scholarly journals Evidence that major 78-44-kD concanavalin A-binding glycopolypeptides in pig epidermis arise from the degradation of desmosomal glycoproteins during terminal differentiation.

1987 ◽  
Vol 105 (6) ◽  
pp. 3053-3063 ◽  
Author(s):  
I A King ◽  
A Tabiowo ◽  
P R Fryer
Keyword(s):  
Stratum Corneum ◽  
Concanavalin A ◽  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Terminal Differentiation ◽  
Con A ◽  
Immune Precipitation ◽  
Membrane Bound ◽  
Spinous Layer ◽  
Binding Component

The major concanavalin A (Con A)-binding component in urea/deoxycholate/mercaptoethanol extracts from pig ear epidermis had an apparent Mr of 78 kD. In indirect immunofluorescence affinity-purified polyclonal antibodies against this glycopolypeptide strongly stained the surface of suprabasal cells in the epidermis of pig and human skin. Immunocytochemical labeling with gold-labeled second antibody localized this staining to externally disposed, trypsin-sensitive components of desmosomes. Western blotting showed that the 78-kD glycopolypeptide was immunologically related to several other Con A-binding components in pig epidermis. Immunoreactive components with Mr of 115 and 100 kD were membrane-bound, appeared to be susceptible to trypsin in intact epidermis, and were absent from the stratum corneum. Immunoreactive components of lower Mr (78-44 kD) were not membrane-bound, were resistant to trypsin in intact tissue, and were present predominantly in the keratinized layers of pig epidermis. The 115-44-kD glycopolypeptides were also recognized by antisera raised against desmoglein II/desmocollin glycoproteins isolated from bovine spinous layer desmosomes. In addition, these antisera reacted with 120- and 105-kD bands that were apparently not recognized by the anti-78-kD glycopolypeptide antiserum in immunoblotting. In immune precipitation the anti-78-kD glycopolypeptide and antidesmoglein II/desmocollin antisera precipitated comparable amounts of the radioiodinated 78-44-kD components. Both antisera also precipitated the 120- and 105-kD components although the anti-78-kD glycopolypeptide serum was less effective. Little reaction with the 115- and 105-kD components was observed in immune precipitation with either serum. Proteolytic peptide mapping confirmed that the various immunoreactive glycopolypeptides were biochemically as well as immunologically related. The results suggest that terminal differentiation in pig epidermis is accompanied by the orderly degradation of desmoglein II/desmocollin glycoproteins resulting in the accumulation of 78-44-kD glycopolypeptides in the stratum corneum. These glycopolypeptides may represent functionally important nonmembranous domains of cell-adhesion molecules in desmosomes.

scholarly journals Further characterization of erythrocyte p80 and the membrane protein defect of In(Lu) Lu(a-b-) erythrocytes

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1475-1481
Author(s):  
MJ Telen ◽  
I Rogers ◽  
M Letarte
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Concanavalin A ◽  
Polyclonal Antibodies ◽  
Erythrocyte Ghosts ◽  
Con A ◽  
Down Regulation ◽  
Regulation Of Expression ◽  
Variable Effect

We have previously shown that the In(Lu) gene down-regulates expression of an erythrocyte protein antigen identified by murine monoclonal antibody (MoAb) A3D8. In the present study we have examined In(Lu) Lu(a- b-) erythrocytes for expression of additional epitopes on the erythrocyte 80 kilodalton protein (p80) bearing the A3D8 antigen. Using a total of seven additional MoAbs that recognize three epitopes on erythrocyte p80, we have shown that In(Lu) Lu(a-b-) erythrocytes exhibit down-regulation of expression of all three epitopes. In(Lu) erythrocytes also showed a reduction in their reactivity to rabbit antibodies produced against purified p80 from either erythrocytes or lymphocytes. Furthermore the reactivity of the MoAbs was not altered by treatment of the cells with neuraminidase but was substantially reduced by treatment of cells with trypsin or chymotrypsin. The polyclonal anti- p80 sera were shown to react with a fragment of 50,000 daltons, still associated with erythrocyte ghosts, following treatment of the cells with trypsin or chymotrypsin. Treatment of erythrocytes with the thiol- reactive reagent AET decreased their reactivity with the MoAbs but had a variable effect on their reactivity with polyclonal antibodies. Erythrocyte p80 is a glycoprotein with N-linked oligosaccharides, as demonstrated by its binding to concanavalin A (Con A) and Len culinaris lectins. Following Endoglycosidase F treatment, erythrocyte p80 underwent a reduction in apparent mol wt of 11,000. The presence of a reduced amount of an intact p80 glycoprotein, seen by a decrease in reactivity with MoAbs directed at three distinct epitopes and with two different polyclonal antibodies, suggests that the In(Lu) gene interferes with expression by erythrocytes of the entire p80 glycoprotein.

