scholarly journals Identification of an epidermal cell-adhesion glycoprotein

1985 ◽  
Vol 232 (1) ◽  
pp. 67-70 ◽  
Author(s):  
G P Roberts ◽  
J Brunt
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Epidermal Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Keratinocytes ◽  
Intercellular Adhesion ◽  
Polyacrylamide Gel ◽  
Galactose Oxidase ◽  
Surface Labelling

Glycoproteins which mediate intercellular adhesion were studied by comparing the effects of trypsin and the neutral proteinase, Dispase, on human keratinocytes metabolically labelled with D-[1-14C]glucosamine or L-[1-3H]fucose. Whereas digestion of keratinocytes with trypsin/EDTA resulted in loss of both cell-substratum and intercellular adhesion, only cell-substratum adhesion was disrupted by incubation with Dispase. Analysis of the radiolabelled glycoproteins by polyacrylamide-gel electrophoresis revealed that a glycoprotein of Mr 126 000 was cleaved by trypsin/EDTA, but not by Dispase. Surface labelling of keratinocytes with galactose oxidase/NaB3H4 confirmed that this glycoprotein was exposed on the cell surface. Addition of lmM-Ca2+ prevented dispersion of keratinocytes by trypsin and concomitantly protected the glycoprotein of Mr 126 000 from digestion. These results indicate that this glycoprotein has an important role in mediating intercellular adhesion of keratinocytes.

scholarly journals Differentiation-related changes in glycoprotein synthesis by human keratinocytes

1986 ◽  
Vol 237 (2) ◽  
pp. 519-525 ◽  
Author(s):  
G P R Roberts ◽  
J Brunt
Keyword(s):  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Keratinocytes ◽  
Polyacrylamide Gel ◽  
Galactose Oxidase ◽  
Glycoprotein Synthesis ◽  
Surface Labelling ◽  
Triton X

Human keratinocytes were cultured in media in which the Ca2+ concentration controlled the stage of differentiation. In media containing less than 0.1 mM-Ca2+ keratinocytes grew as a monolayer, but in the presence of 2mM-Ca2+ the cells differentiated and formed stratified colonies. Glycoproteins of both stratified and unstratified cells were radiolabelled by metabolic incorporation of radioactive precursors and by cell-surface labelling using galactose oxidase/NaB3H4. The radiolabelled keratinocytes were extracted with 0.5% Triton X-100, and the glycoproteins in both the Triton X-100-soluble and Triton X-100-insoluble fractions were analysed by polyacrylamide-gel electrophoresis in the presence of SDS. Two Triton X-100-soluble glycoproteins with high Mr values (greater than 200,000) were major glycoproteins in stratified keratinocytes, but were present in only trace amounts in unstratified keratinocytes. Characterization of these glycoproteins by examination of the effect of tunicamycin on their synthesis and the effect of neuraminidase on their migration characteristics showed that they were cell-surface sialoglycoproteins containing O-glycosidically linked oligosaccharides. Analysis of the adherent cytoskeletons left after Triton X-100 extraction of stratified and unstratified keratinocytes revealed that a glycoprotein of Mr 184,000 was decreased in stratified keratinocytes. Incubation of unstratified keratinocytes in high-Ca2+ medium resulted in a rapid modification of the glycoprotein of Mr 184,000, and it is suggested that this event may be related to desmosome formation and stratification.

scholarly journals Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

1981 ◽  
Vol 198 (2) ◽  
pp. 331-338 ◽  
Author(s):  
Ian A. King ◽  
Anne Tabiowo
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Cell Adhesion ◽  
Epidermal Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Membrane Glycoproteins ◽  
Glycosaminoglycan Synthesis ◽  
Core Proteins

1. When pig ear skin slices were cultured for 18h in the presence of 1mug of tunicamycin/ml the incorporation of d-[3H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5mug of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-14C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[3H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[3H]glucosamine but not of (U-14C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[3H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[3H]glucosamine and 35SO42− into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.

scholarly journals Glycoproteins and glycosaminoglycans synthesized by human keratinocytes in culture. Their role in cell-substratum adhesion

1983 ◽  
Vol 212 (2) ◽  
pp. 355-363 ◽  
Author(s):  
G P Roberts ◽  
L Jenner
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Keratinocytes ◽  
Cytoskeletal Proteins ◽  
Galactose Oxidase ◽  
Surface Labelling ◽  
Triton X ◽  
Sulphated Glycosaminoglycan

