Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B³H₄ (c) pyridoxal phosphate/B³H₄ and (d) periodate/B³H₄. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated.The LETS protein was also labelled with [¹⁴C]glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with [³H]fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.     

Enzymatic and chemical oxidation of gangliosides in cultured cells: effects of choleragen.

Cell surface glycolipids of normal human fibroblasts and NCTC2071 cells (transformed mouse fibroblasts) were labeled by incubating the intact cells with either galactose oxidase or sodium periodate, followed by reduction of the oxidized sugar residues with NaB3H4. In intact human fibroblasts, incorporation of 3H was increased with increasing time of exposure to galactose oxidase prior to treatment with NaB3H4. Following limited exposure to galactose oxidase, more label was incorporated into the larger glycolipids. Although labeling of the monosialoganglioside GM1 was maximal by 16 h, not all of the GM1 in the intact cells appeared to be accessible to galactose oxidase, since 10 to 12 times more GM1 was labeled when cells were disrupted before incubation with the enzyme. The human fibroblasts contained approximately 8 X 10(6) molecules of GM1 per cell. Maximal binding of choleragen (5 X 10(5) molecules of [125I]choleragen per cell) completely prevented cholevented oxidation of GM1 in intact fibroblasts by galactose oxidase but only partially protected the sialic acid moiety of GM1 from oxidation by periodate. Choleragen had little effect on the enzymatic or chemical oxidation of other glycolipids. NCTC 2071 cells do not contain endogenous GM1 but incorporate exogenous GM1 from the culture medium. When bound to NCTC 2071 cells, exogenous GM1 was protected by choleragen from oxidation by galactose oxidase or whether endogenous or taken up from the incubation medium, are, after interaction with choleragen, less accessible to oxidation by periodate or galactose oxidase.     

Differences between the carbohydrate units of cell-surface glycoproteins of moust B- and T-lymphocytes.

Carbohydrate units of cell-surface glycoproteins of mouse B- and T-lymphocytes, labelled in their sialic acid residues by the periodate/NaB3H4 method and in their galactose residues by the galactose oxidase/NaB3H4 method after neuraminidase treatment, have been studied. Glycopeptides were prepared from the labelled cells by Pronase digestion and fractionated by concanavalin A affinity chromatography into two fractions (A and B). Alkali-labile oligosaccharides were isolated after mild NaOH/NaBH4 treatment by gel filtration. The alkali-labile oligosaccharides were further analysed by t.l.c. To study the relative proportion of neutral mannose-rich carbohydrate units (fraction C) in lymphocyte glycoproteins, glycopeptides were also prepared from unlabelled cells and subjected to concanavalin A affinity chromatography after N-[3H]acetylation of their peptide moiety. The major alkali-labile oligosaccharide component of both cell types was identified as galactosyl-(beta 1 leads to 3)-N-acetylgalactosaminitol. T-Lymphocytes were characterized by a high proportion of this oligosaccharide and a lower proportion of alkali-stable fraction A glycopeptides, whereas the opposite was observed for B-lymphocytes. The relative proportions of the concanavalin A-binding fractions B and C were similar in both cell types. The differences observed may correlate with the different surface properties of B- and T-lymphocytes.     

The glycoprotein of measles virus. External radioactive labelling of its carbohydrate and partial characterization of the glycopeptide.

Measles virus was propagated in VERO cells and purified from the culture supernatants by two successive tartrate-density-gradient centrifugations. Surface carbohydrates were labelled both in vitro and in vivo with 3H after treatment with galactose oxidase/NaB3H4 or with [3H]glucosamine. The major labelled glycoprotein in measles virions had a mol.wt. of 79 000. After labelling with periodate/NaB3H4, which would result in specific labelling of sialic acid residues, the 79 000-mol.wt. glycoprotein was very weakly labelled. This suggests that there is no or a very low amount of sialic acid in the virions. Further analysis of the glycoprotein showed that galactose is the terminal carbohydrate unit in the oligosaccharide, and the molecular weight of the glycopeptide obtained after Pronase digestion is about 3000. The oligosaccharide is attached to the polypeptide through an alkali-stable bond, indicating a N-glycosidic asparagine linkage.     

