Highly active amino-terminal sialoglycopenta-, hexa- and heptapeptides from human erythrocyte NM antigens.

Abstract

Blood group Mand N-specific structures on human erythrocytes are carried by the hydrophilic :~-1 glycopeptides of the M and N glycoproteins [1 3]. Like the blood group ABH(O)-specific haptens [4, 5], those of the MN antigens are oligosaccharides as shown by mild acid hydrolysis [6], hemagglutination inhibition [2] and biosynthesis [7]. Most but not all Mand Nspecific groupings are located at the NH2terminus of the M and N antigens [2]. We describe here isolation and characterization of highly blood-group-active NH2terminal sialoglycopenta-, hexa-, and heptapeptides (SGPs). These novel SGPs were prepared by proteolysis with trypsin and pronase followed by column chromatography. From NN erythrocytes were obtained a sialoglyco(SG)-hexaand a SGheptapeptide, from MM red blood cells a SG-pentapeptide. All hydroxyamino acids of these three SGPs have glycosidically linked carbohydrate substituents. The Mand N-specific alkali-labile carbohydrate structures from Nand M-derived SGPs are different [2, 3] as is their amino acid composition [3, 8]. The M-derived SGP possesses, in addition to the tetrasaccharide, predominant in M and N glycoproteins, which was first described by Thomas and Winzler [9] (W.T.), one larger oligosaccharide chain. This chain, unless re-N-acetylated after the mild fl-elimination, appeared as two components on chromatographic separation. This large oligosaccharide amounted to ca. 35% of the total carbohydrate. Preparation of tryptic glycopeptides from whole erythrocytes and isolation of the c~-i SGPs was similar to our earlier procedure but was done on a larger scale. All serological and chemical .values given in the text and Tables represent arithmetic averages of at least three determinations; all carbohydrate analyses were carried out by two different standard analytical procedures [2, 6]. After rechromatography the c~-I SGP preparation moved as one component on polyacrylamide and thin-layer electrophoresis (TLE). The average yield of M-derived ~-1 SGP exceeded that of the ~-1 originating from N erythrocytes by 11% (wt.); in addition M-derived ~-1 SGP had 9% more NAN per unit weight (15 consecutive preparations of each c~-I N and e-1 M). Proteolysis of the ~-1 SGPs was with pronase (after its prior autodigestion to de,stroy possibly contaminating glycosidases). The digest was separated on two interconnected Sephadex G150 and G100 columns [2]. Five major fractions (I-V) were obtained, of which the fourth, because of its high activity of both Mas well as N-derived material and its relatively small molecular size, was studied in detail. Neither the Mnor the N-derived fractions IV were homogeneous by chromatography on Sephadex G-50 columns. Four and 5 elution peaks (SGP IV1_ 5) for Mand N-derived materials, respectively, were observed; their elution patterns were similar only for peak 1. SGP IV2M (peak 2) had highest M activity and had the NH2-terminal Ser; it had ca. 3.5% of ~-1 M SGP's total weight as well as NAN content (eight preparations average). In contrast, among the N-derived GP IV fractions, peak 1 had the highest N activity; it amounted to ca. 6% of e-1 SGP N by weight and had ca. 4% of its NAN (six preparations). The still impure SGP IV2M and IVIN were further purified by repeated DEAE-Sephadex A-25 ion exchange column chromatography using 0.I-1.0 M NH4HCO3, pH 7.8 gradient elution. SGP IV2M and SGP IV1N each yielded 5 subfractions, a-e. The most active fraction from SGP IV2M was c and from IV,N, e. Both amounted to 40 50% of the weight of the recovered materials.