Band 4.1-like proteins of the bovine lens. Effects of differentiation, distribution and extraction characteristics.

Bovine lens epithelium, cortex and nucleus were screened for the presence of red-cell-membrane band 4.1-like proteins by using an immunoblot method. Lens epithelial cells were found to contain proteins of Mr 78 000 and higher (approximately 150 000) that cross-reacted with anti-(protein 4.1) sera. Fibre cells of the superficial cortex were also found to contain these two proteins, as well as an additional protein of approx. 80 000 Mr. In contrast, deep layers of the cortex and the lens nucleus contained no detectable cross-reactive protein at these Mr values. Treatment of a crude membrane fraction prepared from superficial bovine cortices with a low-ionic-strength buffer resulted in release of the high-Mr band 4.1-like protein. The 80 000- and 78 000-Mr proteins remained with the membrane fraction in low-ionic-strength buffer, but were released into solution by high-ionic-strength-buffer treatment. We have also demonstrated that the human red-blood-cell membrane, like lens epithelial cells and fibre cells, also contains a high-Mr band 4.1-like protein that is released from membranes by low-ionic-strength-buffer treatment.     

Spectrin, fodrin and protein 4.1-like proteins in differentiating rat germ cells

The presence and the distribution of proteins of the membrane skeleton in differentiating germ cells of the rat has been investigated. Immunofluorescence and immunoblotting analysis, performed using monoclonal and polyclonal antibodies to human erythroid alpha-spectrin and protein 4.1 and to brain spectrin (fodrin), demonstrated the presence of analogues of spectrin and fodrin in spermatocytes and round spermatids and of protein 4.1-like molecules in spermatocytes, spermatids and spermatozoa. Spectrin and fodrin showed molecular weights comparable to those of their analogues in erythrocytes but a distinct intracellular distribution. Fodrin was localized along the plasma membrane while spectrin appeared associated with the regions of the Golgi apparatus and of the developing acrosome. Antibodies to protein 4.1 recognized molecules with a molecular weight not comparable with that in erythrocytes, and their presence in spermatozoa was confined to specific regions of the head and of the tail.     

Presence and localization of proteins immunologically related to erythrocyte protein 4.1 in human skin

Summary Analogues of human erythrocyte protein 4.1 have been examined in the human skin by immunochemical techniques using anti-human erythrocyte protein 4.1 antibodies. Immunoblot analysis revealed that human epidermis contains 4.1-like proteins of 80 kDa and 78 kDa that cross react with anti-protein 4.1 antibodies.Analysis with immunofluorescence microscopy revealed that the plasma membrane of the human epidermal keratinocyte was stained intensively in the basal cells, whereas spinous cells were moderately stained. It is noted that eccrine sweat gland cells and ductal cells were also stained in the peripheral cytoplasma. Taken together, these results demonstrate that 4.1-like proteins are present in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells. The present findings enable us to suggest that a membrane skeletal protein lattice might exist in these cells.     

Presence and localization of proteins immunologically related to erythrocyte protein 4.1 in human skin

Analogues of human erythrocyte protein 4.1 have been examined in the human skin by immunochemical techniques using anti-human erythrocyte protein 4.1 antibodies. Immunoblot analysis revealed that human epidermis contains 4.1-like proteins of 80 kDa and 78 kDa that cross react with anti-protein 4.1 antibodies. Analysis with immunofluorescence microscopy revealed that the plasma membrane of the human epidermal keratinocyte was stained intensively in the basal cells, whereas spinous cells were moderately stained. It is noted that eccrine sweat gland cells and ductal cells were also stained in the peripheral cytoplasma. Taken together, these results demonstrate that 4.1-like proteins are present in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells. The present findings enable us to suggest that a membrane skeletal protein lattice might exist in these cells.     

