Effect of endoglycosidase F-peptidyl N-glycosidase F preparations on the surface components of the human erythrocyte

Abstract

Endo- N-acetyl-β- d-glucosaminidase F-Peptidyl N-glycosidase F preparations (abbreviated Endo F) and endo-β- d-galactosidase were used to study the major human erythrocyte membrane glycoproteins and the components carrying the blood group A, B, Rhesus (D), and Duffy (Fy a) antigens. The results are consistent with the known presence of an N-glycosyl-linked oligosaccharide on sialoglycoprotein α and the absence of such an oligosaccharide from sialoglycoprotein δ. Under the conditions used, only a portion of the N-glycosyl-linked oligosaccharides on band 3 molecules were cleaved by Endo F alone or by Endo F in combination with endo-β- d-galactosidase. Immunoblotting experiments showed that treatment of red cells with Endo F alone had little effect on the components carrying blood group A and B antigen activity. However, Endo F used in combination with endo-β- d-galactosidase caused a substantial reduction in the binding of monoclonal anti-A and anti-B antibodies. The results clearly show that sialoglycoproteins α and δ carry little or no blood group A or B activity. Endo F alone, or in combination with endo-β- d-galactosidase, had no effect on the electrophoretic mobility of the Rh(D) polypeptide, supporting previous suggestions that this membrane polypeptide is unusual in not being glycosylated. Endo F had a dramatic effect on the electrophoretic mobility of the component(s) carrying blood group Fy a activity. The diffuse Fy a component of M r 38 500–90 000 was sharpened to a band of M r 26 000. Either endo-β- d-galactosidase or neuraminidase treatment reduced the M r of the Fy a component(s) but did not significantly sharpen the bands, suggesting that the Fy a component contains between 40–50% by mass of N-glycosyl-linked oligosaccharides.