scholarly journals Ubiquitin-like polypeptide conjugates to acceptor proteins in concanavalin A- and interferon γ-stimulated T-cells

1998 ◽  
Vol 330 (2) ◽  
pp. 683-688 ◽  
Author(s):  
Morihiko NAKAMURA ◽  
Yoshinori TANIGAWA
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Concanavalin A ◽  
Cell Activation ◽  
Polyclonal Antibodies ◽  
Con A ◽  
Antibody Treatment ◽  
Suppressor Factor

Monoclonal non-specific suppressor factor (MNSF), a lymphokine produced by a murine T-cell hybridoma, possesses pleiotrophic non-specific suppressive functions. MNSFβ (a subunit of MNSF) is a 14.5 kDa fusion protein consisting of a protein with 36% homology with ubiquitin and ribosomal protein S30. The ubiquitin-like segment of MNSFβ (Ubi-L) is an 8 kDa polypeptide with MNSF-like activity. Since the amino acids critical for the ubiquitination process are conserved in Ubi-L, we examined whether Ubi-L may conjugate with intracellular proteins in a manner similar to the ubiquitin system. Rabbit polyclonal antibodies specific for Ubi-L detected the induction of Ubi-L conjugations, including 33.5 kDa and 70 kDa molecules in concanavalin A (Con A)-stimulated T-cells, but not in lipopolysaccharide-stimulated B-cells and macrophages. High-molecular-mass conjugates were consistently present in pan-T-cells. However, free Ubi-L could not be observed in all the cells tested. Con A-activated CD8+ T-cells, but not CD4+ T-cells, induced the 70 kDa Ubi-L adduct, which was recognized by an anti-MNSF monoclonal antibody. Treatment of CD8+ T-cells with interferon (IFN) γ also caused the expression of the 70 kDa Ubi-L adduct, whereas the responses to IFNα and IFNβ were nil. Antigen- and Con A- stimulated D.10 G4.1, a murine T helper clone type 2, induced the 33.5 kDa, but not the 70 kDa, adduct. These results suggest a role for Ubi-L conjugation in the regulation of T-cell activation.

scholarly journals Further characterization of erythrocyte p80 and the membrane protein defect of In(Lu) Lu(a-b-) erythrocytes

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1475-1481 ◽  
Author(s):  
MJ Telen ◽  
I Rogers ◽  
M Letarte
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Concanavalin A ◽  
Polyclonal Antibodies ◽  
Erythrocyte Ghosts ◽  
Con A ◽  
Down Regulation ◽  
Regulation Of Expression ◽  
Variable Effect

Abstract We have previously shown that the In(Lu) gene down-regulates expression of an erythrocyte protein antigen identified by murine monoclonal antibody (MoAb) A3D8. In the present study we have examined In(Lu) Lu(a- b-) erythrocytes for expression of additional epitopes on the erythrocyte 80 kilodalton protein (p80) bearing the A3D8 antigen. Using a total of seven additional MoAbs that recognize three epitopes on erythrocyte p80, we have shown that In(Lu) Lu(a-b-) erythrocytes exhibit down-regulation of expression of all three epitopes. In(Lu) erythrocytes also showed a reduction in their reactivity to rabbit antibodies produced against purified p80 from either erythrocytes or lymphocytes. Furthermore the reactivity of the MoAbs was not altered by treatment of the cells with neuraminidase but was substantially reduced by treatment of cells with trypsin or chymotrypsin. The polyclonal anti- p80 sera were shown to react with a fragment of 50,000 daltons, still associated with erythrocyte ghosts, following treatment of the cells with trypsin or chymotrypsin. Treatment of erythrocytes with the thiol- reactive reagent AET decreased their reactivity with the MoAbs but had a variable effect on their reactivity with polyclonal antibodies. Erythrocyte p80 is a glycoprotein with N-linked oligosaccharides, as demonstrated by its binding to concanavalin A (Con A) and Len culinaris lectins. Following Endoglycosidase F treatment, erythrocyte p80 underwent a reduction in apparent mol wt of 11,000. The presence of a reduced amount of an intact p80 glycoprotein, seen by a decrease in reactivity with MoAbs directed at three distinct epitopes and with two different polyclonal antibodies, suggests that the In(Lu) gene interferes with expression by erythrocytes of the entire p80 glycoprotein.

