scholarly journals The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein.

1994 ◽  
Vol 127 (3) ◽  
pp. 803-811 ◽  
Author(s):  
R A Bloodgood ◽  
N L Salomonsky
Keyword(s):  
Monoclonal Antibody ◽  
Signaling Pathway ◽  
Live Cells ◽  
Cross Linking ◽  
Carbohydrate Binding ◽  
Membrane Glycoprotein ◽  
Channel Blockers ◽  
Membrane Glycoproteins ◽  
Flagellar Membrane

Cross-linking of Chlamydomonas reinhardtii flagellar membrane glycoproteins results in the directed movements of these glycoproteins within the plane of the flagellar membrane. Three carbohydrate-binding reagents (FMG-1 monoclonal antibody, FMG-3 monoclonal antibody, concanvalin A) that induce flagellar membrane glycoprotein crosslinking and redistribution also induce the specific dephosphorylation of a 60-kD (pI 4.8-5.0) flagellar phosphoprotein (pp60) that is phosphorylated in vivo on serine. Ethanol treatment of live cells induces a similar specific dephosphorylation of pp60. Affinity adsorption of flagellar 32P-labeled membrane-matrix extracts with the FMG-1 monoclonal antibody and concanavalin A demonstrates that pp60 binds to the 350-kD class of flagellar membrane glycoproteins recognized by the FMG-1 monoclonal antibody. In vitro, protein phosphatase 2B (calcineurin) removes 60% of the 32P from pp60; this correlates well with previous observations that directed flagellar glycoprotein movements are dependent on micromolar calcium in the medium and are inhibited by calcium channel blockers and calmodulin antagonists. The data reported here are consistent with the dephosphorylation of pp60 being a step in the signaling pathway that couples flagellar membrane glycoprotein cross-linking to the directed movements of flagellar membrane glycoproteins.

scholarly journals Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A.

1986 ◽  
Vol 102 (5) ◽  
pp. 1797-1812 ◽  
Author(s):  
R A Bloodgood ◽  
M P Woodward ◽  
N L Salomonsky
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
High Molecular Weight ◽  
Concanavalin A ◽  
Live Cells ◽  
Surface Phenomenon ◽  
Carbohydrate Binding ◽  
Membrane Glycoproteins ◽  
Western Blots ◽  
Flagellar Membrane

Two carbohydrate-binding probes, the lectin concanavalin A and an anti-carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross-react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres.

Calcium influx regulates antibody-induced glycoprotein movements within the Chlamydomonas flagellar membrane

1990 ◽  
Vol 96 (1) ◽  
pp. 27-33 ◽  
Author(s):  
R.A. Bloodgood ◽  
N.L. Salomonsky
Keyword(s):  
Calcium Channel ◽  
Calcium Channel Blockers ◽  
Calcium Influx ◽  
Gliding Motility ◽  
Free Calcium ◽  
Membrane Glycoprotein ◽  
Channel Blockers ◽  
Membrane Glycoproteins ◽  
Whole Cell ◽  
Flagellar Membrane

The Chlamydomonas flagellar surface exhibits a number of dynamic membrane phenomena associated with whole-cell gliding locomotion and the early events in fertilization. Crosslinking of a specific population of flagellar surface-exposed glycoproteins with the lectin concanavalin A or an anti-carbohydrate mouse monoclonal antibody, designated FMG-1, results in a characteristic pattern of glycoprotein redistribution within the plane of the flagellar membrane. Recent evidence suggests that flagellar membrane glycoprotein movements are associated with both whole-cell gliding motility and the early events in mating. It is of interest to determine the transmembrane signaling pathway whereby crosslinking of the external domains of flagellar glycoproteins activates the intraflagellar machinery responsible for translocation of flagellar membrane glycoproteins. The redistribution of flagellar membrane glycoproteins requires micromolar levels of free calcium in the medium; lowering the free calcium concentration to 10(−7) M results in complete but reversible inhibition of redistribution. Redistribution is maximal in the presence of 20 microM free calcium in the medium. Redistribution is inhibited in the presence of 20 microM free calcium by the calmodulin antagonists trifluoperazine, W-7 and calmidazolium, the calcium channel blockers diltiazem, methoxyverapamil (D-600) and barium chloride, and the local anesthetics, lidocaine and procaine. The actions of all of these agents can be interpreted in terms of a requirement for calcium in the signaling mechanism associated with flagellar glycoprotein redistribution. In particular, the requirement for micromolar calcium in the external medium and the effects of specific calcium channel blockers suggest that flagellar membrane glycoprotein crosslinking may induce an increase in calcium influx, which may be the initial trigger for activating the flagellar machinery responsible for active movement of flagellar membrane glycoproteins.

