The Role of Free Amino Groups in the Blood Group Activity of M and N Mucoids

Abstract

1. The M and N blood group substances were treated with different amino-group reagents, such as : acetic-, succinic-, and phthalic-anhydrides, benzyloxycarbonylchloride, dinitrofluorobenzene, nitrous acid and 0-methyl-isourea.2. It was shown that these reagents blocked or destroyed 2-4 free amino groups (mainly lysine E-NH, groups) per one subunit of M or N blood group substance of mol. wt. 30,000. No other effects were found under the conditions used.3. The blocking or decomposition of amino groups, connected with the loss of their positive charge, lowered the blood group activity of M and N mucoids and did not decrease their activity towards phytoagglutinins and the inhibitory activity of viral hemagglutination. The guanidylation of amino groups did not destroy the blood group activity of M and N mucoids.The M and N blood group substances from human erythrocytes have been known as the sialoglycoproteins [l]. They exhibit three types of serological activity : (a) the specific blood group activity towards anti-M and anti-N rabbit immune sera; (b) the less specific activity towards anti-M and anti-N phytoagglutinins; (c) the inhibitory activity of hemagglutination by influenza viruses. The activities (a) and (c) were lost when terminally situated N-acetylneuraminic acid (NANA) was set free by neuraminidase or by mild acid hydrolysis [2,3], but activity towards phytoagglutinins was even increased after removal of NANA [4,5]. I n this paper the evidence is presented for the specific role of free amino groups, especially of free 8-NH, groups of lysine residues, in the blood group activity of M and N blood group substances. MATERIALS AND METHODSThe M, N, and MN blood group mucoids were obtained from human erythrocytes of blood group OM, ON, or OMN respectively, by phenol extraction of ghosts [I].Acetic-, succinic-, and phthalic-acid anhydrides, benzyloxycarbonylchloride and dinitrofluorobenzene were commercially available products. 0-methylisourea was a gift of dr. K. Nowak from the Dept. Enzyme. Neuraminidase (EC 3.2.1 .IS).Neuraminidase from Vibrio cholerae was the product of Behringwerke.Anti-M and anti-N rabbit immune sera were supplied by the State Laboratory of Sera and Vaccines (Warsaw). The extract from the seeds of Vicia granzinea, containing anti-N phytoagglutinins, was prepared according to van Wageningen and Nijenhuis IS]. The M and N blood group activity and the N , activity, (activity towards V . graminea anti-N phytoagglutinins), were determined by an inhibition of hemagglutination : the various dilutions of the studied preparation were incubated for 30 minutes a t a room temperature with equal volumes of anti-serum or V . graminea extract, followed by the addition of double volumes of 2 O/ , , erythrocyte suspension; the degree of agglutination was recorded after 30 minutes. The activity was expressed as the minimal concentration (mglml) of the blood group substance, giving the complete inhibition of serum or V . graminea extract of the titre 1/8-1/16. Influenza virus Lee was obtained from the Dept. o...

Modification of Amino Groups of Human‐Erythrocyte Glycoproteins and the New Concept on the Structural Basis of M and N Blood‐Group Specificity

“…The measurements of absorbance at 420 nm after modification with 2-methoxy-5-nitrotropone revealed that 9-11 nitrotroponyl microequivalents were introduced to 100mg of the glycoprotein. These values were in accord with our previous data [13]. The release of reversibly blocking residues was achieved in 50-70% of the total number of modified amino groups.…”

“…The small amount of specific receptors among many unspecific oligosaccharide chains could justify the fact that no conclusive chemical evidence for the difference in structure of oligosaccharides of M and N glycoproteins has been reported so far. We found [13] that modification of amino groups of the glycoproteins abolished their capacity to inhibit rabbit anti-M and anti-N sera, and this finding was confirmed later by Ebert et ul. [4].…”

The Degradation of M and N Blood Group Glycoproteins and Glycopeptides with Alkaline Borohydride

“…The reactivity with rabbit immune sera was found to be dependent on the presence of free amino groups of lysine residues, but the reactivity with phytoagglutin i n s from V . graminea was not affected by the modification of amino groups [20]. Although lysine was found in peptides liberated during the alkaline degradation the loss of both activities suggested the participation of the described trisaccharide and disaccharide structures in the reaction with rabbit sera and phytoagglutinins, respectively.…”

“…Determination of the blood group activity with rabbit immune sera and anti-N phytoagglutinins from Vicia gramineu was described earlier [20]. The inhibition of viral hemagglutination was estimated by the method of Tamm and Horsfall [21], influenza virus A, was obtained from the Dept.…”

Modifications of N-Acetylneuraminic Acid and Their Influence on the Antigen Activity of Erythrocyte Glycoproteins

“…Arabitol was used as internal standard. The chromatographic separation was performed with the liquid phase methylphenylsilicone (OV 17) 20/, (w/w) on Chromosorb G. Temperature ("c) phate buffer, p H 7, according to Lisowska and Morawiecki [8], modified by Merz and Roelcke [9]. Electrophoresis on cellulose acetate strips was carried out in sodium veronal buffer, pH 8.6, 25 min a t 250 V, 5 mA.…”