scholarly journals A type VI collagen-related glycopolypeptide is the major concanavalin A-binding component in pig skin

1989 ◽  
Vol 257 (1) ◽  
pp. 79-86 ◽  
Author(s):  
I A King ◽  
A Tabiowo ◽  
P R Fryer ◽  
F M Pope
Keyword(s):  
Mast Cells ◽  
Molecular Mass ◽  
Blood Vessels ◽  
Concanavalin A ◽  
Type Vi Collagen ◽  
Pig Skin ◽  
Metabolic Labelling ◽  
Immune Precipitation ◽  
Tissue Form ◽  
Binding Component

The major concanavalin A-binding component in urea/deoxycholate/mercaptoethanol extracts of pig skin was a collagenous disulphide-cross-linked glycopolypeptide with an apparent molecular mass of 150 kDa and a pI of 5.5. Antiserum against the electrophoretically purified glycopolypeptide gave strong dermal staining similar to that seen with fluorescent concanavalin A. Immunocytochemical labelling showed prominent labelling of 3-4 nm dermal microfilaments, particularly those associated with dermal blood vessels and mast cells. Immunoblotting with authentic antiserum indicated that the major skin glycopolypeptide was probably identical with collagen-like glycoprotein, the tissue form of the alpha 1/alpha 2 subunits of type VI collagen. This was confirmed by immunoblotting of authentic type VI collagen from pepsin-treated pig skin. Immunoblotting, metabolic labelling with [3H]glucosamine and immune precipitation showed that an immunoreactive collagenous glycopolypeptide was synthesized and secreted by cultured pig skin fibroblasts. The results suggest that type VI collagen is the major concanavalin A-binding component in pig skin.

scholarly journals Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase.

1987 ◽  
Vol 104 (3) ◽  
pp. 461-472 ◽  
Author(s):  
E Damiani ◽  
A Margreth ◽  
A Furlan ◽  
A S Dahms ◽  
J Arnn ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Integral Membrane Protein ◽  
Amino Acid Residues ◽  
Con A ◽  
Transverse Tubule ◽  
Immunological Relationship

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.

Paracrystalline Aggregates of Psoriatic Keratin and Their Relation to Normal Keratin Structure

1985 ◽  
Vol 43 ◽  
pp. 680-681
Author(s):  
S. Trachtenberg ◽  
P.M. Steinert ◽  
B.L. Trus ◽  
A.C. Steven
Keyword(s):  
Cell Death ◽  
Stratum Corneum ◽  
Intermediate Filament ◽  
Skin Disease ◽  
Amorphous Matrix ◽  
Terminal Differentiation ◽  
Proteolytic Degradation ◽  
Horny Layer ◽  
Chronic Skin ◽  
Chronic Skin Disease

During terminal differentiation of vertebrate epidermis, certain specific keratin intermediate filament (KIF) proteins are produced. Keratinization of the epidermis involves cell death and disruption of the cytoplasm, leaving a network of KIF embedded in an amorphous matrix which forms the outer horny layer known as the stratum corneum. Eventually these cells are shed (desquamation). Normally, the processes of differentiation, keratinization, and desquamation are regulated in an orderly manner. In psoriasis, a chronic skin disease, a hyperkeratotic stratum corneum is produced, resulting in abnormal desquamation of unusually large scales. In this disease, the normal KIF proteins are diminished in amount or absent, and other proteins more typical of proliferative epidermal cells are present. There is also evidence of proteolytic degradation of the KIF.

