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Updated: Jun 10, 2026
Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction
Published on: April 26, 2011
The lipid-modulating effects of a CD4-specific recombinant antibody correlate with ZAP-70 segregation outside
Myriam Chentouf1, Maxime Rigo, Soufiane Ghannam
1IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier F-34298, France.
Insights
The antibody 13B8.2 alters T-cell lipid rafts by increasing ceramide and decreasing phosphatidylserine, impacting CD4 and ZAP-70 localization for anti-tumoral effects.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- The anti-tumoral effects of antibody 13B8.2 involve CD4 retention in membrane rafts and altered T-cell receptor signaling.
- Previous studies linked these effects to the exclusion of ZAP-70 kinase and downstream signaling molecules from rafts.
Purpose of the Study:
- To investigate whether antibody 13B8.2 affects the lipid composition of membrane rafts.
- To elucidate the role of lipid changes in the anti-tumoral mechanism of 13B8.2.
Main Methods:
- Treatment of Jurkat T cells with antibody 13B8.2.
- Analysis of lipid composition (neutral lipids, cholesterol, phosphatidylserine, fatty acids) in GM1-positive membrane rafts.
- Measurement of ceramide release and acid sphingomyelinase (ASMase) activity.
- Inhibition studies using imipramine, myriocin, and fumonisin B1.
- Assay of bacterial sphingomyelinase activity.
Main Results:
- 13B8.2 treatment did not alter neutral lipids or cholesterol in membrane rafts but decreased phosphatidylserine synthesis.
- Concomitant increase in C18:0 saturated fatty acid and ceramide release within membrane rafts.
- Antibody-induced ceramide release was mediated by enhanced ASMase activity and blocked by imipramine.
- Ceramide release was independent of de novo synthesis pathways.
- Bacterial sphingomyelinase mimicked 13B8.2 effects on ceramide release and ZAP-70 segregation.
Conclusions:
- Antibody 13B8.2 modulates T-cell membrane raft lipid composition by decreasing phosphatidylserine and increasing ceramide via ASMase activation.
- This lipid remodeling, particularly ceramide increase, is crucial for structuring raft platforms and modulating CD4/ZAP-70 localization.
- The acid sphingomyelinase/ceramide pathway activation is a key event in antibody-mediated anti-tumoral effects.
Abstract:
We previously reported that the anti-tumoral effects of the recombinant IgG(1) antibody 13B8.2, which is directed against the CDR3-like loop on the D1 domain of CD4, are linked to accumulation/retention of CD4 inside membrane rafts, recruitment of signaling molecules of the TCR/CD3 pathway and raft exclusion of the ZAP-70 kinase and its downstream targets Vav-1, PLCγ1 and SLP-76. We thus wanted to assess whether this compartmentalization could be related to a possible effect of 13B8.2 on the lipid composition of rafts. Here we show that 13B8.2 treatment of Jurkat T cells did not affect neutral lipids and particularly cholesterol content in GM1-positive membrane rafts, but decreased phosphatidylserine synthesis. C18:0 saturated fatty acid level in GM1-positive membrane rafts and ceramide release were concomitantly increased following treatment with 13B8.2. Antibody-induced ceramide release in membrane rafts occurred through enhanced acid sphingomyelinase activity and was blocked by the acid sphingomyelinase inhibitor imipramine, but was not affected by inhibitors of de novo ceramide synthesis, myriocin and fumonisin B1. Similarly to 13B8.2, addition of bacterial sphingomyelinase increased ceramide release and segregated ZAP-70 outside GM1-positive membrane rafts from Jurkat T cells. Besides CD4/ZAP-70 modulation in membrane rafts, the 13B8.2-induced activation of the acid sphingomyelinase/ceramide pathway is an important event for structuring raft platforms and transducing CD4-related intracellular signals, which can further fine-tune antibody-triggered tumoral effects.