Identification of exposed surface glycoproteins of four human bladder carcinoma cell lines

Three cell surface protein-specific methods were used to radiolabel the major glycoproteins of four human bladder carcinoma cell lines: The well-differentiated lines RT112 and TR4 and more anaplastic lines T24 and EJ. Five acidic glycoproteins iodinated in all lines by the lactoperoxidase/125I method were designated CP-175/5.8-6.0 (apparent molecular weight X 10(-3)/pl of iodoprotein), GP-155/5.0-5.3, GP-145/4.9-5.2, GP-130/4.8-5.5 and GP-110/4.9-5.3. Another iodinated glycoprotein, GP-200/5.5-6.0, was prominently labelled in RT112 and RT4 but was not detected in T24 or EJ. GP-200 as well as GP-175, GP-155 and GP-145 were not detected by the galactose oxidase/NaB(3H)4 method and were poorly labelled by the neuraminidase-galactose oxidase/NaB(3H)4 and NaIO4/NaB(3H)4 labelling methods. The major sialogalactoproteins identified in the four lines by the neuraminidase-galactose oxidase/NaB(3H)4 and NaIO4/NaB(3H)4 methods were GP-130, and a duplet of GP-90 and GP-80 which were poorly iodinated by lactoperoxidase/125I. The galactose oxidase/NaB(3H)4 reaction was increased by between 4- and 10-fold and many additional glycoproteins were labelled after neuraminidase treatment, indicating that the cell surface galactose and N-acetylgalactosamine residues of glycoproteins are highly sialylated. In cell lines RT112 and RT4 there was prominent labelling of very high molecular weight sialogalactoconjugates that was not present in extracts of T24 and EJ.     

A survey of surface glycoproteins of four human squamous cell carcinoma lines

The major cell surface glycoprotein components of four new cell lines derived from human squamous cell carcinomas of the head and neck (TR126, TR131, TR138, TR146, Rupniak, H. T. et al., JNCI 75, 621-635, 1985) were identified by three complementary labelling methods. The profile of labelled glycoprotein components was very similar in the four cell lines, although quite large quantitative differences in individual bands were seen. Two galactoproteins, designated GPC-130 and GPC-80 (apparent molecular weight X 10(-3)) were labelled by galactose oxidase/NaB [3H]4 but in all four lines only GPC-130 was prominent. The cell surface galactose and N-Acetylgalactosaminyl residues of glycoproteins were quite highly sialylated, as the galactose oxidase/NaB [3H]4 reaction was increased by between 3- and 6-fold after neuraminidase treatment. The neuraminidase-galactose oxidase/NaB [3H]4 and NaIO4/NaB [3H]4 methods identified a complex profile of glycoprotein components, with very high molecular weight sialogalactoconjugates being prominent. The major sialoglycoproteins were GPC-205, GPC-175, GPC-155, GPC-90 and GPC-70 and in addition, GPC-130 and GPC-80 showed enhanced labelling. Lactoperoxidase catalyzed the iodination of a similar profile of high molecular weight glycoprotein components, with GPC-205 and GPC-175 being prominent in TR126, TR131 and TR146 but less evident in TR138. Overall, the profile of labelled glycoprotein components was similar to the pattern seen in the well differentiated transitional carcinoma lines RT112 and RT4 (Steele, J. G. et al., Biochim. Biophys. Acta. 732, 219-228, 1983).     

