David J. Anstee

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

Endo-N-acetyl-beta-D-glucosaminidase F-Peptidyl N-glycosidase F preparations (abbreviated Endo F) and endo-beta-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 (Fya) antigens. The results are consistent with the known presence of an N-glycosyl-linked oligosaccharide on sialoglycoprotein alpha and the absence of such an oligosaccharide from sialoglycoprotein delta. 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-beta-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-beta-D-galactosidase caused a substantial reduction in the binding of monoclonal anti-A and anti-B antibodies. The results clearly show that sialoglycoproteins alpha and delta carry little or no blood group A or B activity. Endo F alone, or in combination with endo-beta-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 Fya activity. The diffuse Fya component of Mr 38,500-90,000 was sharpened to a band of Mr 26,000. Either endo-beta-D-galactosidase or neuraminidase treatment reduced the Mr of the Fya component(s) but did not significantly sharpen the bands, suggesting that the Fya component contains between 40-50% by mass of N-glycosyl-linked oligosaccharides.

Mutations in the second zinc finger of human EKLF reduce promoter affinity but give rise to benign and disease phenotypes

Mutations in the human erythroid Krüppel-like factor (EKLF) can lead to either anemia or the benign InLu phenotype. To elucidate the relationship between these mutations and the differing phenotypes, we prepared recombinant forms of wild-type and 5 mutant EKLF proteins and quantitated their binding affinity to a range of EKLF-regulated genes. Missense mutants (R328H, R328L, and R331G) from persons with InLu phenotype did not bind DNA. Hence, as with the heterozygous loss of function nonsense (L127X, S270X, and K292X) and frameshift (P190Lfs and R319Efs) EKLF mutations, monoallelic loss of EKLF does not result in haploinsufficiency at all loci. In contrast, K332Q has a slightly reduced DNA binding affinity (∼ 2-fold) for all promoters examined but exhibits a phenotype only in a compound heterozygote with a nonfunctional allele. E325K also has a reduced, but significant, binding affinity, particularly for the β-globin gene but results in a disease phenotype even with the wild-type allele expressed, although not as a classic dominant-negative mutant. E325K protein may therefore actively interfere with EKLF-dependent processes by destabilizing transcription complexes, providing a rational explanation for the severity of the disease phenotype. Our study highlights the critical role of residues within the second EKLF zinc finger domain.

Transient reduction in erythrocyte membrane sialoglycoprotein β associated with the presence of elliptocytes

Erythrocyte membranes from an anaemic patient receiving gold therapy for rheumatoid arthritis had reduced β‐sialoglycoprotein (β‐SGP) content but normal expression of sialoglycoproteins α, δ and γ. Elliptocytes were present in the peripheral blood. The serum of the patient contained anti‐β‐SGP which did not appear to bind to her own cells. It reacted with all erythrocytes apart from β‐SGP deficient Leach phenotype cells. The antibody was inhibited by purified β‐SGP from normal red cells, bound to β‐SGP on immunoblots and also reacted with the abnormal β‐related‐SGP in erythrocyte membranes of both the Gerbich type and Yus type of Gerbich negative. Two years later the patient was no longer anaemic, no elliptocytes were seen in her peripheral blood film and her erythrocyte membranes had normal β‐SGP content. Antibody was no longer present in her serum and antibody from the earlier sample now reacted with the patients erythrocytes.

Normal membrane function of abnormal β-related erythrocyte sialoglycoproteins

Summary. Red cells totally deficient in β and γ sialoglycoproteins (the Leach type of Gerbich‐negative) are elliptocytic and have altered membrane physical properties as evidenced by marked decreases in both membrane mechanical stability and membrane deformability. Red cells from individuals who are of the Gerbich and Yus phenotypes of Gerbich‐negative are also deficient in β and γ sialoglycoproteins, but possess abnormal β‐related sialoglycoproteins. In order to determine if these β‐related sialoglycoproteins can functionally substitute for normal sialoglycoproteins, we measured membrane deformability and stability of red cells of the Gerbich and Yus phenotypes. In contrast to the red cells of the Leach phenotype, cells of Gerbich and Yus phenotypes were found to have normal membrane deformability and stability. Moreover, flow cytometric analysis using a monoclonal anti‐β antibody revealed that the Gerbich and Yus phenotype red cells expressed the β‐related sialoglycoprotein to the same extent as its normal counterpart on normal cells. Based on these data, we suggest that the abnormal β‐related sialoglycoproteins can functionally substitute for normal β and γ sialoglycoproteins.