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S R Carlsson

Publications and source records attributed to S R Carlsson.

33 records · Page 2Linked to original sources
PubMed1988

Characterization of a hydrophilic form of Thy-1 purified from human cerebrospinal fluid.

Thy-1 is a developmentally regulated cell surface glycoprotein in nervous tissue. An inositol-containing glycolipid structure is covalently attached to its carboxyl terminus, which anchors the protein to the cell membrane. In the present paper we report the characterization of a water-soluble form of Thy-1, purified from human cerebrospinal fluid (CSF). In contrast to the membrane-bound form of Thy-1 (M-Thy-1) isolated from human brain cerebral cortex, CSF-Thy-1 behaved like a completely hydrophilic glycoprotein, as analyzed by charge-shift electrophoresis in the presence of detergents and by liposome incorporation experiments. CSF-Thy-1 displayed a slightly higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than M-Thy-1. Digestions with endoglycosidases demonstrated that this difference in size was correlated to different processing of the three N-linked oligosaccharides, and the mobilities of the deglycosylated molecules were indistinguishable in sodium dodecyl sulfate gels. A Pronase-resistant carboxyl-terminal fragment was isolated from the CSF-Thy-1 after trypsin digestion and compared with the corresponding structure of M-Thy-1, obtained by treatment either with bacterial phosphatidylinositol-specific phospholipase C or with human serum (as a source of phosphatidylinositol-specific phospholipase D). The major fragment from CSF-Thy-1 behaved identically, with respect to size and charge, to the carboxyl-terminal fragment from M-Thy-1 solubilized by phospholipase D. These findings suggest an in vivo release of phosphatidylinositol-anchored Thy-1 glycoprotein from brain cells by the action of an endogenous phospholipase D.

Antigens, Surface↗
PubMed1988

Molecular cloning of cDNAs encoding lamp A, a human lysosomal membrane glycoprotein with apparent Mr approximately equal to 120,000.

Although several lysosomal membrane glycoproteins have been characterized by using specific antibodies, none of the studies so far elucidated the amino acid sequence of a lysosomal membrane glycoprotein. Here we describe cDNA clones encoding for one of the lysosome-associated membrane proteins with apparent Mr approximately equal to 120,000, lamp A. The amino acid sequence based on the fully coded cDNA shows that as many as 18 potential N-glycosylation sites can be found in the total of 385 amino acid residues. The results obtained by endoglycosidase F digestion support the conclusion that this glycoprotein contains 18 N-glycans. These N-glycosylation sites are clustered in two domains; one contains 10 and the other contains 8 N-glycosylation sites. These domains are separated by a (proline-serine)-rich region that has a distinct homology to the IgA hinge structure. The first N-glycosylated domain is elongated to a potential leader peptide toward the NH2-terminal end. The second N-glycosylated domain, on the other hand, is connected to a putative transmembrane portion consisting of hydrophobic amino acids. This segment, in turn, is elongated to a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end.

Amino Acid Sequence↗
PubMed1986

Isolation and characterization of leukosialin, a major sialoglycoprotein on human leukocytes.

A major sialoglycoprotein (previously called gp105) on the human erythroleukemic cell line K562 was purified, and specific antibodies were raised in a rabbit. A number of different hematopoietic cell lines belonging to erythroid, myeloid, T-lymphoid, and B-lymphoid cell lineages were found to possess glycoproteins that were immunoprecipitated by these antibodies. However, the apparent molecular weights differed between cell lines, ranging from 113,000 to 150,000. In almost all cases, the immune precipitated molecule corresponded to the major sialoglycoprotein of the respective cell. Pulse-chase experiments showed that all cells produced an early precursor form of the molecule of 54 kDa, which was susceptible to endo-beta-N-acetylglucosaminidase H to give an apoprotein of 52 kDa. Neuraminidase treatment of the mature forms resulted in a characteristic decrease of the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (apparent molecular weights from 150,000 to 183,000). Amino acid analysis of the glycoprotein isolated from HL-60 cells showed a high content of serine, threonine, and proline, and the carbohydrate composition was compatible with the presence of a large number (approximately 90) of O-linked carbohydrate chains. The name leukosialin is proposed for this sialoglycoprotein, which seems to be widely distributed, but differently glycosylated, on leukocytes with diverse functions. In the following paper (Carlsson, S.R., Sasaki, H., and Fukuda, M. (1986) J. Biol. Chem. 261, 12787-12795), we demonstrate that the structures of O-linked oligosaccharides vary significantly depending on the cells from which leukosialin was isolated.

Animals↗
PubMed1986

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines.

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.

Antigens, CD↗
PubMed1986

Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells.