Properties of red cell membrane proteins: mechanism of spectrin and band 4.1 interaction

Interactions between human red cell's band 4.1 and spectrin were studied by fluorescence resonance energy transfer and batch microcalorimetry techniques. The association constant (Ka = 8.6 X 10(7) M-1), the stoichiometry (one molecule of band 4.1 to one molecule of spectrin), the reversibility, and the enthalpy (delta H = -6 kcal/mol) were determined. A proton uptake was observed to take place as a result of the spectrin-band 4.1 complex formation. In addition to the protonation of the reaction products, the entropic contribution (-T delta S) has been observed to be responsible for approximately 50% of the binding free energy. We concluded that the environment plays a significant role in the stabilization of the complex. Since band 4.1 has been required for the maintenance of the cytoskeletal stability, small alterations of the binding energies or the degree of interaction could have a pronounced effect on the structure of the erythrocyte membrane.     

Membrane proteins related to anion permeability of human red blood cells

Summary (³H)DIDS (4,4-diisothiocyano-2,2-ditritiostilbene-disulfonate) was used as a convalent label for membrane sites involved in anion permeability. The label binds to a small, superficially located population of sites, about 300,000 per cell, resulting in almost complete inhibition of anion exchange. The relationship of biding to inhibition is linear suggesting that binding renders each site nonfunctional. In the inhibitory range less than 1% of the label is associated with lipids but at higher concentrations of DIDS, the fraction may be as high as 4%. In ghosts, however, treatment with (³H)DIDS results in extensive labeling of lipids. In cells, a protein fraction that behavens on SDS acrylamide gels as thought its molecular weight is 95,000 daltons (95K) is predominatly labeled by (³H)DIDS. The only other labeled protein is the major sialoglycoprotein which contains less than, 5% of the total bound (³H)DIDS. Because of the linear relationship of binding to inhibition and the unique architecture of the site, it is suggested that the (³H)DIDS-binding site of the 95K protein is the substrate binding site of the anion transport system. The 95K protein is asymmetrically arranged in the membrane with the sites arranged on the outer face accessible to agent in the medium. In leaky ghost, only a few additional binding sites can be reached from the inside of the membrane in the 95K protein, in contrast to the extensive labeling of other membrane proteins in ghosts as compared to cells.Abbreviations DADS 4,4-Diamino-2,2-dihydrostilbene disulfonic acid - DIDS 4,4-Diisothiocyano-2,2-stilbene disulfonic acid - (³H)DADS 4,4-Diamino-2,2-ditritiostilbene disulfonic acid - (³H)DIDS 4,4-Diisothiocyano-2,2-ditritiostilbene disulfonic acid     

Selective solubilization of proteins from red blood cell membranes by protein perturbants

Isolated human erythrocyte membranes were exposed to a series of reagents known to modify or perturb proteins; these included sodium hydroxide, lithium diiodosalicylate, acid anhydrides, and organic mercurials. Each reagent liberated the same set of relatively polar polypeptides from the membrane, while the other, more hydrophobic species invariably remained associated with the membrane residue. The selective elution pattern was precisely that seen previously with 6 M guanidine hydrocloride. The released polypeptides, comprising half of the membrane protein mass, contained no carbohydrate; current evidence indicates that all of these components are confined to the cytoplasmic surface of the membrane. The residue contained all the lipids and all the glycoproteins. The latter are accessible to the outer membrane surface and, in at least two cases, seem to extend asymmetrically across the thickness of the membrane. Thus, the distinctive elution behavior which defines these two groups of polypeptides relates both to their chemical composition and their organizational disposition in the membrane.     

Localization of 4.1 related proteins in cerebellar neurons

Localization of 4.1 related proteins in neurons was studied with immunofluorescence microscopy and with immunoelectron microscopy on ultrathin cryosections. In rat cerebellum, 4.1 immunoreactive proteins were demonstrated in Purkinje cell bodies, dendrites and other neurons in the cerebellar cortex. Some glial cells showed staining, but no labeling was found in myelinated axons of the white matter and of the glomeruli in the granule cell layer. At the ultrastructural level, the 4.1 related proteins were localized mainly in the cytoplasmic matrix, while some labeling was found underneath the plasma membrane. To determine whether 4.1 related proteins in neuronal cytoplasm exist as part of the cytoskeleton or not, PC12 cells cultured in the presence of nerve growth factor were stained with the anti-4.1 antibody. Since cytoplasmic staining was retained after detergent treatment, the 4.1 related proteins seem to exist as a component of the neural cell cytoskeleton. Localization of 4.1 related proteins during the postnatal development of the cerebellum was also studied. In Purkinje cells, localization of 4.1 related proteins changed according to the stages of the postnatal development. The present data suggest that 4.1 related proteins in neurons localized mainly in the cytoplasm and may play some role in organizing cytoskeletal networks in the cytomatrix. Their distribution is developmentally regulated in some neurons, possibly in relationship to their maturation in the cytoskeleton.     

