligosaccharide Determinant of Blood Group Cad Specificity"

Glycophorin A and B from Cad erythrocyte membranes are the carriers of the blood group Cad deter- minants. They are characterized by a significant increase in molecular mass, as compared to the corre- sponding glycophorins from control erythrocytes (Car-tron, J.-p., and Blanchard, D. (1982) Biochem. J. 207, 497-504). Lipid-free glycophorin A, purified from Cad red cells, showed an increased GalNAc content in comparison to blood group B, Cad-negative, control cells. Alkaline-borohydride treatment of this Cad glycophorin A released as a predominant species a penta- saccharide; its structure was determined, by methylation analysis and by 500-MHz 'H-NMR studies, to be: This novel oligosaccharide inhibited

Institut National de la Santb et de la Recherche MBdicale, by the Netherlands Foundation for Chemical Research with financial aid from the Netherlands Organization for the Advancement of Pure Research, and by Grant UUKC-OC 79-13 from the Netherlands Foundation for Cancer Research. 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.
$ To whom all correspondence should be addressed. I All sugars are of the D-configuration, unless otherwise indicated.
ficities (4)(5)(6)(7). Following the observation that all Cad samples reacted strongly with anti-Sd" antibodies, which define an antigen of varying strength present on more than 90% of Caucasian red cells, Sanger et al. (3) suggested that Cad was in fact a very strong form of Sd". In order to prove this assumption, the chemical structure of both the Cad and the Sd" determinants has to be established. Preliminary investigations have shown that Cad determinants are carried by the main red cell membrane sialoglycoproteins (glycophorin A and B). This was deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and affinity binding on immobilized D. biflorus lectin (8,9). The carbohydrate composition of highly purified lipid-free glycophorin A molecules prepared from Cad erythrocytes indicated an increased GalNAc content and suggested that these residues form part of alkali-labile oligosaccharide chains. In order to clarify the chemical structure of the Cad determinant, the predominant oligosaccharide chains obtained by alkaline-borohydride treatment of purified glycophorin A molecules were isolated and analyzed.
Based on methylation analysis followed by GLC-MSZ as well as independently on 500-MHz 'H-NMR spectral studies, the complete structure of a pentasaccharide bearing the Cadspecific determinant was identified. The novel structure shares the terminal, nonreducing sequence GalNAcS( I+ 4)GalP( 1+. ) with blood group Sd" determinants (10).

EXPERIMENTAL PROCEDURES
The red cells from the original Cad individual (group B) were kindly provided by Monique Monis, Centre de Transfusion Sanguine de Montpellier, France (1). Control red cells were collected from blood donors of the Centre National de Transfusion Sanguine, Paris, France and were typed as group B, Cad negative. The major red cell membrane sialoglycoprotein (glycophorin A) was purified from lipid-free sialoglycoproteins obtained after fractionation of 60 ml of packed red cells as described previously (9). Alkaline-borohydride treatment was performed on lipid-free glycophorin A essentially as described by Aminoff et al. (11). Briefly, 5 to 10 mg of purified material was incubated in a medium containing 1 M KBH,, 0.1 M KOH, and 1.5 to 3 mCi of NaBI3HI4 (7 Cilmmol, New England Nuclear) for 20 h at 45 "C. Samples were neutralized by addition of Dowex 50 x 8 (H' form) and filtered through glass wool. Borate salts were partially * The abbreviations used are: GLC-MS, gas-liquid chromatography combined with mass spectrometry; GLC, gas-liquid chromatography; TLC, thin layer chromatography; NMR, nuclear magnetic resonance; Ac, acetyl; Me, methyl. eliminated as methyl derivatives by concentration in uacuo after addition of methanol. The resulting products of the @-elimination procedure were separated on a Bio-Gel P-6 column (1.5 x 50 cm) equilibrated with 1% acetic acid. TLC was performed on 20 X 20 cm Kieselgel plates (Merck, Darmstadt) in ethano1:water:butanol: pyridine:acetic acid (100:30:10103, v/v) for 6 h at room temperature (12). Carbohydrates were stained with orcinol/sulfuric acid reagent at 105 "C for 10 min (13). The carbohydrate composition of purified glycophorin A and its alkaline degradation products was determined by GLC after methanolysis (0.5 M HCl/methanol, 24 h, 80 "C) and pertrifluoroacetylation (14). 200 pg of pure oligosaccharide from Cad sialoglycoprotein (Cad fraction 11) were methylated according to Finne et al. (15). The permethylated oligosaccharide was methanolyzed and the products were identified by GLC-MS after peracetylation (16 (17). Chemical shifts are given for a neutral solution at 300 K, relative to internal sodium 4,4-dimethyl-4-silapentane-l-sulfonate, but were actually measured by reference to internal acetone: 6 = 2.225 ppm, with an accuracy of 0.002 ppm.
Cad blood group activity of 0-eliminated oligosaccharides has been checked by agglutination-inhibition tests carried out as described (9) using the D. biflorus lectin (Serva Laboratory).