O-Linked oligosaccharides were isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells by alkaline borohydride treatment. Oligosaccharides were fractionated by Sephadex G-50 gel filtration and QAE-Sephadex column chromatography, and their structures were elucidated by fast atom bombardment-mass spectrometry after permethylation and methylation analysis before and after specific exoglycosidase treatments. Results show that normal granulocytes and chronic myelogenous leukemia cells contain a series of O-linked oligosaccharides with the following structure, (formula: see text) where, in normal granulocytes n = 0 is major and n = 1 or 2, and thus polylactosaminyl oligosaccharides are present as minor components. However, these polylactosaminyl oligosaccharides were barely detectable in chronic myelogenous leukemia cells. On the other hand, acute myelogenous leukemia cells, which represent poorly differentiated myeloid cells, mainly contain short O-linked oligosaccharides with 2----6-linked sialic acid as follows. (formula: see text) These results suggest that structures of O-linked oligosaccharides vary in the different maturation stages along the same cell lineage.

Carbohydrate Conformation↗
PubMed1986

Regional and subcellular distribution of Thy-1 in human brain assayed by a solid-phase radioimmunoassay.

A solid-phase radioimmunoassay, specific for the monomeric form of human Thy-1, was developed and used for quantitation of the Thy-1 antigen in human brain tissue. Determination of Thy-1 in homogenates of 12 anatomically defined brain regions showed that Thy-1 is present throughout the human brain. However, significant variation was found in the expression of the glycoprotein in different regions. Thy-1 appears to be generally enriched within gray matter: caudate nucleus, cerebral cortex, and putamen were found to contain the highest Thy-1 concentration (approximately 2.5 micrograms Thy-1/mg protein). Interestingly, the cerebellar cortex contained only 25% of the Thy-1 concentration of cerebral gray matter. Cerebral subcortical white matter contained half the amount of Thy-1 compared to cerebral cortex. Determination of Thy-1 in subcellular fractions prepared from human brain biopsy tissue indicated that the highest relative concentration of Thy-1 is associated with synaptosomal membranes and myelin/axonal membrane fractions.

Adult↗
PubMed1986

Leukosialin, a major sialoglycoprotein on human leukocytes as differentiation antigens.

Most blood cells derived from the bone-marrow are known to possess only a limited number of heavily sialylated glycoproteins. We have recently isolated a major sialoglycoprotein on leukocytes and found that this glycoprotein, termed leukosialin, is ubiquitously present on various human leukocytes, granulocytes, monocytes/macrophages and T- and B-lymphocytes. Our studies showed that leukosialin is significantly glycosylated by O-linked oligosaccharides (90 chains/molecule). The structures of those O-linked oligosaccharides are characteristic to each cell lineage and maturation stage. The polypeptide portion of these molecules are, however, apparently the same, with a molecular size of 52 KDa. So it will be interesting to explore the possibility that leukosialin expresses different functions by having different O-glycosylation in a variety of hematopoietic cells.

Antigens, CD↗
PubMed1985

Changes in glycan branching and sialylation of the Thy-1 antigen during normal differentiation of mouse T-lymphocytes.

The glycans of the Thy-1 antigen present on thymocytes and lymph-node T-lymphocytes were investigated after external labelling of the cells. Neuraminidase, endoglycosidase H and endoglycosidase F were used in combination with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing in order to characterize the nature of the glycans on 125I-labelled and immunoprecipitated Thy-1. Glycopeptides were prepared from Thy-1 obtained from cells labelled by periodate/boro[3H]hydride treatment. The glycopeptides were separated by affinity chromatography on concanavalin A-Sepharose and analysed by gel filtration. The results show that both types of cells possess Thy-1 molecules with three N-linked carbohydrate chains, of which one is of 'high-mannose' type and the other two of triantennary and biantennary 'complex' type. The ratio of triantennary/biantennary chains was decreased on Thy-1 of mature cells compared with that of immature cells, but instead more sialic acid was present on these chains. Deglycosylated Thy-1 appeared to be of the same size regardless of origin, indicating that only the carbohydrate moiety differs between Thy-1 molecules of the two cell types.

Animals↗
PubMed1985

Activation of Lyt-2+ T cells by antibodies towards brain-associated antigens. I. Accessory cell requirement and role of Fc receptors in the induction of reactivity to interleukin 2.

The present report describes a system where essentially all Lyt-2+ T cells are selectively activated by rabbit anti-mouse brain antibodies (RaMB) to interleukin 2 (IL 2) reactivity. High efficiency of RaMB-mediated induction was obtained by a 5 h incubation with antibodies at high cell density of Sephadex G-10-nonadherent spleen cells. No in situ production of IL 2 by RaMB-treated cells was detected, and proliferative responses were entirely dependent on exogeneous IL 2. RaMB-induced IL 2 reactivity was found to require accessory cells which are Fc receptor positive, and clearly distinct from those required to induce T cell proliferation in mixed lymphocyte cultures. We conclude that Lyt-2+ T cells are triggered to IL 2 reactivity by Fc receptor-mediated presentation of RaMB antibodies. The mechanism of induction by RaMB antibodies is discussed.

Animals↗
PubMed1984

Partial characterization of the oligosaccharides of mouse thymocyte Thy-1 glycoprotein.