Primary structure of barwin: a barley seed protein closely related to the C-terminal domain of proteins encoded by wound-induced plant genes.

Barwin is a basic protein with pI above 10 and molecular mass 13.7 kDa isolated from aqueous extracts of barley seed. The complete amino acid sequence of 125 residues has been determined by a combination of conventional protein sequencing, plasma desorption mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. Three disulfide bridges have been localized as Cys31-Cys63, Cys52-Cys86, and Cys66-Cys123 both by 1H nuclear magnetic resonance spectroscopy and by plasma desorption mass spectrometry. The N-terminal residue was identified as pyroglutamate. Barwin is closely related to a peptide segment of 122 residues at the C-terminal region of the proteins encoded by two wound-induced genes in potato plants, win1 and win2, and a protein encoded by the hevein gene of rubber tree. In 77 sequence positions of 125 the barwin, win1, win2, and hevein protein sequences have amino acid sequence identity, when two gaps--one of two residues allowing for the insert of Gly23 and Ala24 and one allowing for the insert of Thr97 in the barwin sequence--are introduced in the latter three. The close sequence similarity with the proteins encoded by the wound-induced potato and rubber tree genes and the ability of the protein to bind saccharides suggest that barwin might belong to a group of proteins involved in a common defense mechanism in plants.     

The human RD protein is closely related to nuclear RNA-binding proteins and has been highly conserved

We have isolated cDNA clones encoding the human RD protein, which is closely related to several known nuclear RNA-binding proteins. The RD protein contains a 60-amino acid (aa) tract almost entirely of alternating basic and acidic aa, (RD)_n, primarily arginine (Arg; R) and aspartic acid (Asp; D). The protein also contains an ‘RNP sequence domain’. Arg-rich tracts and the RNP sequence domain are both features of nuclear RNA-binding proteins. However, we have been unable to detect RNA-binding by the human RD protein. The very strong evolutionary conservation of the mammalian RD protein as sequence suggests that it plays an important role in the cell.     

Glutamic-pyruvic transaminase activity related to red blood cell age.

Red blood cell glutamic-pyruvic transaminase (GPT) phenotypes and catalytic activities were studied in normal subjects and in patients with various hemolytic syndromes associated with reticulocytosis. To assess the effect of cell age of GPT activity, young cells were separated from older cells by centrifugation, and the catalytic activities were compared. In normal blood, there was a progressive fall in GPT activity from the top layer (younger cells) to the bottom layer (older cells), with a mean ratio of 1.90 plus or minus 0.42. Similarly, in the blood of patients with reticulocytosis, the enzyme activity of the reticulocyte-rich layer was higher than that of the layer containing older cells (mean ratio 1.94 plus or minus 0.95).     

Calcium-dependent peripheral localization of 4.1-like proteins and fodrin in cultured human keratinocytes

Summary— Recently, several proteins immunologically related to erythrocyte membrane skeletal proteins, such as protein 4.1 and fodrin (non-erythroid spectrin), have been found in keratinocytes. In the present study, in order to investigate the roles of these proteins in cell-cell contact, we analyzed the distribution of non-erythroid protein 4.1, β-fodrin and actin in cultured human keratinocytes at low (0.15 mM) and standard (1.85 mM) Ca²⁺ concentrations. Immunofluorescence microscopy revealed that immunoreactive forms of protein 4.1, β-fodrin and actin filaments were present in the cytoplasm of cells cultured in low Ca²⁺ medium, while in cells in the standard Ca²⁺ medium, these proteins were localized at the cell boundary and partially in the cytoplasm. When cells in the low-Ca²⁺ medium were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 1 h, these proteins were also present at the cell boundary. Increasing extracellular Ca²⁺ concentration from low to standard in the medium induces cell-cell contact among the cultured human keratinocytes, accompanied by the translocation of protein 4.1 and β-fodrin from the cytoplasm to the membrane. On the basis of the present study, movement of membrane skeletal proteins from the cytosol to the membrane suggests that either these proteins or the membrane skeletal lattice plays an important role in the regulation of cell-cell intergigitations in response to changes in the Ca²⁺ concentrations in culture medium, and that phosphorylation of these skeletal proteins might be involved in the regulation of the membrane skeletal proteins of keratinocytes in response to Ca²⁺.     