scholarly journals Proteolytic processing of a protein involved in sperm-egg fusion correlates with acquisition of fertilization competence.

1990 ◽  
Vol 111 (1) ◽  
pp. 69-78 ◽  
Author(s):  
C P Blobel ◽  
D G Myles ◽  
P Primakoff ◽  
J M White
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Developmental Stages ◽  
Biochemical Properties ◽  
Proteolytic Processing ◽  
Molecular Forms ◽  
Membrane Glycoprotein ◽  
Processing Step

A protein located on the surface of guinea pig sperm (PH-30) has been implicated in the process of sperm-egg fusion (Primakoff, P., H. Hyatt, and J. Tredick-Kline. 1987. J. Cell Biol. 104:141-149). In this paper we have assessed basic biochemical properties of PH-30 and have analyzed the molecular forms of PH-30 present at different stages of sperm maturation. We show the following: (a) PH-30 is an integral membrane glycoprotein; (b) it is composed of two tightly associated and immunologically distinct subunits; (c) both subunits are made as larger precursors; (d) processing of the two subunits occurs at different developmental stages; (e) the final processing step occurs in the region of the epididymis where sperm become fertilization competent; (f) processing can be mimicked in vitro; (g) processing exposes at least two new epitopes on PH-30-one of the newly exposed epitopes is recognized by a fusion-inhibitory monoclonal antibody. These results are discussed in terms of the possible role of PH-30 in mediating fusion with the egg plasma membrane.

scholarly journals CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 682-688 ◽  
Author(s):  
RB Jenkins ◽  
WL Nichols ◽  
KG Mann ◽  
LA Jr Solberg
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Protein Synthesis ◽  
Bone Marrow Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Glycoprotein ◽  
Factor X ◽  
Membrane Glycoproteins ◽  
Molecular Weight Range

Abstract Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1–1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1–1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1–1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.

scholarly journals In Vitro Characterization of the Carbohydrate-Binding Agents HHA, GNA and UDA as Inhibitors of Influenza a and B Virus Replication

2020 ◽  
Author(s):  
Evelien Vanderlinden ◽  
Nathalie Van Winkel ◽  
Lieve Naesens ◽  
Els J. M. Van Damme ◽  
Leentje Persoons ◽  
...  
Keyword(s):  
Influenza A ◽  
Inhibitory Effect ◽  
H3n2 Virus ◽  
Influenza B ◽  
Carbohydrate Binding ◽  
Membrane Glycoproteins ◽  
Influenza A H1n1 ◽  
Binding Agents ◽  
Continuous Presence

Here, we report on the anti-influenza virus activity of the mannose-binding agents, Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), and the (N-acetylglucosamine)n-specific Urtica dioica agglutinin (UDA). These carbohydrate-binding agents (CBA) strongly inhibited various influenza A(H1N1), A(H3N2) and B viruses in vitro, with EC50 values ranging from 0.016 to 83 nM, generating selectivity indexes up to 125,000. Somewhat less activity was observed against the A/PR/8/34 and an A(H1N1)pdm09 strain. In time-of-addition experiments, these CBA lost their inhibitory activity when added 30 min p.i. Interference with virus entry processes was also evident from strong inhibition of virus-induced hemolysis at low pH. However, a direct effect on acid-induced refolding of the viral hemagglutinin (HA) was excluded by the tryptic digestion assay. Instead, HHA treatment of HA-expressing cells led to a significant reduction of plasma membrane mobility. Crosslinking of membrane glycoproteins, through interaction with HA, could also explain the inhibitory effect on the release of newly formed virions when HHA was added at 6 h p.i. These CBA presumably interact with one or more N-glycans on the globular head of HA, since their absence led to reduced activity against mutant influenza B viruses and HHA-resistant A(H1N1) viruses. The latter condition only emerged after 33 cell culture passages in the continuous presence of HHA, and the A(H3N2) virus even retained full sensitivity after 50 passages. Thus, these CBA qualify as potent inhibitors of influenza A and B viruses in vitro with a pleiotropic mechanism of action and a high barrier for viral resistance.

