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The erythrocyte receptors for S-fimbriated Escherichia coli, which causes sepsis and meningitis in newborn infants, were investigated. Neuraminidase and trypsin treatments of erythrocytes abolished the hemagglutination ability of the bacteria. To identify the receptor glycoproteins, we separated erythrocyte membrane proteins by gel electrophoresis, blotted them to nitrocellulose, and incubated them with 1251-labeled bacteria. The only bacterium-binding bands identified corresponded to glycophorin A dimer and monomer, and the binding was abolished by neuraminidase treatment of the blot. Radiolabeled bacteria also bound to purified glycophorin A adsorbed to polyvinyl chloride microwells, and the binding was inhibited by other sialoglycoproteins and isolated sialyloligosaccharides containing the NeuAcoa2-3Gal sequence. Oligosaccharides which contain the NeuAca2-3GalPi1-3GalNAc and NeuAcat2-3Gal1-3(NeuAca2-6)GalNAc sequence and which are identical to the 0-linked saccharides of glycophorin A were twofold more effective inhibitors of binding than were other oligosaccharides containing the NeuAca2-3Gal sequence. The replacement of sialic acid in asialoerythrocytes with a purified GalP1-3GalNAc a2-3 sialyltransferase, which forms the 0-linked NeuAcx2-3GalP1-3GalNAc sequence in asialoglycophorins, restored bacterial hemagglutination. These results indicated that the major erythrocyte receptor for S-fimbriated E. coli is the NeuAca2-3Gal1-3GalNAc sequence of the 0-linked oligosaccharide chains of glycophorin A.

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Peptides are designated according to the sialoglycoprotein (MN, Ss or D = glycophorins A, B and C, respectively) from which they were derived by trypsin (= T), chymotrypsin (= C), Staphylococcus aureus V8 protease (= V) or cyanogen bromide (= B) digestion, the elution during gel filtration (1 etc) and the MN or Ss blood type of the red cells. Presumable genotypes are given in italics. Some peptides were desialylated (= d) prior to a second enzyme treatment. The terms A and B are used to designate peptides of different lengths derived from the same part of the sequence.

Experimental data are discussed which indicate that one alkali-labile tetrasaccharide present on each MN-glycoprotein subunit is part of the Mand N-antigen receptor site. It is concluded therefore that the difference between Mand N-antigens resides in the amino acid sequence of the glycoprotein. Based on the different topographical distribution of the MNSs-receptors within the membrane an explanation for the cross-reaction of anti-N reagents with ordinary M-erythrocytes is proposed. E 0.8 -0 PAS-I '0 0.6 -m m u1 GL T 0 0 0.2 0.4 0.6 0.8 I .o Apparent mol, Wt X I O -~ 95 70 50 41 37 35 28 24 Relative mobility Designation A: commassie band III PAS-I =dimeric MN-glycoprotein B broad rather weakly PAS-staining region r

SUMMARY En(a‐) and EnaEn red cells from different sources were studied using biochemical and serological methods. The results suggest that Finnish En(a–) erythrocytes contain only ‘N’ but no M antigenic properties. Data on the members of the English En(a‐) family suggest that English En(a‐) red cells exhibit a combination of three rare alterations. The English En(a–) individuals are apparently heterozygous for the defects En and Mk. In addition, the ‘N’ receptor on the Ss glycoprotein is, to all appearances, converted to a M (‘M’) antigen. These extraordinary data are brought to light by investigations on the children of the propositus (G.P. and J.P.), whose red cells are M‐N+S‐s+‘N’+‘M’‐ and M‐N+S+s+‘N’+‘M’+ respectively. Sodium dodecylsulphate polyacrylamide gel electrophoretic results indicate that the MN glycoprotein content is decreased by about 50 % in all English S+s+EnaEn red cells, the Ss glycoprotein being normal. G.P. erythrocytes, however, have only about half of the normal MN and Ss glycoprotein content.