Loss of covalently labeled glycoproteins and glycolipids from the surface of newly transformed schistosomula of Schistosoma mansoni

Schistosomula of Schistosoma mansoni were labeled by oxidation with galactose oxidase or with periodate followed by reduction with NaB3H4 to study the loss of the surface membrane of these parasites in vitro. Grain counts of light microscope autoradiographs (LMARG) of radiolabeled schistosomula show that both galactose oxidase and periodate specifically label the surface of the organisms. Galactose oxidase labels 11 glycoproteins on the surface of skin and mechanical schistosomula, ranging in apparent molecular weight from 17,000 to greater than 105,000. These glycoproteins are lost from the surface of schistosomula with a halftime of 10-15 h in culture in defined medium. Most of these glycoproteins appear to be shed intact from the surface of the schistosomula rather than endocytosed and degraded, because greater than 50% of each of the lost proteins can be recovered by trichloroacetic acid precipitation of the culture medium and because there is no internalization of the radiolabels into cultured schistosomula examined by LMARG. In addition to glycoproteins, periodate labels at least seven glycolipids on the surface of mechanical schistosomula. After culture for 15 h, more than half of each of these periodate-labeled proteins and lipids are lost from the schistosomula, and their abundance relative to each other remains similar to that of freshly labeled organisms. Since both proteins and lipids are lost from the surface of the schistosomula at the same rate, we believe that we are observing a general loss of the parasite surface membrane.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.     

External labelling of glycoproteins from first-trimester human placental microvilli.

The brush-border glycoproteins of first-trimester human placentas were investigated by using two external labelling techniques: (1) sequential digestion with neuraminidase and galactose oxidase, followed by reduction with NaB3H4, which 3H-labels terminal galactose and galactosamine residues; and (2) sequential treatment with periodate and NaB3H4, which 3H-labels terminal sialic acid residues. The labelling procedures were performed on intact tissue so that the results would more closely approximate the topography of the brush border in vivo. The microvilli were isolated, subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the [3H]glycoproteins detected by fluorography. Densitometer scans of the fluorograms of the [3H]galactoproteins showed that, under reducing conditions, 90% of the protein-associated radioactivity was incorporated into two glycoproteins. The major [3H]galactoprotein of early placental microvilli had an estimated molecular mass of 92 kDa (desialylated) and migrated as a diffuse band. A minor 180 kDa glycoprotein was less consistently labelled. No change in the apparent molecular mass of either component was detected in the absence of beta-mercaptoethanol, suggesting that the 180 kDa component was not a dimer of the 92 kDa glycoprotein. The remaining 10% the radioactivity was equally distributed among several minor membrane components. Densitometer scans of the fluorograms of the [3H]sialoproteins showed that, under either reducing or non-reducing conditions, 90% of the 3H was preferentially incorporated into the 92-110 kDa region of the gel. Although no distinct bands were visible, the higher-molecular-mass region of this area was always most heavily labelled. A minor 180 kDa glycoprotein was also 3H-labelled. The pattern of brushborder [3H]glycoproteins from first-trimester placentas differed markedly from that of term placental microvilli and from placental fibroblast plasma membranes that were 3H-labelled by identical external labelling techniques. These results indicate that: (1) the glycoprotein determinants of brush-border topography change during pregnancy; (2) within the placenta, the major 92 kDa (desialylated) determinant, which has not been previously described, is unique to the trophoblastic cells.     

Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein.

Transformed cells lack a large, external, transformation-sensitive (LETS) glycoprotein which is a major surface component of their normal counterparts. Addition of LETS glycoprotein isolated from normal cells to transfomed cells restores certain morphological features and adhesive properties characteristic of normal cells. LETS protein is detected on the cell surface both by iodination using lactoperoxidase and by immunofluorescent staining. The surface distribution pattern detected by immunofluorescence is strikingly similar to that of normal cells. After addition of LETS protein, transformed cells also exhibit well defined actin cables which are not seen in untreated, transformed cells. All these alterations can be blocked by treating LETS protein with specific antisera or by subjecting it to mild trypsinization prior to addition to transformed cells. The effects are rapidly reversible by mild trypsinization, which removes the added LETS protein. The high rate of uptake of 2-deoxyglucose, characteristic of transformed cells, is not affected by LETS protein. These results suggest that LETS protein may have a role in cell attachment and spreading, and affect the organization of cytoskeleton.     