Concanavalin A-induced Aggregation of Human Blood Platelets

1975 ◽  
Vol 33 (02) ◽  
pp. 354-360 ◽  
Author(s):  
Heinrich Patscheke ◽  
Reinhard Brossmer
Keyword(s):  
Concanavalin A ◽  
Binding Capacity ◽  
Specific Binding ◽  
Blood Platelets ◽  
Residual Effect ◽  
Independent Effect ◽  
Con A ◽  
Experimental Conditions ◽  
Main Effect ◽  
Induced Aggregation

SummaryConcanavalin A (CON A) causes platelets to aggregate. A Ca++-independent effect of CON A could be separated from a main effect which depends on Ca++. The main effect probably is a consequence of the CON A-induced platelet release reaction and therefore is platelet-specific. The weak residual effect observed in the presence of Na2EDTA may be due to a similar mechanism as has been demonstrated for CON A-induced aggregations of several other normal and malignant transformed animal cells.Na2EDTA did not inhibit the carbohydrate-specific binding capacity of CON A. Therefore, Na2EDTA appears not to demineralize the CON A molecules under these experimental conditions.α-methyl-D-glucoside inhibits the Ca++-independent as well as the Ca++-dependent effect of CON A.Pretreatment by neuraminidase stimulated the platelet aggregation induced by CON A. It is possible that removal of terminal sialic acid residues makes additional receptors accessible for the binding of CON A.

scholarly journals Visualization of intracellular concanavalin A binding sites in retinal photoreceptors.

1978 ◽  
Vol 26 (10) ◽  
pp. 822-828 ◽  
Author(s):  
I Nir
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Photoreceptor Cells ◽  
Con A ◽  
Thin Sections ◽  
Glycol Methacrylate ◽  
Outer Segments ◽  
Retinal Photoreceptors ◽  
Carbohydrate Components ◽  
The Relationship

Localization of carbohydrate components in retinal photoreceptor cells and membranes was studied. Frog and rat retinas were fixed with glutaraldehyde and embedded in glycol methacrylate or in a mixture of glycol methacrylate, glutaraldehyde and urea. Thin sections were incubated with ferritin-labeled concanavalin A (F-Con A) and stained with osmium vapors. Intensive binding was observed in both rod and cone outer segments. In the rod inner segment, differential binding of F-Con A was demonstrated. While numerous ferritin granules were observed in the myoid zone, only a few were seen in the ellipsoid zone, except for a local accumulation along the plasma membrane. In the rod outer segment, Con A binding sites were closely associated with the disk membranes. Ferritin granules were observed on both sides of the membranes. The relationship between the localization of Con A binding sites and the orientation of visual pigment molecules within the rod outer segments disk membranes was discussed.

Factors Influencing the Reaction of α1-Fetoprotein with Concanavalin A and Lens Culinaris Agglutinin in Crossed Affinoimmunoelectrophoresis

1992 ◽  
Vol 38 (8) ◽  
pp. 1418-1424 ◽  
Author(s):  
D Magne ◽  
N Seta ◽  
D Lebrun ◽  
G Durand ◽  
D Durand
Keyword(s):  
Concanavalin A ◽  
Lens Culinaris ◽  
Biological Fluids ◽  
Con A ◽  
Cellular Origin ◽  
Lens Culinaris Agglutinin ◽  
Lentil Lectin ◽  
Wide Range ◽  
Minimal Quantity ◽  
Antibody Ratio

Abstract Concanavalin A (Con A) and lentil lectin (LCA) analysis of alpha-fetoprotein (AFP) glycosylation heterogeneity is used in a variety of clinical situations. We studied the influence of analytical conditions on the separation of AFP glycoforms by using lectin-crossed affinoimmunoelectrophoresis, regardless of the AFP concentration, which can vary over a wide range in biological fluids. We defined the optimal concentration of Con A (2 g/L) and LCA (0.35 g/L) in the first-dimension gel, together with the optimum antigen (AFP)/antibody ratio in the second-dimension gel. The presence of protein in the diluent used for AFP samples was found to change the shape of crossed affinoimmunoelectrophoresis patterns without changing the percentage composition of AFP fractions. The within-run CV was less than 4% for both lectins, and the between-run CV was less than 6.3%. The minimal quantity of AFP that provided a visible pattern with both lectins was 4 ng, corresponding to 50 microL of an 80 micrograms/L AFP sample. These technical conditions allow the cellular origin of AFP to be determined, regardless of the concentration in the sample. Typical AFP lectin patterns of secreting tumors are compared with fetal and cord serum AFP.

Sign in / Sign up

Export Citation Format

Share Document