scholarly journals Lamp-1 does not acquire the large polylactosaminoglycans characteristic of F9 cells

1993 ◽  
Vol 296 (1) ◽  
pp. 253-257 ◽  
Author(s):  
P A Romero ◽  
T Way ◽  
A Herscovics
Keyword(s):  
Monoclonal Antibodies ◽  
Retinoic Acid ◽  
Membrane Protein ◽  
Acid Treatment ◽  
Datura Stramonium ◽  
Lysosomal Membrane ◽  
Retinoic Acid Treatment ◽  
F9 Cells ◽  
Lysosomal Membrane Proteins ◽  
Lysosomal Membrane Protein

Although F9 cells labelled with [3H]glucosamine synthesize many glycoproteins that bind to Datura stramonium agglutinin-agarose, only a small proportion of these were immunoprecipitated with monoclonal antibodies to lamp-1 and lamp-2 (lamp = lysosomal membrane protein). Differentiation of F9 cells by retinoic acid increased labelling of all Datura stramonium-bound glycoproteins, including lamp-1 and lamp-2. Although the large polylactosaminoglycans excluded from Bio-Gel P-6 that are characteristic of F9 cells were obtained from total glycoproteins, little of these large polylactosaminoglycans was found on lamp-1 and lamp-2. There was no increase in large polylactosaminoglycans of lamp-1 and lamp-2 after retinoic acid treatment, but an increase in the size of small polylactosaminoglycans (included on Bio-Gel P-6) and tri- and tetra-antennary complex oligosaccharides. Therefore, other factors besides the expression of specific glycosyltransferases determine the extent of elongation of polylactosaminoglycans on lysosomal membrane proteins.

scholarly journals Expression of Sec61alpha in F9 and P19 teratocarcinoma cells after retinoic acid treatment

2003 ◽  
Vol 63 (2) ◽  
pp. 245-252 ◽  
Author(s):  
L. R. Ferreira ◽  
C. E. E. Velano ◽  
E. C. Braga ◽  
C. C. Paula ◽  
H. Martélli-Junior ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Acid Treatment ◽  
Secretory Proteins ◽  
P19 Cells ◽  
Retinoic Acid Treatment ◽  
Teratocarcinoma Cell ◽  
F9 Cells ◽  
Teratocarcinoma Cells ◽  
Differentiation Stage

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (RE) membrane through a protein-conducting channel called the translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as essential for translocation of polypeptides chains into the cisterns of the ER. However, it is not known whether Sec61alpha is ubiquitously expressed in collagen producing teratocarcinoma cells. Furthermore, the production, expression, and utilization of Sec61alpha may depend on the cell differentiation stage. Stem cells from many cultured teratocarcinoma cell lines such as F9 and P19 cells are capable of differentiation in response to low retinoic acid concentrations. This differentiation of the tumorigenic stem cells results in tumorigenicity loss. For this study, mouse F9 and P19 teratocarcinoma cells were grown in culture medium treated with or without retinoic acid. Expression of Sec61alpha was determined by reverse trancriptase polimerase chain reaction (RT-PCR). In untreated conditions, F9 cells expressed undetected Sec61alpha amounts. It was also demonstrated that Sec61alpha expression is stimulated in F9 cells after retinoic acid treatment for 72 hours. No changes were found in Sec61alpha expression in P19 cells after retinoic acid treatment. These data indicate that the expression of Sec61alpha is enhanced with retinoic acid induced differentiation of F9 teratocarcinoma cells.

scholarly journals NCU-G1 is a highly glycosylated integral membrane protein of the lysosome

2009 ◽  
Vol 422 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Oliver Schieweck ◽  
Markus Damme ◽  
Bernd Schröder ◽  
Andrej Hasilik ◽  
Bernhard Schmidt ◽  
...  
Keyword(s):  
Amino Acid ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mouse Liver ◽  
Lysosomal Membrane ◽  
Molecular Forms ◽  
Type I ◽  
Calculated Molecular Mass ◽  
Sorting Motif ◽  
Lysosomal Membrane Proteins

Until recently, a modest number of approx. 40 lysosomal membrane proteins had been identified and even fewer were characterized in their function. In a proteomic study, using lysosomal membranes from human placenta we identified several candidate lysosomal membrane proteins and proved the lysosomal localization of two of them. In the present study, we demonstrate the lysosomal localization of the mouse orthologue of the human C1orf85 protein, which has been termed kidney-predominant protein NCU-G1 (GenBank® accession number: AB027141). NCU-G1 encodes a 404 amino acid protein with a calculated molecular mass of 39 kDa. The bioinformatics analysis of its amino acid sequence suggests it is a type I transmembrane protein containing a single tyrosine-based consensus lysosomal sorting motif at position 400 within the 12-residue C-terminal tail. Its lysosomal localization was confirmed using immunofluorescence with a C-terminally His-tagged NCU-G1 and the lysosomal marker LAMP-1 (lysosome-associated membrane protein-1) as a reference, and by subcellular fractionation of mouse liver after a tyloxapol-induced density shift of the lysosomal fraction using an anti-NCU-G1 antiserum. In transiently transfected HT1080 and HeLa cells, the His-tagged NCU-G1 was detected in two molecular forms with apparent protein sizes of 70 and 80 kDa, and in mouse liver the endogenous wild-type NCU-G1 was detected as a 75 kDa protein. The remarkable difference between the apparent and the calculated molecular masses of NCU-G1 was shown, by digesting the protein with N-glycosidase F, to be due to an extensive glycosylation. The lysosomal localization was impaired by mutational replacement of an alanine residue for the tyrosine residue within the putative sorting motif.

scholarly journals Antibodies to platelets in patients with anti-phospholipid antibodies

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2655-2659 ◽  
Author(s):  
HJ Out ◽  
PG de Groot ◽  
M van Vliet ◽  
GC de Gast ◽  
HK Nieuwenhuis ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Lysosomal Membrane ◽  
Immunofluorescence Method ◽  
Exact Role ◽  
Flow Cytofluorometry ◽  
Healthy Donors ◽  
Lysosomal Membrane Proteins ◽  
Lysosomal Membrane Protein ◽  
Platelet Autoantibodies

Abstract Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme- linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.

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