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Biomedical subjects

C G Gahmberg

Publications and source records attributed to C G Gahmberg.

At least 127 records · Page 7Linked to original sources
PubMed1982

Role of sialic acid in the mobility of membrane proteins containing O-linked oligosaccharides on polyacrylamide gel electrophoresis in sodium dodecyl sulfate.

The major sialoglycoprotein of the human red-cell membrane, glycophorin A, contains 15 O-glycosidically linked oligosaccharides and one N-glycosidic oligosaccharide. The protein shows a decreased mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate after neuraminidase treatment of the non-denatured protein. The molecular mechanism behind this phenomenon has been elucidated. Neuraminidase treatment of glycophorin A in intact cells or after solubilization in buffers containing Triton X-100 resulted in conversion of the predominant tetrasaccharide N-acetylneuraminosyl alpha 2-3galactosyl beta 1-3(N-acetylneuraminosyl alpha 2-6)-N-acetylgalactosamine to the trisaccharide galactosyl beta 1-3(N-acetylneuraminosyl alpha 2-6)-N-acetylgalactosamine and the disaccharide galactosyl beta 1-3-N-acetylgalactosamine. After denaturation with sodium dodecyl sulfate, Vibrio cholerae neuraminidase also liberated the N-acetylgalactosamine-bound sialic acids. Such treatment resulted in increased electrophoretic mobility. The results show that distal sialic acids linked to galactose are readily available to neuraminidase, and that their negative charge gives an increased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In contrast, most of the N-acetylgalactosamine-linked sialic acids of glycophorin A are not liberated by neuraminidase without denaturation of the substrate. Like sialic acids of complex-type oligosaccharides the decreased electrophoretic mobility caused by them is exclusively due to their mass while no significant contribution by the charge was seen.

Chemical Phenomena↗
PubMed1982

Acute erythroleukaemia with L3 morphology and the 14q+ chromosome.

L3 morphology according to the FAB classification and the 14q+ chromosome are usually ascribed to the Burkitt type of leukaemia or lymphoma with a B or pre-B cell phenotype. We report here a case of adult acute leukaemia with Burkitt morphology and the 14q+, which did not express lymphoid markers. Instead, the leukaemia was shown to be an acute erythroleukaemia. The erythrocyte marker glycophorin A was present on the surface of the leukemic blasts as shown by immunofluorescence and immunoprecipitation from membrane lysates of surface labeled cells with antiglycophorin A antiserum. Spectrin and fetal hemoglobin appeared after cultivation of the blasts in the presence of sodium butyrate. The present case shows that a short term cultivation is sometimes useful for further characterization of the commitment of acute leukaemias.

Acute Disease↗
PubMed1982

Pericellular matrix and cell surface glycoproteins of virus-transformed mouse epithelial cells.

Mouse embryo Mus musculus castaneous epithelial cells, transformed with Moloney murine sarcoma virus (MSV) or with ecotropic murine leukemia virus (MuLV), were analyzed for production of pericellular matrix glycoproteins. The nontransformed, MSV-transformed, and MuLV-transformed cells produced fibronectin, laminin, type I collagen, and small amounts of type III collagen when studied by immunofluorescence using specific antibodies. The virus-transformed epithelial cells produced enhanced amounts of fibronectin into their growth media. Nontransformed M. musculus castaneous epithelial cells mainly produced type I collagen, as shown by metabolic labeling and polypeptide analysis. A significant increase in the glycoprotein production was seen by the MuLV-transformed cells, whereas small changes in the collagen production were apparent after MSV transformation. MuLV-transformed cells produced increased amounts of type I collagen and also some collagenous polypeptides that comigrated with procollagen type IV chains. The ratio of the procollagen type I chains deposited in the matrix was altered in transformed cells. Radioactive surface labeling of the cells revealed changes of the high-molecular-weight glycoproteins in both the MSV- and the MuLV-transformed cells. Unlike virus-transformed fibroblastic cells, these transformed epithelial cells deposited and retained connective tissue glycoproteins in their pericellular matrices. The results indicate that viral transformation modulates the pericellular matrix and surface glycoproteins of cultured mouse epithelial cels. The ability of virus-transformed epithelial cells to deposit pericellular matrices is a major difference between them and virus-transformed fibroblastic cells.