scholarly journals A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leakage in vivo

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 338-340 ◽  
Author(s):  
KE Arfors ◽  
C Lundberg ◽  
L Lindbom ◽  
K Lundberg ◽  
PG Beatty ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Polymorphonuclear Leukocyte ◽  
Critical Role ◽  
Skin Lesions ◽  
Membrane Glycoprotein ◽  
Glycoprotein Complex ◽  
Plasma Leakage ◽  
And Migration

Previous in vitro findings suggest a critical role for the polymorphonuclear leukocyte (PMN) membrane glycoprotein complex CD18 in PMN adherence and chemotaxis. We examined the effect of the murine monoclonal antibody (MoAb) 60.3, recognizing CD18, on induced PMN accumulation in vivo. Rabbits were pretreated with MoAb 60.3, and the chemotactic factors fMLP, leukotriene (LT)B4, and C5a, as well as histamine, were injected intradermally; 4 hours later, plasma leakage (125I-albumin) and the PMN accumulation (myeloperoxidase) were determined. Both PMN accumulation and PMN-dependent plasma leakage were abolished in the inflammatory skin lesions of rabbits pretreated with MoAb 60.3 as compared with control animals, whereas histamine-induced PMN-independent plasma leakage was unaffected. Intravital microscopy of the rabbit tenuissimus muscle revealed that MoAb 60.3 inhibited both PMN adherence in the venules and migration into the tissue following application of LTB4 and zymosan-activated serum (ZAS). Rolling of PMNs along the venular endothelium was unaffected. Thus, these experiments confirm and extend earlier in vitro findings of the critical role of the membrane glycoprotein complex, CD18, in PMN adherence and chemotaxis.

FAB FRAGMENTS OF MONOCLONAL ANTIBODIES SPECIFIC FOR PORCINE PLATELET MEMBRANE GLYCOPROTEINS GP IB ANDGP IIB/IIIA

1987 ◽  
Author(s):  
H Takami ◽  
W L Nichols ◽  
S E Kaese ◽  
R S Miller ◽  
J A Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments ◽  
Igg1 Subclass ◽  
Von Willebrand

For further study of the porcine hemostatic mechanism, we have prepared murine monoclonal antibodies, and F(ab')2 and Fab fragments, specific for porcine platelet membrane glycoproteins GP lb and GP Ilb/IIIa. To avoid production of antibodies to von Willebrand factor (vWF), mice were immunized with platelets obtained from pigs with severe von Willebrand,s disease. One monoclonal antibody (PP3-4C), of IgG1 subclass, caused 85% inhibition of Ristocetin-induced platelet binding of 125I-vWF (porcine) at ≥12 µg IgG/ml. PP3-4C did not affect ADP or collagen-induced platelet aggregation nor inhibit 125I-fibrinogen (porcine) binding. Pepsin and papain digestion, respectively, were used to prepare PP3-4C F(ab')2 and Fab fragments. PP3-4C F(ab')2 at concentrations ≥12 µg/ml caused 80% inhibition of washed platelet agglutination in the presence of vWF and Ristocetin, whereas Fab fragments at concentrations ≥10 µg/ml caused 60% inhibition. Another monoclonal antibody (PP3-3A), of IgG1 subclass, completely inhibited ADP or collagen-induced platelet aggregation at an IgG concentration of 6 µg/ml. At 10 µg IgG/ml PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated porcine platelets. PP3-3A did not affect vWF-dependent Ristocetin-induced platelet agglutination, nor 125I-vWF binding to platelets in the presence of Ristocetin. PP3-3A did not bind to platelets which were treated with 10 mM EDTA at 37°C for 60 min. F(ab')2 and Fab fragments were isolated from PP3-3A pepsin or papain digests. Both types of PP3-3A fragments caused 100% inhibition of ADP-induced platelet aggregation, at concentrations ≥6 yg/ml. Immunoprecipitation of surface-radiolabeled porcine platelets and subsequent SDS-PAGE demonstrated that PP3-4C recognized a glycoprotein with molecular weight of 140,000 (under reducing conditions), and 165,000 (non-reduced). PP3-3A recognized glycoproteins with molecular weights of 115,000 and 100,000 (reduced), and 130,000 and 80,000 (non-reduced). Neither monoclonal antibody bound to human platelets. These monoclonal antibodies to porcine platelet membrane glycoproteins which are analogues of human GP lb and GP Ilb/IIIa will be useful for in vitro and in vivo studies to further understanding of mammalian hemostatic mechanisms.

