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 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 The characterization of protein 4.1 Presles, a shortened variant of RBC membrane protein 4.1

Blood ◽  
1985 ◽  
Vol 65 (6) ◽  
pp. 1511-1517 ◽  
Author(s):  
L Morle ◽  
M Garbarz ◽  
N Alloisio ◽  
R Girot ◽  
I Chaveroche ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Limited Proteolysis ◽  
Genetic Data ◽  
Compound Heterozygote ◽  
Heterozygous State ◽  
Protein 4.1 ◽  
Rbc Membrane ◽  
The Family

Abstract In a previous report (Blood 60:265, 1982), we described a family with an abnormal RBC membrane protein doublet, which we considered a shortened protein 4.1 on the basis of biochemical and genetic data. Using an anti-4.1 monoclonal antibody, we confirm here that the shortened protein derives from protein 4.1. One of the members of the family contemporaneously displayed the 4.1 (-) trait, eg, the heterozygous state of this variety of hereditary elliptocytosis that lacks protein 4.1. The 4.1a/4.1b ratio was low whenever the 4.1 trait was present, regardless of the type of protein 4.1 involved. The RBCs of the compound heterozygote, containing only the shortened species of protein 4.1, made it possible to analyze without interference the contact between shortened protein 4.1 and sialoglycoprotein beta, or glycoconnectin. Shortened protein 4.1 did not alter the amount of glycoconnectin in the ghosts nor did it change its extractability into the Triton shells. Limited proteolysis of shortened polypeptides 4.1a and 4.1b showed that they are sequence related. It is conflicting that the persons carrying the shortened protein 4.1 are devoid of specific clinical and morphological abnormalities, apart from those pertaining to the 4.1- trait, when the latter is present.

Characterization of a Monoclonal Antibody Against Concanavalin A Binding Antigen of Aspergillus Fumigatus

Hybridoma ◽  
1991 ◽  
Vol 10 (3) ◽  
pp. 387-393 ◽  
Author(s):  
VISWANATH P. KURUP ◽  
NANCY ELMS ◽  
JORDAN N. FINK
Keyword(s):  
Monoclonal Antibody ◽  
Aspergillus Fumigatus ◽  
Concanavalin A

scholarly journals Generation and characterization of LPA-KIV9, a murine monoclonal antibody binding a single site on apolipoprotein (a)

2020 ◽  
Vol 61 (9) ◽  
pp. 1263-1270
Author(s):  
Ayelet Gonen ◽  
Xiaohong Yang ◽  
Calvin Yeang ◽  
Elena Alekseeva ◽  
Marlys Koschinsky ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Risk Factor ◽  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Basic Research ◽  
Murine Monoclonal Antibody ◽  
Therapeutic Approaches ◽  
Apolipoprotein A ◽  
Monoclonal Antibody Binding

Lipoprotein (a) [Lp(a)] is a risk factor for CVD and a target of therapy, but Lp(a) measurements are not globally standardized. Commercially available assays generally use polyclonal antibodies that detect multiple sites within the kringle (K)IV2 repeat region of Lp(a) and may lead to inaccurate assessments of plasma levels. With increasing awareness of Lp(a) as a cardiovascular risk factor and the active clinical development of new potential therapeutic approaches, the broad availability of reagents capable of providing isoform independence of Lp(a) measurements is paramount. To address this issue, we generated a murine monoclonal antibody that binds to only one site on apo(a). A BALB/C mouse was immunized with a truncated version of apo(a) that contained eight total KIV repeats, including only one copy of KIV2. We generated hybridomas, screened them, and successfully produced a KIV2-independent monoclonal antibody, named LPA-KIV9. Using a variety of truncated apo(a) constructs to map its binding site, we found that LPA-KIV9 binds to KIV9 without binding to plasminogen. Fine peptide mapping revealed that LPA-KIV9 bound to the sequence 4076LETPTVV4082 on KIV9. In conclusion, the generation of monoclonal antibody LPA-KIV9 may be a useful reagent in basic research studies and in the clinical application of Lp(a) measurements.

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.

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