RESULTS AND DISCUSSION
Lipid-free glycophorin A purified from Cad red cells (9) inhibited strongly the agglutination of AI red cells by D.
biflorus lectin (0.002 pg of substance inhibited four hemagglutinating doses of lectin) and exhibited a high GalNAc content as compared to glycophorin A from control cells (Table I).
Purified glycophorin A preparations from Cad and control red cells were submitted to alkaline-borohydride treatment. The reduced oligosaccharides obtained were fractionated on a Bio-Gel P-6 column equilibrated with 1% acetic acid (Fig.  1). The six fractions eluted from this column were lyophilized, redissolved in water, and analyzed by TLC. Fraction I, which eluted in the void volume of the Bio-Gel P-6 column, contained the residual glycoprotein which does not migrate on TLC. Fraction I1 gave a single spot on TLC, both when the reduced sugars from control and Cad glycoproteins were examined. However, the mobility of the control and Cad fraction I1 oligosaccharides was clearly distinct (namely, Real = 0.60 and 0.45, for control and Cad, respectively), indicating a difference in structure. Fraction I11 contained, as a predominant species, the same oligosaccharide which is present in fraction 11 (RG,, = 0.60 and 0.45, for control and Cad, respectively) together with minor components of larger mobility on TLC. Interestingly, three of these minor saccharides had a similar mobility (Real = 0.74, 0.92, and 1.02) in material derived from control and Cad cells, suggesting a possible identity of structure. In addition, another oligosaccharide (Real = 0.58) with a mobility virtually identical with that of the pure oligosaccharide found in fraction I1 from control cells (Real = 0.60) was also identified in the Cad sample.
Fractions IV, V, and VI contained salts, but no detectable sugar.
In hemagglutination-inhibition assays using group AI red cells and the D. biflorus lectin, 1.0 pg of fraction I1 or 111 from Cad was sufficient to inhibit agglutination, whereas the fractions from control cells were inactive.
The sugar composition of the products obtained after Pelimination is given in Table I. The predominant oligosaccharide in fraction I11 from control (RGal = 0.60) has a carbohydrate composition close to that found for the sialic acid-rich  (14) using a Varian 1400 gas chromatograph equipped with flame ionization detector. Trifluoroacetyl derivatives of methylglycosides were separated on a glass column (300 X 0.6 cm) filled with OV-210 5% silicon on chromosorb W (HP) DMCS, 100-200 mesh. Nitrogen flux was 15 ml/min, and the column temperature was raised at 2 "C/min, from 100 to 210 "C. Only traces of L-FUC and Glc were detected.
The @-elimination products obtained after treatment of the glycophorin A preparation were fractionated on a Bio-Gel P-6 column as described under "Experimental Procedures" and as illustrated in Elution pattern of glycophorin A oligosaccharidealditols from Bio-Gel P-6 column. The borate-free products obtained after alkaline-borohydride treatment of glycophorin A from control (0) and Cad (0) erythrocyte membranes were solubilized in 1 ml of 1% acetic acid and applied to Bio-Gel P-6 column (1.5 X 50 cm) equilibrated in 1% acetic acid. Fractions of 1 ml were collected and monitored for radioactivity. I to VI, the six fractions which were pooled, concentrated, and further processed for analysis. tetrasaccharide structure isolated by Thomas and Winzler (18) from the main human red cell membrane glycoprotein (see Table I). Fraction 111 isolated from Cad cells contains nonreduced GalNAc in addition to the sugars of control fraction 111. The carbohydrate composition of Cad fraction I11 (Table I) indicates that the main species of this fraction might represent a pentasaccharide structure. Fraction I1 from Cad cells has a carbohydrate composition essentially identical with that of fraction 111. Since Cad fraction I1 contained a single oligosaccharide species ( R m = 0.45), this fraction was further studied by methylation analysis and 'H-NMR spectroscopy.