Periodate oxidation and Smith degradation of desialized human erythrocyte glycoproteins lead to an almost complete destruction of galactose and some loss of galactosamine and glucosamine. Neutral sugar and hexosamine content of native MN substances are only slightly influenced by periodate oxidation. Destruction of galactose is accompanied by an uncovering of A-like receptor sites detectable by agglutinins from plants and snails, which are directed against terminal N-acetylgalactosamine. From the specificity of these antibody-like substances, it is deduced that the amino sugar functioning as receptor is a-glycosidically linked. Destruction of the receptor sites by subsequent alkaline borohydride treatment suggests that the N-acetylgalactosamine is unmasked by degradation of the alkali-labile tetrasaccharide of human erythrocyte glycoproteins. Periodate-oxidized and Smith-degraded NANA-free erythrocyte and ovarian cyst glycoproteins treated by acetolysis are potent inhibitors of anti-Tn from man, animals, and plants, suggesting that Tn-antigen is represented by terminal, a-glycosidic, peptide-bound N-acetylgalactosamine. It is concluded that Tn-antigen is probably a cryptic determinant of the alkali-labile tetrasaccharide, which has been isolated from human erythrocyte glycopeptides.

The amino acid sequence of the N-terminal tryptic glycopeptide from a minor human erythrocyte membrane sialoglycoprotein (component D or glycophorin C) was determined by manual sequencing. The glycosylation sites were identified by a new procedure for the detection of the glycosylated derivatives released by Ednian degradation. The fragment, comprising 47 residues, was found to contain an average of about 12 0-glycosidically linked oligosaccharides and one asparagine-linked carbohydrate chain. An identical hexapeptide sequence occurring in two regions of the glycopeptide provides evidence that it has developed by an internal gene duplication during evolution. In addition, a part of its structure shows a striking similarity to the sequence of a certain region of the M N and Ss erythrocyte membrane sialoglycoproteins (glycophorins A and B), suggesting that the molecules might be related. Preparation of PeptidesThe tryptic glycopeptide DTI was isolated as described previously [3,9,10]. Reduction followed by CNBr cleavage and protease treatments were performed as in earlier publications [9,10]. Carboxypeptidase Y digestion was carried out in 0.1 M ammonium acetate buffer (pH 5.5 for peptides Bl A1 and B3A1; pH 6.5 for DTI and B4ii) at an enzyme: substrate ratio of 1 :25 for up to 24 h at room temperature. N-Acetylation of peptides was performed in 0.1 M N-ethylmorpholine/acetate buffer (pH 9.0) by the slow addition of 10 pl of acetic acid anhydride (diluted with 100 PI of methanol) per ml. The pH was maintained at a value of about 9.0 by the addition of NaOH solution. Desialylation was carried out in 0.1 M HCI for 80 min at 80 "C, under nitrogen. Under these conditions, the Asp-Pro bonds in DTI were found to be cleaved to an extent of about 40 "/;:. Incubation for 4 h or treatment with 75 n~i (viv) formic acid for 72 h at 45 C [I 71 was used for cleavage of these bonds. Carbodiimidejglycine ethyl ester treatment of DTI was carried out according to Hoare and Koshland [18]. The reference DABTH derivatives of glycine-amidated aspartic and glutamic acid were prepared by treatment of N-tert-butoxycarbonyl-L-aspartic acid n-benzyl ester or the corresponding derivative of L-glutamic acid (Sigma Chem. Corp., St Louis, USA). The protecting groups were removed by incubation in 66% (v/v) pyridine and trifluoroacetic acid for 24 h and 15 min, respectively. at 5 2 ' C . The DABTH derivatives were prepared and separated by thin-layer chromatography (19,201. Gel filtration of peptides was performed as described [lo]. Peptides were also separated by DEAE-cellulose chromatography. The sample in 10 mM pyridine/acetate buffer (pH 7.0) was applied to a column (1.6 x 5.0 cm) equilibrated with the same buffer. The column was eluted with a linear gradient of pyridine/acetate (pH 7.0; total volume 200 ml) up to 0.1 M. Subsequently, a pH gradient was applied, which consisted of 200 ml of pyridine/acetate (0.1 M) in the mixing vessel (pH 7.0) and reservoir (pH 5.0). Column effluents were assayed for absorbance at 225 nm. tryptophan flu...