Kathryn M. John

ANION EXCHANGER 1 (BAND 3) IS REQUIRED TO PREVENT ERYTHROCYTE MEMBRANE SURFACE LOSS BUT NOT TO FORM THE MEMBRANE SKELETON

The red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1-lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis.

On the Protein Defect in Abetalipoproteinemia

Abstract The plasma lipoproteins from a patient with abetalipoproteinemia were delipidated and fractionated; the lipid-free proteins (apoproteins) were compared by physical, chemical and immunologi...

A Genetic Defect in the Binding of Protein 4.1 to Spectrin in a Kindred with Hereditary Spherocytosis

Indirect evidence suggests that the genetic defect in hereditary spherocytosis lies in the erythrocyte membrane skeleton, a submembranous meshwork of proteins (principally spectrin, actin, and protein 4.1) responsible for membrane shape and structural stability. To test this premise we systematically assayed the interactions of spectrin, the major skeletal protein, in six kindreds with autosomal dominant hereditary spherocytosis. In one these kindreds, enhancement of spectrin-actin binding by protein 4.1 was reduced, owing to a 39 +/- 4 per cent decrease (mean +/- S.D) in the binding of normal protein 4.1 by spectrin, in all of four members with the disorder. The defective spectrin was separated into two populations by affinity chromatography on immobilized normal protein 4.1. One population (41 +/- 2 per cent) lacked the ability to bind 4.1, but the other functioned normally. Presumable, the nonfunctional spectrin was the product of the autosomal dominant gene responsible for the hereditary spherocytosis in this kindred.

Mild spherocytosis and altered red cell ion transport in protein 4.2–null mice

Protein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4. 2-null (4.2(-/-)) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2(-/-) RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4. 2(-/-) RBCs are normal. Band 3 and band 3-mediated anion transport are decreased. Protein 4.2(-/-) RBCs show altered cation content (increased K+/decreased Na+)resulting in dehydration. The passive Na+ permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4. 2(-/-) RBCs are significantly increased. Protein 4.2(-/-) RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2(-/-) RBCs compared with controls. The increased passive Na+ permeability of 4.2(-/-) RBCs is also dependent on cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport.

A New Spectrin, βIV, Has a Major Truncated Isoform That Associates with Promyelocytic Leukemia Protein Nuclear Bodies and the Nuclear Matrix

We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10–16 of β-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length β-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin βIV, thus encodes a full-length minor isoform (SpβIVΣ1) and a truncated major isoform (SpβIVΣ5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpβIVΣ5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpβIVΣ5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin βIV is the first β-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds.

Effects of free fatty acid on the fluorescence of bovine serum albumin

Abstract The ultraviolet fluorescence emission spectrum of bovine serum albumin was altered when free fatty acid (FFA) was added to the protein. This occurred when FFA was taken up from aqueous soap solutions, n -heptane solutions, rat epididymal fat pads, or fat emulsions undergoing enzymatic hydrolysis. As the molar ratio of FFA to albumin increased, the maximum emission was shifted to a shorter wave length and the fluorescence intensity was decreased. The blue shift did not exceed 7 mμ, and the maximum reduction in fluorescence intensity was less than 50% even when the molar ratio of FFA to albumin was high. Qualitatively similar results were obtained with FFA of 6–22 C atoms, but smaller effects occurred when FFA of less than 10 C atoms were used. FFA produced little or no change in the fluorescence intensity when the pH was less than 5.5 or greater than 10. FFA produced changes in the fluorescence spectrum of porcine albumin but had little effect on that of human, rabbit, equine or canine albumins.

Identification of the lipid-binding cyanogen bromide fragment from the cystine-containing high density apolipoprotein, ApoLP-Gln-II

Abstract ApoLP-Gln-II is one of the major protein constituents of human plasma high density lipoproteins (HDL). This protein and its two CNBr fragments, C-III (carboxyl-terminal) and C-IV (amino-terminal) were tested for ability to bind phosphatidyl choline by the formation of lipid-protein complexes of density 1.063 to 1.210 gm/ml, by changes induced in circular dichroism, and by the inhibition of the reactivation of delipidated mitochondrial β-hydroxybutyric dehydrogenase. In all three of these experimental procedures, C-III but not C-IV retained the ability to bind phosphatidyl choline. These findings suggest that the phospholipid binding site(s) of apoLP-Gln-II may be localized in the carboxyl-terminal portion of the molecule.

Further characterization of the polymorphic forms of a human high density apolipoprotein, apoLP-Gln-I (apoA-I)

Abstract The major apoprotein of human high density lipoproteins that has previously been designated apoLp-Thr, on the basis of its proposed carboxylterminal amino acid, was isolated and fractionated into 2–6 polymorphic forms. The subfractions had identical amino acid compositions, electrophoretic mobilities, molecular weights, and were immunochemically identical. They had aminoterminal aspartic acid and carboxylterminal glutamine. According to the carboxylterminal convention of nomenclature, “apoLp-Thr” and apoLp-Gln (a second major high density apolipoprotein) should be redesignated apoLp-Gln-I and apoLp-Gln-II, respectively.

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.

THE ROLE OF SPECTRIN AND ACTIN IN IRREVERSIBLY SICKLED CELLS: UNSICKLING OF “IRREVERSIBLY” SICKLED GHOSTS BY CONDITIONS WHICH INTERFERE WITH SPECTRIN-ACTIN POLYMERIZATION

We have previously shown that irreversibly sickled cells (ISCs) form ISC-shaped ghosts and ISC-shaped membrane skeletons which suggests that an alteration in the membrane skeleton of the ISC may be responsible for its abnormal shape. In the present studies we observe that ISC-shaped ghosts become round in hypertonic media (>400 mM NaCl), hypotonic media (10 to 50 mM NaCl), and isotonic NaCl containing small amounts of Zn 2+ (0.1 to 0.5 mM). This ISC-shape reversal is time and temperature dependent and does not correlate with the elution or proteolysis of any membrane protein(s) detectable on SDS-gels. Conditions which promote ISC ghosts reversal also inhibit the formation of spectrin-actin complex(es). This suggests that ISCs may be stabilized by abnormal interactions between the spectrin and/or actin components of the membrane skeleton and that disruption of these bonds may allow the skeleton to resume a normal shape.