scholarly journals Comparative study of glycophorin A derived O-glycans from human Cad, Sd(a+) and Sd(a-) erythrocytes

1985 ◽  
Vol 232 (3) ◽  
pp. 813-818 ◽  
Author(s):  
D Blanchard ◽  
C Capon ◽  
Y Leroy ◽  
J P Cartron ◽  
B Fournet
Keyword(s):  
Blood Group ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Red Cells ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Methylation Analysis ◽  
Dolichos Biflorus ◽  
Major Acid

Glycophorin A was purified from the erythrocyte membranes of blood group Cad, Sd(a+) and Sd(a-) donors and the oligosaccharide alditols, obtained after alkaline borohydride degradation, separated by h.p.l.c. on an alkylamine silica gel column, were characterized by sugar analysis. Structure determination of the major acid components by methylation analysis, g.l.c.-m.s. and 1H-n.m.r. indicated that the three blood group Cad red cells under study (samples Cad., Bui. and Des.) carry the same pentasaccharide GalNAc(beta 1-4)[NeuAc(alpha 2-3)]Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc -ol(Cad determinant) but in different amounts. This pentasaccharide, however, was absent from glycophorin A of Sd(a+) and Sd (a-) donors, suggesting that the Sda determinant is not associated with glycophorins. It was calculated that glycophorin A from the original Cad donor (Cad.) carries about 12 O-glycosidically linked pentasaccharide chains per molecule whereas only 2-3 of these chains were present in the samples from the two other unrelated Cad individuals (Bui. and Des.) It is well known from quantitative agglutination studies that the proportion of red cells which can be agglutinated by the Dolichos biflorus lectin varies from one Cad blood sample to another. Some are completely agglutinated (Cad. donor) whereas others are only partially agglutinated (Bui. and Des. donors) suggesting that some red cells might not carry the Cad determinants. From the results presented above and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis studies it is suggested that Cad red cells from Bui. and Des. do not carry a mixture of glycophorin A molecules with or without the Cad pentasaccharides but a spectrum of glycoprotein molecules with varying amounts of Cad determinants.

scholarly journals Glycophorin A in two patients with congenital dyserythropoietic anemia type I and type II is partly unglycosylated.

2000 ◽  
Vol 47 (3) ◽  
pp. 773-779 ◽  
Author(s):  
E Zdebska ◽  
M Adamczyk-Popławska ◽  
J Kościelak
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glycosylation Site ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Type I ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Significant Deficit ◽  
Preliminary Extraction

Glycophorins A from erythrocyte membranes of two patients with congenital dyserythropoietic anemia type I and type II (CDA type I and II) were analyzed for carbohydrate molar composition employing a modification of the recently published method that allowed simultaneous determination of carbohydrates and protein in electrophoretic bands of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Zdebska & Kościelak, 1999, Anal Biochem., 275, 171-179). The modification involved a preliminary extraction of erythrocyte membranes with aqueous phenol, subsequent electrophoresis and analysis of the extracted glycophorins rather than electrophoresis and analysis of the glycophorin from intact erythrocyte membranes. The results showed a large deficit of N-acetylgalactosamine, galactose, and sialic acid residues in glycophorin A from patients with CDA type I and type II amounting to about 45% and 55%, respectively. The results strongly suggest that glycophorin A in these patients is partly unglycosylated with respect to O-linked glycans. In addition, glycophorin A from erythrocytes of a patient with CDA II but not CDA I exhibited a significant deficit of mannose and N-acetylglucosamine suggesting that its N-glycosylation site was also partly unglycosylated.

scholarly journals Miltenberger Class I and II erythrocytes carry a variant of glycophorin A

1983 ◽  
Vol 213 (2) ◽  
pp. 399-404 ◽  
Author(s):  
D Blanchard ◽  
A Asseraf ◽  
M J Prigent ◽  
J P Cartron
Keyword(s):  
Blood Group ◽  
Rare Variants ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Class I ◽  
Glycophorin A ◽  
M Gene ◽  
Periodic Acid Schiff ◽  
Unusual Variant

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.

scholarly journals Association of human erythrocyte membrane glycoproteins with blood-group Cad specificity

1982 ◽  
Vol 207 (3) ◽  
pp. 497-504 ◽  
Author(s):  
J P Cartron ◽  
D Blanchard
Keyword(s):  
Blood Group ◽  
Erythrocyte Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Receptors ◽  
Erythrocyte Membranes ◽  
Molar Ratio ◽  
Membrane Glycoproteins ◽  
Acetylneuraminic Acid ◽  
Group B