The molar ratios of the monosaccharide methyl ethers obtained by methanolysis and acetylation of the permethyl- GalNAcNMe-ol were found in the molar ratios of 2.1:1.2:0.9:1. From the nature of the latter derivative it can be concluded that GalNAc-ol bears substituents at C-3 and C-6. Moreover, the oligosaccharide from Cad fraction I1 contains 1 GalNAc residue and 2 NeuAc residues in terminal, nonreducing positions. The identification of the methyl 2,6-di-OMe-3,4-di-OAc-galactoside indicates that the Gal residue is substituted at positions C-3 and C-4.
The second NeuAc residue present shows its H-3 signals at 6 = 1.933 ppm (H-3.J and 6 = 2.681 (H-3eq). It should be noted that the H-3,, triplet is partly obscured by the singlet at 6 = 1.908 ppm stemming from contaminating acetate (see Fig. 2). The N-acetyl singlet of this NeuAc residue coincides with that of the aforementioned a(2+6)-linked residue (6 = 2.032 ppm). From comparison with the data for the ganglioside oligosaccharide I13NeuAc-GgOse3 (see Table 11), it can be inferred that this very typical set of H-3 chemical shifts, in particular, 6H-3,, = 1.93 ppm, points to the occurrence of a so-called internal sialic acid residue (19,25). That means NeuAc is a(2+3)-linked to a Gal residue that bears also a 0- According to sugar and methylation analysis, a GalNAc residue should be the substituent at C-4 of Gal. This is supported by the presence of an N-acetyl signal in the NMR spectrum at 6 = 2.025 ppm (see Fig. 2). However, at 300 K, only one anomeric signal could be observed (at 6 = 4.561 ppm) instead of the two expected; thus, no H-1 doublet for the GalNAc residue was observable. In order to attempt visual- The former signal is attributed to the GalNAc anomeric proton, on the basis of comparison with the ganglioside oligosaccharides (see Table 11). In consequence, the latter doublet belongs to Gal H-1.
Thus, the Cad I1 pentasaccharide-alditol can be conceived as an extension of the disialo tetrasaccharide-alditol with a GalNAc residue in p( 14)-linkage to Gal. Attachment of this GalNAc residue results in a downfield shift for H-1 of the substituted Gal (A6 = 0.02 ppm). For a similar extension, namely, that from G a l p ( l 4 ) G l c (lactose) (26) to GgOse3, hardly any shift effect was observed for H-1 of Gal. The aberrant shift effect observed in this study might be due to the crowdedness of the C-3,C-4-disubstituted Gal residue in the Cad pentasaccharide.
The results from the present investigation demonstrate clearly that the blood group Cad specificity is associated with a pentasaccharide, the structure of which could be deduced from 500-MHz 'H-NMR analysis in combination with methylation analysis: GalNAc~(14)[NeuAca(2+3)]Gal@(l-t

3)[NeuAca(24)]GalNAc-ol.
This oligosaccharide represents a novel mucin-type structure, originally being attached via an 0-glycosidic linkage from GalNAc to Ser and/or Thr residues of glycophorin A. It is not known how many of such pentasaccharide chains are present on a single glycophorin A molecule. However, the 3,000-dalton increase in apparent molecular mass of glycophorin A from Cad erythrocytes in comparison to controls (8,9) strongly suggests that most of the 15 Ser/Thr-linked 0-glycosidic oligosaccharide chains normally present on this glycoprotein (27) might be of that novel type. Nevertheless, isolation of minor components after alkalineborohydride treatment indicates that also some chains with a lower content in NeuAc or GalNAc, or both, might be present.
Since the pentasaccharide isolated from Cad red cells is a potent inhibitor of D. biflorus lectin, the specificity of this lectin reported to be highly specific for terminal a-linked GalNAc (28)