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S R Carlsson

Publications and source records attributed to S R Carlsson.

9 recordsLinked to original sources
PubMed1992

The lysosomal membrane glycoprotein lamp-1 is transported to lysosomes by two alternative pathways.

It has been demonstrated that lysosomal membrane proteins are directed to lysosomes by a tyrosine-containing structural motif in their cytoplasmic tails. It is presently unclear whether lysosomal membrane proteins are directly transported to lysosomes or first taken to the plasma membrane and then directed to the lysosomes via the endocytic pathway. In the present study, the transport pathways taken by one of the members of highly glycosylated lysosomal membrane proteins, lamp-1, were examined in human HL-60 cells. Pulse-chase labeling, combined with cell surface biotinylation and Percoll density gradient fractionation, was used to measure the kinetics of transport to the cell surface and lysosomes. The results show that the majority of lamp-1 is directly transported to lysosomes by a fast pathway (half-time 60 min), which involves sorting at an intracellular site, presumably in the trans-Golgi network. A minor part of lamp-1 is transported out to the cell surface, where it is internalized and eventually delivered to lysosomes. Transport by this pathway requires a long transit time (half-time greater than 2 h). After granulocytic differentiation of HL-60 cells by dimethyl sulfoxide, the synthesis of lamp-1 was increased approximately twofold. In these cells, the sorting in the Golgi apparatus is more effective, leaving only a minute fraction of lamp-1 for the bulk flow to the cell surface. This study establishes that the majority of lamp-1 is directly transported to lysosomes and that, in certain cells, the minority of the molecules is transported to lysosomes via the cell surface.

Antigens, CD↗
PubMed1991

Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse.

We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809-815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.

Amino Acid Sequence↗
PubMed1990

The polylactosaminoglycans of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Localization on the peptide backbones.

Lysosome membrane glycoproteins, lamp-1 and lamp-2, have been shown to contain 18 and 16 N-glycans, some of which are modified by poly-N-acetyl-lactosamine. We have localized the polylactosaminoglycans to specific sites on lamp-1 and lamp-2 purified from human chronic myelogenous leukemia cells. Polylactosaminoglycan-containing glycopeptides, obtained by trypsin, pepsin, and V8 protease digestion of the glycoproteins, were isolated by Datura stramonium agglutinin affinity chromatography, gel filtration, and reverse phase high performance liquid chromatography. The poly-N-acetyllactosaminyl structures of isolated glycopeptides were confirmed by the susceptibility of their released oligosaccharides to endo-beta-galactosidase. Amino acid analysis and sequencing demonstrated that polylactosaminoglycans were located at Asn-34, Asn-93 and/or Asn-102, and Asn-195 and/or Asn-200 in lamp-1, and at Asn-4 and/or Asn-10, and Asn-279 in lamp-2. These results indicated that only certain glycosylation sites can be selectively modified by poly-N-acetyllactosamine, and those sites may confer the requirement by beta 1----3-N-acetylglucosaminyl transferase.

Amino Acid Sequence↗
PubMed1989

Decrease in polylactosaminoglycans associated with lysosomal membrane glycoproteins during differentiation of CaCo-2 human colonic adenocarcinoma cells.

The proportion of labeled polylactosaminoglycans found in glycoproteins decreases during spontaneous differentiation of CaCo-2 human colonic adenocarcinoma cells to enterocytes in culture (A. Youakim and A. Herscovics, Biochem. J., 247: 299-306, 1987). To identify polylactosaminoglycan-containing glycoproteins, CaCo-2 cells were incubated with [3H]glucosamine or [3H]fucose, for 24 h, and membrane glycoproteins solubilized with 0.5% Nonidet P-40 were fractionated by affinity chromatography on Datura stramonium (DSA)-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that a restricted set of glycoproteins with a molecular weight of about 100,000 bound to DSA-agarose. These labeled glycoproteins were shown to contain polylactosaminoglycans by DSA-agarose chromatography and endo-beta-galactosidase digestion of Pronase-derived glycopeptides. Immunoprecipitation of the [3H]glucosamine-labeled Nonidet P-40 extract with polyclonal antibodies to the lysosomal membrane proteins h-lamp-1 and h-lamp-2 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography also revealed a band with a molecular weight of about 100,000. The immunoprecipitates were digested with Pronase, and the resulting glycopeptides were first fractionated on Bio-Gel P-6 into excluded (Fraction I) and included (Fraction II) glycopeptides, and then by DSA-agarose affinity chromatography. A much greater proportion of labeled glycopeptides in undifferentiated cells (3 to 5 days in culture) than in differentiated cells (19 to 27 days in culture) was recovered in Fraction I; these glycopeptides were bound to DSA-agarose and were sensitive to endo-beta-galactosidase. This decrease in polylactosaminoglycans was associated primarily with h-lamp-1. These results indicate that h-lamp-1 of CaCo-2 cells contains polylactosaminoglycans and that it undergoes a change in glycosylation with differentiation.

Adenocarcinoma↗
PubMed1989

Structure of human lysosomal membrane glycoprotein 1. Assignment of disulfide bonds and visualization of its domain arrangement.

The amino acid sequence of one of the major lysosomal membrane glycoproteins, lysosome-associated membrane protein 1 (lamp-1), was deduced from its cDNA sequence (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928). This amino acid sequence suggests that lamp-1 contains a hinge-like structure and could form disulfide bridges that are observed in the immunoglobulin superfamily. To test this possibility, we have determined the positions of the disulfide bridges by isolating and sequencing cystine-containing peptides which contain disulfide bridges. The results indicate that disulfide arrangement of lamp-1 is different from that of immunoglobulins. Each molecule contains, in total, four loops formed by disulfide bonds, and each loop contains 36-39 amino acid residues. However, none of the disulfide bonds connects two domains that are separated by a hinge-like structure. The results indicate that the hinge region has no ordered structure, and the relative positions of the two domains can be altered in space. Examination of the ultrastructure of lamp-1 by electron microscopy showed that the hinge-like structure actually functions as a hinge. These results indicate that the lamp-1 molecule represents a novel family of glycoproteins with unique structural properties.

Amino Acid Sequence↗
PubMed1989

Characterization of cDNAs encoding human leukosialin and localization of the leukosialin gene to chromosome 16.

We describe the isolation and characterization of cDNA clones encoding human leukosialin, a major sialoglycoprotein of human leukocytes. Leukosialin is very closely related or identical to the sialophorin molecule, which is involved in T-cell proliferation and whose expression is altered in Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disease. Using a rabbit anti-serum to leukosialin, a cDNA clone was isolated from a lambda gt11 cDNA library constructed from human peripheral blood cells. This lambda gt11 clone was used to isolate longer cDNA clones that correspond to the entire coding sequence of leukosialin. DNA sequence analysis reveals three domains in the predicted mature protein. The extracellular domain is enriched for Ser, Thr, and Pro and contains four contiguous 18-amino acid repeats. The transmembrane and intracellular domains of the human leukosialin molecule are highly homologous to the rat W3/13 molecule. RNA gel blot analysis reveals two polyadenylylated species of 2.3 and 8 kilobases. Southern blot analysis suggests that human leukosialin is a single-copy gene. Analysis of monochromosomal cell hybrids indicates that the leukosialin gene is not X chromosome linked and in situ hybridization shows leukosialin is located on chromosome 16. These findings demonstrate that the primary mutation in WAS is not a defect in the structural gene for leukosialin.

Amino Acid Sequence↗
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↗