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of erythrocyte membranes from a blood-group-B individual with the rare Cad phenotype indicates a lower-than-normal mobility of the main sialoglycoproteins, suggesting an increase in apparent molecular mass of 3kDa and 2kDa respectively for glycoprotein alpha (synonym glycophorin A) and glycoprotein delta (synonym glycophorin B). Since the chief structural determinant of Cad specificity is N-acetylgalactosamine, the membrane receptors have been isolated by affinity binding on immobilized Dolichos biflorus (horse gram) lectin. The predominant species eluted from the gel was the abnormal glycoprotein alpha, whereas in control experiments no material could be recovered from the adsorbent incubated with group-B Cad-negative erythrocyte membranes. After partition of the membranes with organic solvents, the blood-group-Cad activity was found in aqueous phases containing the sialoglycoproteins, but not in the organic phases containing simple or complex glycolipids, which, however, retained the blood-group-B activity. The carbohydrate composition of highly purified lipid-free glycoprotein alpha molecules prepared from Cad and control erythrocytes was determined. Interestingly the molar ratio of N-acetylneuraminic acid to N-acetylgalactosamine was equal to 2:1 in the case of controls and equal to 1:1 in the case of Cad erythrocytes. Taken together these results suggest that Cad specificity is defined by N-acetylgalactosamine residues carried by the alkali-labile oligosaccharide chains attached to the erythrocyte membrane sialo-glycoproteins.

scholarly journals Multiplicity of molecules carrying blood-group-I antigen on erythrocyte membranes

1979 ◽  
Vol 181 (3) ◽  
pp. 533-538 ◽  
Author(s):  
R A Childs ◽  
T Feizi ◽  
Y Tonegawa
Keyword(s):  
Electrophoretic Mobility ◽  
Blood Group ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Galactose Oxidase ◽  
Erythrocyte Membranes ◽  
Band 3 Protein ◽  
Group I ◽  
Membrane Components

A human serum containing a monoclonal anti-(blood-group I) antibody was used to investigate the distribution of blood-group-I antigen on erythrocyte membrane components. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis profiles of immuneprecipitates by using 3H-labelled (by the galactose oxidase/NaB3H4 method) and 125I-labelled solubilized stroma were compared. Different radioactive profiles were revealed by the two radiolabelling methods. In the immunoprecipitates the predominant 125I radioactivity within the gel had the electrophoretic mobility of Band-3 protein (apparent mol.wt. 90 000–100 000), whereas the 3H radioactivity revealed a diffusely migrating component(s) (apparent mol.wt. range 40 000–70 000) in addition to radioactivity compatible with glycolipids at the dye front. The diffusely migrating 3H-labelled component was shown to have a similar electrophoretic mobility to a subpopulation of erythrocyte poly(glycosyl)ceramides with blood-group-I activity.

Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4802-4807 ◽  
Author(s):  
Chandrashekhara Manithody ◽  
Philip J. Fay ◽  
Alireza R. Rezaie
Keyword(s):  
Protein C ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Factor Va ◽  
Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

Enzymatic Determination of the Anomeric Structures of Two Blood-Group B Active Glycosphingolipids Recombined with Apolipoproteins

1978 ◽  
Vol 33 (1-2) ◽  
pp. 73-78 ◽  
Author(s):  
Peter Hanfland ◽  
Gerd Assmann ◽  
Heinz Egge
Keyword(s):  
Blood Group ◽  
Water Solubility ◽  
Erythrocyte Membranes ◽  
High Density Lipoproteins ◽  
Composition Analysis ◽  
Glycosidic Linkage ◽  
Anomeric Configuration ◽  
Enzymatic Determination ◽  
Group B

Abstract Anomeric configuration of oligosaccharides usually is established by specific glycosidases. For this purpose detergents achieving water solubility of primarily insoluble glycosphingolipids as substrates have been replaced by delipidated hum an serum high density lipoproteins. The new method, tested by several well characterized glycosphingolipids and glycosidases, finally was applied to the evaluation of anomeric structures of two blood-group B active glycosphingolipids [ceramide hexa-saccharide (B-I) and ceramide octasaccharide (B -II)] from hum an erythrocyte membranes. In both B-I and B-II, α-glycosidic linkage was dem onstrated for the term inal galactose and fucose residues. β-glycosidic linkage has been evaluated for backbone saccharides. Together with the results pre­ viously obtained by composition analysis, linkage analysis and sequence analysis the following complete structure can be established:B -I: Galα1 → 3Gal (2 ← 1αFuc)β1 → 4GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 1Cer;B-II: Galα1 → 3Gal (2 ← 1αFuc)β1 → 4GlcNAcβ1 → 3Galβ1 → 4GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 1Cer.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

scholarly journals Metabolic dependence of protein arrangement in human erythrocyte membranes. I. Analysis of spectrin-rich complexes in ATP-depleted red cells