Detection of glycoproteins in the Acanthamoeba plasma membrane

In the present study we have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by 125I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB3H4 and galactose oxidase/NaB3H4 labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with Mr of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with [35S]methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.     

The structure of a glycopeptide purified from porcine thyroglobulin

1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, alpha-l-fucosidase, beta-d-galactosidase, beta-N-acetyl-d-glucosaminidase and alpha-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid-->galactose-->N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose-->N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, alpha-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide.     

Effects of cytochalasin B and colchicine on attachment of a major surface protein of fibroblasts

We have investigated the effects of the drugs cytochalasin B and colchicine on the surface levels of the large, external, transformation-sensitive (LETS) glycoprotein. Colchicine neither removed LETS protein from the surface, nor inhibited its regeneration after removal by mild trypsinization. Cells treated with cytochalasin B, however, showed both a 2–3-fold increase in the turnover rate of their surface LETS protein and a marked inhibition in its regeneration. Inhibition of regeneration was not due to inhibition of synthesis or transport to the surface. In fact, in the presence of cytochalasin B, increased quantities of LETS protein were released into the medium. The results are consistent with the idea of an association of LETS protein with the actin-containing microfilaments. However, other possible explanations, such as effects on cellular morphology or on transport of sugar precursors cannot yet be excluded.     

Generation of a soluble IFN-gamma inducer by oxidation of galactose residues on macrophages

Depletion of macrophages from human peripheral blood mononuclear cells (PBMC) caused a marked decrease in galactose oxidase and sodium periodate, but not a calcium ionophore, stimulated Interferon-gamma (IFN-gamma) production. Reconstitution of such depleted cultures with galactose oxidase treated macrophages, but not lymphocytes, restored IFN-gamma levels to those of control nonfractionated PBMC. Thus, galactose oxidase seemed to act on macrophages which in turn stimulated lymphocyte production of IFN-gamma. Unlike human cells which have terminal galactose residues on glycoproteins, murine cell glycoproteins terminate their oligosaccharide component in the order N-acetyl-neuraminic acid followed by D-galactose, N-acetyl-glucosamine, and glycoprotein. Galactose oxidase or sodium periodate only activated murine macrophages to stimulate lymphocyte IFN-gamma production after exposing D-galactose residues by the removal of the terminal N-acetyl-neuraminic acid residues with neuraminidase. Removal of such exposed terminal galactose residues with beta-galactosidase inhibited the effect of galactose oxidase on murine macrophages. Taken together, these results strongly suggest that oxidation of terminal galactose residues on macrophages is the initial site of action of galactose oxidase and sodium periodate. Studies with Boyden chambers have shown that galactose oxidase-treated macrophages released a soluble factor which stimulates lymphocyte production of IFN-gamma. Based on these findings, it appears that the oxidation of terminal galactose residues on the surface of macrophages leads to the induction and transmission of a soluble signal for lymphocyte production of IFN-gamma.     

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].     

Isolation and partial characterization of “galactoprotein a” (LETS) and “galactoprotein b” from hamster embryo fibroblasts

The cell surface glycoproteins of hamster NIL cells, labeled with galactose oxidase and NaB³H4, were selectively solubilized by sequential extraction with Tris buffer containing 1) sucrose-ATP-EDTA, 2) zwitterionic detergent (Empigen BB), and 3) 8 M urea. The previously reported “galactoprotein b” $\text{(Gap b)}$ and “galactoprotein a” ($\text{Gap a}$ or LETS) were isolated by affinity chromatography on insoluble Ricinus communis lectin colums (RCA column) from extracts 2) and 3), respectively. The affinity-purified $\text{Gap a}$ contained an actin-like protein, whereas the other affinity-purified galactoproteins did not contain the actin-like protein. The isolated $\text{Gap b}$ was heterogeneous, and an additional glycoprotein, specific for NILpy cells was copurified on RCA-column with $\text{Gap b}$.     