Animals↗
PubMed1981

Organization of membrane lipids and proteins in human En(a-) erythrocytes that lack the major sialoglycoprotein, glycophorin A. A spin-label study.

Membrane fluidity was studied by electron-spin-resonance techniques in human En(a-) erythrocytes that lack the major membrane sialoglycoprotein, glycophorin A. By using stearic acid spin labels with a doxyl group in the C-12 or C-15 positions, we demonstrated that the hydrophobic core in these cells was more fluid than in normal cells. Surface-located regions in isolated En(a-) membranes, when probed with stearic acid labelled in the C-5 position, appeared more stable than in normal membranes. In isolated En(a-) membranes, protein motion was decreased when probed with a nitroxide derivative of maleimide. After incubation with anti-(glycophorin A) antibodies protein motion and membrane fluidity were increased in normal membranes. This effect was observed also after spectrin depletion, which by itself increased protein motion but decreased membrane fluidity in the hydrophobic core of the membrane. The results show that membrane proteins influence the fluidity of membrane lipids.

Blood Group Antigens↗
PubMed1981

Biosynthesis of the major human red cell sialoglycoprotein, glycophorin A. A review.

The human leukemia cell line K562 is erythroid and expresses the major red cell sialoglycoprotein, glycophorin A. With this cell line we have studied the biosynthesis of glycophorin A after pulse-chase labeling with [35S] methionine. Using lectin-Sepharose affinity chromatography and immune precipitation with specific anti-glycophorin A antiserum followed by polyacrylamide slab gel electrophoresis a precursor of glycophorin A was visualized. This had an apparent molecular weight of 37000 and contained an incompleted N-glycosidic oligosaccharide and unfinished O-glycosidic oligosaccharides. After chase for 10 min, the completed glycophorin A with an apparent molecular weight of 39000 was seen and it appeared at the cell surface in about 30 min. Using tunicamycin N-glycosylation was inhibited but not O-glycosylation. The absence of the N-glycosidic oligosaccharide did not affect the migration of the protein to the cell surface but the yield of glycophorin A was diminished. Translation of glycophorin A messenger-RNA was achieved in a rabbit reticulocyte cell-free system. This yielded a non-glycosylated protein with an apparent molecular weight of 19500, which exceeded that of the glycophorin A apoprotein with about 5000. This indicates the presence of a "signal sequence" in the preprotein. When the translation was performed in the presence of microsomal membranes from dog pancreas the glycophorin A apoprotein aws both N-and O-glycosylated and the apparent molecular weight (37000) of the synthesized protein was identical to that of the precursor obtained from cells.

Amino Acid Sequence↗
PubMed1981

Blood-group A and B determinants are located in different polyglycosyl peptides isolated from human erythrocytes of blood-group AB.

The distribution of blood-group A and B determinants was studied by isolating blood-group ABH-active polyglycosyl peptides from delipidated human blood-group AB erythrocyte membranes after extensive digestion with pronase followed by chromatography on Bandeiraea simplicifolia I (BsI) lectin coupled to Sepharose. 20% of the polyglycosyl peptides were bound to BsI lectin. The glycopeptides bound were further fractionated using the blood-group-A-specific lectin from Vicia cracca (Vc). Approximately half of these were bound to the Vc lectin. The glycopeptides, which were bound to the Vc column, were not bound to the blood-group-B-specific isolectin from B. simplicifolia (BsIB4) whereas the Vc-unbound glycopeptides readily bound. The results indicate that in the polyglycosyl peptides isolated from AB erythrocytes A and B determinants are located in different carbohydrate chains. The polyglycosyl peptides, which did not bind to BsI lectin, were composed on the average of 30 monosaccharide units and those that bound contained on the average 55 monosaccharide units. The sugar composition was similar in both fractions except that N-acetylgalactosamine was found only in the BsI-bound glycopeptides. The substitution patterns of the monosaccharides were quite similar in both fractions except 2,3-O-linked galactose, which was enriched 7.5-fold in the BsI-bound glycopeptides and 3,6-O-linked galactose, which also enriched in the BsI-bound glycopeptides suggesting that these have a more branched structure than the BsI-unbound glycopeptides. Glycopeptides derived from bands 3 and 4.5 were prepared from A1B-blood-group erythrocyte membranes and fractionated as above. 25% of the glycopeptides were bound to BsI-lectin from both samples. 70% of the BsI-bound material from band 3 was bound to Vc lectin and 60% from band 4.5. The results indicate heterogeneity in the glycosylation of these bands.

