Complete amino acid sequence and homologies of human erythrocyte membrane protein band 4.2.

Abstract

The complete amino acid sequence for human erythrocyte band 4.2 has been derived from the nucleotide sequence of a full-length 2.35-kilobase (kb) cDNA. The 2.35-kb cDNA was isolated from a human reticulocyte cDNA library made in the expression vector Agtll. Of the 2348 base pairs (bp), 2073 bp encode 691 amino acids representing 76.9 kDa (the SDS/PAGE molecular mass is 72 kDa). RNA blot analysis of human reticulocyte total RNA gives a message size for band 4.2 of 2.4 kb. The amino acid sequence of band 4.2 has homology with two closely related Ca2+-dependent crosslinking proteins, guinea pig liver transglutaminase (proteinglutamine y-glutamyltransferase; protein-glutamine:amine vglutamyltransferase, EC 2.3.2.13) (32% identity in a 446-amino acid overlap) and the a subunit of human coagulation factor XIII (27% identity in a 639-amino acid overlap), a transglutaminase that forms intermolecular y-glutamyle-lysine bonds between fibrin molecules. The region of greatest identity includes a 49-amino acid stretch of band 4.2, which is 69% and 51% identical with guinea pig liver transglutaminase and the a subunit offactor XIII, respectively, within the regions that contain the active sites of these enzymes. Significantly, within the five contiguous consensus residues of the transglutaminase active site, Gly-Gln-Cys-Trp-Val, band 4.2 has an alanine substituted for cysteine (which is apparently essential for activity). Consistent with this active site substitution, erythrocyte membranes or inside-out vesicles, which contain band 4.2, show no evidence of transglutaminase activity by two types of in vitro assay.Band 4.2 is a major protein (5% by weight) of the human erythrocyte membrane (1). This protein has an apparent mass of 72 kDa on SDS gels but forms oligomers both in solution and on the erythrocyte membrane (2, 3). Previous studies from our laboratory have shown that band 4.2 binds to the 43-kDa cytoplasmic domain of band 3, the erythrocyte anion transport protein (3,4). While this binding is likely responsible for the association of band 4.2 with the cytoplasmic surface of the membrane, band 4.2 also has other, lessunderstood associations. We have shown that band 4.2 can bind to purified erythrocyte ankyrin in solution with a Kd -10-7 M and may also associate with band 4.1 and spectrin (4). Binding measurements and competition studies suggest that ankyrin and band 4.2 bind to distinct sites on the cytoplasmic domain of band 3 (4). While we and others have not found any effect of band 4.2 on ankyrin association with erythrocyte membranes (4-6), other studies suggest that band 4.2 may stabilize ankyrin-band 3 associations (7).While the exact function of band 4.2 on the erythrocyte membrane is not known, individuals whose erythrocytes lack or are deficient in band 4.2 have hemolytic anemia associated with spherocytic or elliptocytic erythrocytes (7,8). Absence of band 4.2 associated with spur or target erythrocytes has also been reported (9). These findings suggest that band 4.2 probably plays an important role ...

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2)

“…6 The peripheral membrane protein, protein 4.2, shares significant homology to transglutaminases but lacks the catalytic triad residues required for transglutaminase activity. [7][8][9] Stable expression of protein 4.2 in erythrocytes depends on its interaction with the band 3 and ankyrin-1.…”

Novel AE1 mutations in recessive distal renal tubular acidosis. Loss-of-function is rescued by glycophorin A.

“…Lux, Harvard Medical School) and glycophorin A (GPA; M-allele) in pSG5 (gift from S.L. Spitalnik, University of Pennsylvania, Philadelphia, PA) were described previously (5,16,17). The G701D AE1 cDNA plasmid was constructed by four-primer PCR methods.…”

Antigen Specificity of the Humoral Immune Response toMycoplasma haemofelisInfection

“…A 71-to 72-kDa protein band was detected in the M. haemofelis and RBC membrane antigen preparations by all preand postinfection plasma samples but was not detected when the secondary antibody was used alone. This protein band has a molecular mass equivalent to that for erythrocyte protein band 4.2 (16,24) and correlated with band 4.2 identified on a Commassie blue-stained polyacrylamide gel of RBC membrane proteins. This band was not of a size that would be consistent with the immunoglobulin light chains or ␥ or heavy chains identified in the blot of the pooled plasma sample.…”