Glycoproteins and proteoglycans synthesized by human keratinocytes in medium containing D-[1-14C]glucosamine were extracted and analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Extraction of the labelled keratinocytes with 0.5% Triton X-100 removed most of the glycoconjugates and left the cytoskeleton and nuclear residue adherent to the substratum. In addition to the cytoskeletal proteins, there was a relatively simple profile of glycoproteins and glycosaminoglycans associated with this adherent cytoskeleton. These consisted of eight glycoproteins in the mol.wt. range 99000-232000, five proteins in the keratin region (mol.wt. 42000-61000), hyaluronic acid and a sulphated glycosaminoglycan. Surface labelling of the keratinocytes with galactose oxidase (with or without neuraminidase)/KB3H4 revealed that many of the glycoproteins were exposed on the cell surface. The importance of the glycoproteins and proteoglycans in attaching the keratinocytes to the substratum was examined by studying their expression after incubation in medium containing tunicamycin and their degradation after digestion with trypsin and hyaluronidase. These studies, together with an examination of the glycoconjugates released by sequential extraction with 0.5% Triton X-100 followed by 0.2% sodium dodecyl sulphate, revealed that the glycoprotein of mol.wt. 232000 has an important role in mediating the attachment of keratinocytes to the substratum.

scholarly journals Surface glycoproteins of human spermatozoa

1986 ◽  
Vol 82 (1) ◽  
pp. 11-22
Author(s):  
M. Kallajoki ◽  
I. Virtanen ◽  
J. Suominen
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Spermatozoa ◽  
Galactose Oxidase ◽  
Sperm Cells ◽  
Neuraminidase Treatment ◽  
Triton X

The surface membrane glycoprotein composition of human spermatozoa has been studied by introducing radioactive label into galactosyl (Gal) and N-acetylgalactosaminyl (GalNAc) residues by using the galactose oxidase/NaB3H4 method. Triton X-100 extracts and Triton X-100-resistant cytoskeletal residues were subjected to analysis by polyacrylamide gel electrophoresis. The distribution of the radiolabel in sperm cells was studied by light-microscopic auto-radiography. The grains were evenly distributed on the cells by the labelling methods used. The Triton X-100 treatment did not affect sperm morphology at the light-microscopic level, but in transmission electron microscopy the plasma membrane covering the acrosome was removed totally, together with most of the acrosomal membranes and acrosomal contents. Plasma membrane residues were, however, always found in the postacrosomal region. Borohydride alone without oxidative pretreatment labelled two polypeptides of molecular weights (Mr) 48,000 and 43,000 in the Triton X-100-soluble fraction. When the Gal/GalNAc residues were labelled by galactose oxidase pretreatment 120,000, 105,000, 78,000 and 68,000 Mr glycoproteins were revealed. When additional neuraminidase treatment was used to remove terminal sialic acid residues, the total labelling intensity was increased two- to fivefold and additional 36,000 and 20,000 Mr glycoproteins were revealed. The Triton X-100-resistant cytoskeletal residue contained 53–75% of the total radioactivity bound in sperm cells. When these components were analysed by polyacrylamide gel electrophoresis, all the major bands found in the Triton X-100-soluble fraction were detected and also some radioactivity was incorporated into the major bands visualized by protein staining. In the present study we describe several human sperm glycoproteins, which seem to be distributed evenly on the sperm cells. Detergent extraction, producing cytoskeletal models, appeared to leave most of the glycoproteins detectable in the extraction residues also with the apparent enrichment of a single 68,000 Mr glycoprotein.

scholarly journals Cell-surface radioiodination with the sparingly soluble catalyst Iodogen. Difference between the dividing and non-dividing populations of rodent thymocytes

1981 ◽  
Vol 194 (1) ◽  
pp. 351-355 ◽  
Author(s):  
J G Salisbury ◽  
J M Graham
Keyword(s):  
Cell Surface ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Surface Proteins ◽  
New Method

The surface proteins of dividing and non-dividing subpopulations of rat and mouse thymocytes have been labelled by using a new method of radioiodination. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography of the labelled proteins shows distinct differences in labelling between the mouse and rat cells and also, in the case of the rat, between the dividing and non-dividing populations.

scholarly journals Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes

1986 ◽  
Vol 233 (3) ◽  
pp. 809-818 ◽  
Author(s):  
Y Sommarin ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Suspension Cultures ◽  
Polyacrylamide Gel ◽  
Side Chains ◽  
Dermatan Sulphate ◽  
Core Proteins

The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.

scholarly journals AN ELECTROPHORETIC DIFFERENCE BETWEEN SURFACE AND SECRETED IGM OF MURINE SPLENOCYTES

1973 ◽  
Vol 138 (5) ◽  
pp. 1282-1287 ◽  
Author(s):  
Ulrich Melcher ◽  
Jonathan W. Uhr
Keyword(s):  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Murine Splenocytes

µ-chains on the surface of murine splenocytes are more heterogeneous on SDS-polyacrylamide gel electrophoresis than both secreted and intracellular µ-chains labeled with [3H]tyrosine. No difference in heterogeneity among cell surface, secreted, or intracellular L chains was detected. The possible role of carbohydrate in µ-chain heterogeneity is discussed.

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