Four glycopeptides (I, IIA, IIB, III) with different oligosaccharide structures were isolated from purified mouse thymocyte Thy-1 glycoprotein. The glycoprotein was digested with Pronase, and the glycopeptide fraction was isolated by gel filtration and acetylated with [3H]acetic anhydride. The different glycan structures were separated by affinity chromatography on concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. Size determinations of intact and exoglycosidase- and endoglycosidase-digested glycopeptides were performed by gel filtration on Bio-Gel P-6, calibrated with glycopeptides of known structure. On the basis of these experiments and on the behaviour of the glycopeptides on the lectin columns, the following structures of the oligosaccharide chains were proposed: I, triantennary 'complex-type' with terminal fucose; IIA, biantennary 'complex-type' without fucose; IIB, biantennary 'complex-type' with fucose; III, a mixture of 'high-mannose' chains containing either five or six mannose residues (approx. 50% of each). Amino acid analysis of the glycopeptides showed that the predominant oligosaccharide at glycosylation-site Asn-23 was of 'high-mannose' type, whereas the other two sites (Asn-75 and Asn-99) were glycosylated with 'complex-type' chains. Both these sites were shown to be variably glycosylated. The major glycans linked to Asn-75 were of structures I and IIB, whereas all three 'complex-type' chains were represented at Asn-99. The results presented explain the previously reported carbohydrate heterogeneity of thymocyte Thy-1 glycoprotein.

Amino Acids↗
PubMed1983

Purification and characterization of the mouse thymocyte Thy-1 glycoprotein.

The thymocyte Thy-1 glycoprotein was purified from mouse strain NMRI (Thy 1.2) thymus. Crude membranes were prepared in Tween 20 and solubilized in deoxycholate. The glycoproteins were isolated by affinity chromatography on concanavalin A-Sepharose, and Thy-1 was further purified by two gel-filtration cycles on Sephacryl S-200. The concentration of Thy-1 in fractions obtained during purification was measured by a solid-phase radioimmunoassay with pure mouse brain Thy-1 as standard. Analysis of the purified preparation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed at least five distinct bands in the apparent-Mr region 25000-30000, the polymorphism probably being due to carbohydrate heterogeneity. Amino-acid-analysis data were compatible with the previously published sequence of mouse brain Thy-1. Sugar content was determined at 31% (w/w), and the carbohydrate composition indicates the presence of 'complex-type' oligosaccharide chains. The mean Mr of mouse thymocyte Thy-1 was calculated to be 18 100.

Amino Acids↗
PubMed1983

Alterations in expression and glycosylation pattern of the Thy-1 glycoprotein during maturation and transformation of mouse T lymphocytes.

The mouse Thy-1 glycoprotein of normal and transformed lymphoid cells was studied with regard to amount per cell, apparent m.w., and glycosylation characteristics. Thy-1 was measured by a solid-phase radioimmunoassay calibrated with pure mouse brain Thy-1. Thymocytes were shown to contain five times the amount of Thy-1 found in lymph node cells (1 X 10(6) vs 2 X 10(5) molecules per cell), whereas the T cell lymphomas studied (P52-127-166, RBL-5, YWA, Y191, Y274, YAC-1, RL male 1, and BW5147) varied in their Thy-1 content. The apparent m.w. of Thy-1, as determined by SDS-PAGE, was in all cases 25,000 to 30,000. However, the appearance of the Thy-1 bands revealed a size heterogeneity that was less pronounced with material from lymph node cells than from thymocytes. This band broadening seemed to be inversely correlated to the affinity for lentil lectin. Whereas half the Thy-1 molecules from thymocytes were bound to the lectin, lymph nodes Thy-1 showed 75% binding. All T lymphomas but one (BW5147) contained Thy-1 also heterogeneous in lentil lectin binding. The charge, previously shown to be dependent on the sialic acid content, was shifted to more acidic forms for lymph node Thy-1 compared to thymocytes. The T lymphomas possessed Thy-1 with charge properties similar to those of the thymocytes; the only exception was BW5147, which showed more basic forms. These results show that the expression and the glycosylation of Thy-1 is altered when thymocytes mature into immunocompetent cells and after malignant transformation of lymphocytes.

Animals↗
PubMed1981

Manifestation and growth of a transplantable osteosarcoma in mice: the effects of thymectomy and thymosin treatment.

Normal and thymectomized CBA mice treated with and without thymosin were injected with 2 x 10(6) viable osteosarcoma cells. The activity and weight of tumors, the cell-mediated and humoral-immune responses against the tumors, and the light microscopic characteristics suggested that thymectomy resulted in a significantly decreased tumor frequency. There were also significant increases in cell-mediated cytotoxic activity in vitro as measured by lysis of 51Cr-labeled tumor cells incubated with spleen cells, and in lymphocytic infiltration around the tumors. While thymosin treatment of thymectomized mice restored these parameters to those of intact animals, thymosin had no demonstrable effects on nonthymectomized animals.

Animals↗