Growth related changes to functional parameters in the bovine lens.

Dimensions, volumes and protein contents were measured for bovine lenses with wet weights ranging from 0.17-3.07 g (2 months gestation to 19 years post-natal). All increase in a non-linear fashion. The lens becomes flatter with age due to a more rapid increase in the equatorial plane, but the ratio of anterior to posterior sagittal distances remains constant (1.19). The radius of curvature increases from 4.9 to 15 for the anterior surface and from 4.4 to 13 for the posterior. Protein content increases more rapidly than volume resulting in an increased average protein concentration from around 18% in the early prenatal lens to nearly 50% in the 19 year old. Total protein content (TPC) was found to be related to wet weight (We) according to the equation, TPC = 0.3We1.33. It is suggested that TPC is a better parameter for describing growth than wet weight or age. The refractive index, in the equatorial plane, increases towards the centre, from 1.38 at the edge of the lens. The maximum index, in the centre, increases with lens size up to 1.474 in the largest lens studied. This corresponds to a protein concentration of 70%. In all lenses, refractive index and protein concentration gradients were superimposable when plotted from the outside towards the centre. The optical performance of the lenses was assessed by measuring the back focal length which increases gradually from 24 to 51.5 mm over the 0.17 to 3.07 g size range. This was attributed to the increased radii of curvature.     

Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily

The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R) is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type), and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH) proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK), non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.     

The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence

The amino acid sequence of wheatwin1, a monomeric protein of 125 residues isolated from wheat kernel (variety S. Pastore), is reported. Wheatwin1 is highly homologous (95%) to barwin, a protein from barley seed, which was shown to be related to the C-terminal domain of two proteins encoded by the wound-induced geneswin1 andwin2 in potato and to a protein encoded by the same domain of the hevein gene (hev1) in rubber tree. Similarly to barwin, wheatwin1 contains six cysteine residues all linked in disulfide bridges and the N-terminal residue is pyroglutamate. Moreover, structural studies performed on wheatwin1 andwin1 protein by predictive methods demonstrated that these proteins and barwin are closely related in the secondary structure also. The high level of homology found with the product ofwin1,win2, andhev1 genes strongly suggests that barwin and wheatwin1 play a common role in the mechanism of plant defence.     

Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structure

Recent studies of chitosan have increased the interest in its conversion to chitooligosaccharides (COSs) because these compounds are water-soluble and have potential use in several biomedical applications. Furthermore, such oligomers may be more advantageous than chitosans because of their much higher absorption profiles at the intestinal level, which permit their facilitated access to systemic circulation and potential distribution throughout the entire human body. In that perspective, it is important to clarify their effect on blood further, namely, on human red blood cells (RBCs). The aim of this work was thus to study the effect of two COS mixtures with different molecular weight (MW) ranges, <3 and <5 kDa, at various concentrations (5.0-0.005 mg/mL) on human RBCs. The interactions of these two mixtures with RBC membrane proteins and with hemoglobin were assessed, and the RBC morphology and surface structure were analyzed by optical microscopy (OM) and atomic force microscopy (AFM). In the presence of either COS mixture, no significant hemolysis was observed; however, at COS concentrations >0.1 mg/mL, changes in membrane binding hemoglobin were observed. Membrane protein changes were also observed with increasing COS concentration, including a reduction in both alpha- and beta-spectrin and in band 3 protein, and the development of three new protein bands: peroxiredoxin 2, calmodulin, and hemoglobin chains. Morphologic evaluation by OM showed that at high concentrations COSs interact with RBCs, leading to RBC adhesion, aggregation, or both. An increase in the roughness of the RBC surface with increasing COS concentration was observed by AFM. Overall, these findings suggest that COS damage to RBCs was dependent on the COS MW and concentration, and significant damage resulted from either a higher MW or a greater concentration (>0.1 mg/mL).