Analysis of erythrocyte membrane proteins in patients with hereditary spherocytosis and other types of haemolytic anaemia (1)
Hereditary spherocytosis (HS) occurs frequently among patients with congenital haemolytic anaemia and is associated with abnormalities of the erythrocyte cell membrane. Band 3 protein is also known to penetrate or traverse the lipid bilayer more than 13 times; 50–60% of the protein is within the erythrocyte membrane and 30–40% of the protein is exposed on the intracellular membrane, with only a limited portion of the protein in extracellular membrane.
All of the patients with haemolytic anaemia had major glycoprotein of spectrin, ankyrin, band 3, protein band 4.1, and band 4.2 by SDS-PAGE and were compared with healthy controls. After deglycosylation by N-glycosidase F, the band 3 patterns of patients with HS pre- or post-splenectomy disappeared at 97 kDa, suggesting band 3 glycoprotein from patients with HS could be easily decomposed by N-glycosidase F.
In patients with HS post-splenectomy, other types of haemolytic anaemia, and in healthy controls, the amount of band 3 was not decreased, as shown by SDS-PAGE. The bands (band 3 and spectrin) for patients with HS nearly disappeared in band 3 only after deglycosylation by N-glycosidase F; however, patients with other types of haemolytic anaemia and healthy controls showed a narrow band of 90 kDa near the region of band 3 (97 kDa) by western blotting.
The intracellular domain of band 3 proteins in patients with HS, pre-, and post-splenectomy were thickly stained in a ring-shaped pattern. However, in patients with other types of haemolytic anaemia and healthy controls, staining in a thin, ring-like pattern was observed.
Similarly, western blotting analysis showed that degraded spectrin fragments were found at 120 and 90 kDa in cases HS1(pre-splenectomy stage), HS3, HS5, and were shown in very small quantity in HS7, HS8, HS9, and HS10 with post-splenectomy cases. The three-dimensional structure of the band 3 polypeptide chain may have undergone a conformational change.