Blood ◽  
1978 ◽  
Vol 51 (3) ◽  
pp. 385-395 ◽  
Author(s):  
J Palek ◽  
SC Liu ◽  
LM Snyder
Keyword(s):  
Human Erythrocyte ◽  
Polyacrylamide Gel Electrophoresis ◽  
Band 3 ◽  
Atp Depletion ◽  
Red Cells ◽  
Erythrocyte Membranes ◽  
Red Cell ◽  
Aerobic Conditions ◽  
Spontaneous Formation ◽  
Human Erythrocyte Membranes

Abstract The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. We investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing two-dimensional SDS- polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited (1) an increase in components 4.5 and 8 and globin subunits, (2) a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9, and (3) a large molecular weight (greater than 10(6) daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH levels with glucose, adenine, and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase- deficient fresh erythrocytes that showed marked GSH depletion but preserved greater than 70% of the original ATP level. However, membranes of these cells did not contain the greater 10(6) dalton aggregate with a high spectrin to band 3 ratio. We concluded that the formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.

scholarly journals The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes

1980 ◽  
Vol 187 (3) ◽  
pp. 677-686 ◽  
Author(s):  
T Tsuji ◽  
T Irimura ◽  
T Osawa
Keyword(s):  
Blood Group ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Band 3 ◽  
Carbohydrate Moiety ◽  
Erythrocyte Membranes ◽  
Gel Chromatography ◽  
Branching Points ◽  
Human Erythrocyte Membranes ◽  
Group A

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1→4GlcNAc beta 1→3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.

scholarly journals Abnormal blood-group-Ss-active sialoglycoproteins in the membrane of Miltenberger class III, IV and V human erythrocytes

1979 ◽  
Vol 183 (2) ◽  
pp. 193-203 ◽  
Author(s):  
D J Anstee ◽  
W J Mawby ◽  
M J A Tanner
Keyword(s):  
Blood Group ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Normal Blood ◽  
Crossing Over ◽  
Class Iii ◽  
Class V ◽  
Unequal Crossing Over

1. We have studied the inherited changes occurring in the sialoglycoproteins of membranes from erythrocytes of type Miltenberger Class III (Mi.III), Miltenberger Class IV (Mi.IV) and Miltenberger Class V (Mi.V) by using sodium dodecyl sulphate/polyacrylamide gel electrophoresis and lactoperoxidase radioiodination. 2. Mi.III erythrocytes lack the normal blood-group-Ss-active sialoglycoprotein but contain an unusual s-active sialoglycoprotein of higher apparent molecular weight. A similar abnormal S-active sialoglycoprotein appears to occur in Mi.IV erythrocytes. 3. The Mi.V condition is associated with the hemizygous absence of both the normal blood-group-MN-active sialoglycoprotein and the normal Ss-active sialoglycorprotein. However, a new sialoglycoprotein component is present in these cells that has properties characteristic of both the MN-active and Ss-active sialoglycoproteins. 4. Our results suggest that the new sialoglycorportein present in Mi.V erythrocytes is a hybrid of the normal MN sialoglycoprotein and an s-active sialoglycoprotein that has properties similar to the s-active sialoglycoprotein found in Mi.III erythrocytes. We suggest that the unusual Mi.V sialoglycoprotein is derived from chromosomal misalignment with unequal crossing-over between the genes for the MN- and Ss-active sialoglycoproteins in a manner similar to that which gives rise to haemoglobin Lepore. 5. Further studies of S-s-erythrocytes confirm that these cells lack normal Ss-active sialoglycoprotein, but contain an unusual component that shows some of the properties of the normal Ss-active sialoglycoprotein. 6. Analysis of erythrocytes of type Mk/Mi.III confirms that, in addition to the known hemizygous lack of the MN-active sialoglycoprotein, the Mk condition is also associated with a loss of the Ss-active sialoglycoprotein. 7. In order to facilitate discussion of the complex changes that occur in these variant erythrocytes, a new unified nomenclature is used for the erythrocyte sialoglycoproteins.

Sign in / Sign up

Export Citation Format

Share Document