Sialic acid, galactose and fucose on the surface of Ehrlich ascites tumor cells grown in glucose-free medium in the presence of uridine

1) The content and accessibility of terminal sialic acid and galactose residues as well as the incorporation of [3H]fucose into glycoconjugates were determined in 48-h cultures of Ehrlich ascites tumor cells in a glucose-free medium supplemented with uridine, a compound which can fulfil the necessary functions of glucose. 2) Sialic-acid residues accessible to sialidase cleavage were reduced from 695 +/- 80 nmol/10(9) cells (controls) to 284 +/- 22 nmol/10(9) cells (43% of controls). In situ labeling using periodate oxidation followed by sodium borotritiide reduction revealed a tritium incorporation of 47 +/- 11% that of controls (= 4.1 x 10(5) cpm/mg protein). 3) Labeling of galactose residues of 80-90% of that of controls was achieved after treatment of the cells with galactose oxidase/sodium borotritiide. A nearly six-fold enhancement of tritium incorporation into galactose of control cells was observed after sialidase/galactose oxidase treatment and sodium borotritiide reduction (1.5----8.8 x 10(5) cpm/mg protein); only a 3.6-fold increase (1.2 x 10(5)----4.3 x 10(5) cpm/mg protein) was found with glucose-free cultured cells. It is concluded that the galactose content of the cell surface is reduced to about 50% of controls. 4) The incorporation of tritium into acid-insoluble precipitate after 24 h incubation with [3H]fucose and the activity of the acid-soluble fraction were enhanced by about 85% as compared to controls. The pattern of inhibition by tunicamycin of [3H]fucose uptake and incorporation was the same in glucose-containing standard medium and in glucose-free uridine medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface distribution of LETS protein in relation to the cytoskeleton of normal and transformed cells

The organization of LETS protein on the surface of NIL8 hamster cells has been examined by immunofluorescence staining. The distribution of LETS protein was found to depend on the culture conditions; in subconfluent, low-serum arrested cultures the LETS protein is predominantly located at the cell-substrate interface and also in regions of cell-cell contact, whereas in dense cultures the cells are surrounded by a network of LETS protein fibrils. Transformed derivatives of these cells exhibit only sporadic staining for LETS protein, in the form of short intercellular bridges. Agents that cause alterations in cell shape and cytoplasmic filaments have been used to explore the relationship of LETS protein to the internal cytoskeletal elements. Reciprocally, perturbations of the cell surface were examined for their effects on internal filaments. The arrangement of microtubules seems to be unrelated to the presence of LETS protein in the cells studied. Actin microfilament bundles and LETS protein respond in a coordinate fashion to some perturbants but independently with respect to others. The patterns of staining for LETS protein are consistent with an involvement in cell-to-cell and cell-to-substrate adhesion.     

Role of disulfide bonds in the attachment and function of large, external, transformation-sensitive glycoprotein at the cell surface

Reduction of disulfide linkages by dithiothreitol removes LETS (large, external, transformation-sensitive) protein from the cell surface. This process is dependent upon the concentration of dithiothreitol and the time and temperature of reaction. At 0 degrees C the release of LETS protein by dithiothreitol is completely blocked, but this is apparently not due to a requirement for metabolic energy. At this temperature, reduction of LETS protein is incomplete. These results suggest that intact disulfide bonds are involved in the retention of this protein on the cell surface. Furthermore, reduction of purified LETS protein interferes with its ability to confer flattened morphology and increased adhesivity when added to transformed cells. It appears, therefore, that disulfide bonds are functionally important at the cell surface.