scholarly journals A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leakage in vivo

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 338-340 ◽  
Author(s):  
KE Arfors ◽  
C Lundberg ◽  
L Lindbom ◽  
K Lundberg ◽  
PG Beatty ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Polymorphonuclear Leukocyte ◽  
Critical Role ◽  
Skin Lesions ◽  
Membrane Glycoprotein ◽  
Glycoprotein Complex ◽  
Plasma Leakage ◽  
And Migration

Abstract Previous in vitro findings suggest a critical role for the polymorphonuclear leukocyte (PMN) membrane glycoprotein complex CD18 in PMN adherence and chemotaxis. We examined the effect of the murine monoclonal antibody (MoAb) 60.3, recognizing CD18, on induced PMN accumulation in vivo. Rabbits were pretreated with MoAb 60.3, and the chemotactic factors fMLP, leukotriene (LT)B4, and C5a, as well as histamine, were injected intradermally; 4 hours later, plasma leakage (125I-albumin) and the PMN accumulation (myeloperoxidase) were determined. Both PMN accumulation and PMN-dependent plasma leakage were abolished in the inflammatory skin lesions of rabbits pretreated with MoAb 60.3 as compared with control animals, whereas histamine-induced PMN-independent plasma leakage was unaffected. Intravital microscopy of the rabbit tenuissimus muscle revealed that MoAb 60.3 inhibited both PMN adherence in the venules and migration into the tissue following application of LTB4 and zymosan-activated serum (ZAS). Rolling of PMNs along the venular endothelium was unaffected. Thus, these experiments confirm and extend earlier in vitro findings of the critical role of the membrane glycoprotein complex, CD18, in PMN adherence and chemotaxis.

scholarly journals CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 682-688 ◽  
Author(s):  
RB Jenkins ◽  
WL Nichols ◽  
KG Mann ◽  
LA Jr Solberg
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Protein Synthesis ◽  
Bone Marrow Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Glycoprotein ◽  
Factor X ◽  
Membrane Glycoproteins ◽  
Molecular Weight Range

Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1–1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1–1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1–1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.

scholarly journals A monoclonal antibody-defined membrane antigen complex is required for neutrophil-neutrophil aggregation

Blood ◽  
1985 ◽  
Vol 65 (6) ◽  
pp. 1553-1556 ◽  
Author(s):  
BR Schwartz ◽  
HD Ochs ◽  
PG Beatty ◽  
JM Harlan
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Glycoprotein ◽  
Myristate Acetate ◽  
Glycoprotein Complex ◽  
Antigen Complex ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Neutrophil Aggregation ◽  
Dose Dependent

Abstract We examined the aggregation responses of normal neutrophils treated with the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produced dose-dependent inhibition of neutrophil aggregation in response to phorbol myristate acetate, zymosan-activated plasma, and N-formyl-methionylleucylphenylalanine. We conclude that the membrane glycoprotein complex recognized by MoAb 60.3--designated CDw18- -is required for neutrophil-neutrophil aggregation in vitro.

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