ABO Blood-Group System↗
PubMed1981

Cell-free synthesis and glycosylation of the major human-red-cell sialoglycoprotein, glycophorin A.

The human erythroid cell line, K562, synthesizes the major red cell sialoglycoprotein, glycophorin A. We have isolated an mRNA fraction which codes for glycophorin A from K562 cells and studied the synthesis of the sialoglycoprotein in a rabbit reticulocyte cell-free system. In the absence of membranes a precursor form of glycophorin A was synthesized. This was identified using specific anti-(glycophorin A) serum. The apparent molecular weight of the carbohydrate-free precursor of glycophorin A was 19 500. This exceeds the molecular weight of the glycophorin A apoprotein by approximately 5000. In the presence of membranes from dog pancreas, the synthesized glycophorin A precursor was N-glycosylated and probably also O-glycosylated. The oligosaccharide chains remained incomplete and the glycoprotein synthesized in vitro corresponded to the glycosylated precursor of glycophorin A obtained in intact cells.

Animals↗
PubMed1981

Expression of blood group A antigens in human bone marrow cells.

We have studied the appearance of blood group A-activity during hematopoiesis in human bone marrow cells by the use of the blood group A-specific lectin from Vicia cracca. Cells that bound the lectin were identified using antiserum against the lectin followed by rosetting with protein A-containing Staphylococcus aureus cells. Only cells of the erythroid lineage from blood group A individuals formed staphylococcal rosettes. A-activity occurred in basophilic normoblasts and later stages of erythropoiesis, whereas pronormoblasts were negative. The appearance of blood group A-activity coincided roughly with the onset of hemoglobin synthesis and slightly later than the expression of the major sialoglycoprotein of erythrocytes, glycophorin A. Glycophorin A did not, however, contain blood group A-activity when analyzed by immunoprecipitation and gel electrophoresis.

ABO Blood-Group System↗
PubMed1980

Rotational diffusion of band 3 proteins in membranes from En(a-) and neuraminidase-treated normal human erythrocytes.

Recent experiments have demonstrated an association between band 3 and glycophorin A in the human eythrocyte membrane (Nigg, E.A., Bron, C., Girardet, M. and Cherry, R.J. (1980) Biochemistry 19, 1887-1893). Here, the influence of sialoglycoproteins on the rotational diffusion of band 3 in the human erythrocyte membrane was investigated by studying membranes from En(a-) and neuraminidase-treated erythrocytres. Rotational diffusion was measured by observing flash-induced transient dichroism of eosin-labeled band 3. Although erythrocytes of the rare phenotype En(a-) lack the major sialoglycoprotein, glycophorin A, no significant difference in band 3 rotation at pH 7.4 and 37 degrees C could be detected between En(a-) and normal erythrocyte membranes. Band 3 rotation at pH 7.4 was also insensitive to the enzymatic removal of sialic acid from the surface of normal erythrocytes. Moreover, the existence of an essentially similar temperature-dependent equilibrium between band 3 proteins with different mobilities was observed in normal, En(a-) and neuraminidase-treated erythrocytes. From these results it is concluded that glycophorin A contributes less than 15% to the cross-sectional diameter of the band 3-glycophorin A complex in the plane of the normal membrane. The rotation of the complex at pH 7.4 is not significantly influenced by either steric or electrostatic interactions involving the oligosaccharide moiety of glycophorin A.

Diffusion↗
PubMed1980

Isolation and characterization of the blood group A-specific lectin from Vicia cracca.

We have isolated the blood group A-specific lectin from Vicia cracca by affinity chromatography on immobilized porcine blood group A/H substance. A molecular weight of 100 000 was obtained by gel filtration and analytical ultracentrifugation. The subunit size when determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 29 000. The lectin contains no half-cystine or methionine. It is a glycoprotein apparently with one oligosaccharide per subunit. The oligosaccharide contains mainly D-mannose and D-glucosamine but no D-galactose or sialic acid.

ABO Blood-Group System↗