A cholesterol-dependent CD20 epitope detected by the FMC7 antibody

Polyak, Maria J.; Ayer, Linda M.; Szczepek, Agnieszka J.; Deans, Julie P.

doi:10.1038/sj.leu.2402978

Cited by 24 publications

(19 citation statements)

References 37 publications

(42 reference statements)

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“…Thus, the resistance of CD20 to CHAPS may indicate that its constitutive raft localization is mediated by interactions with lipids rather than proteins. Consistent with this notion, the conformation of CD20 was recently shown to be sensitive to the level of membrane cholesterol, suggesting a potentially direct CD20-cholesterol interaction (27).…”

Section: Discussionsupporting

confidence: 61%

The CD20 Calcium Channel Is Localized to Microvilli and Constitutively Associated with Membrane Rafts

Ayer

Polyak

et al. 2004

Journal of Biological Chemistry

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CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein.CD20 is a B cell-specific tetraspan protein that assembles into oligomeric complexes and forms or regulates a store-operated calcium entry channel that is responsive to B cell receptor (BCR) 1 signaling (1). CD20 is also an effective target for in vivo depletion of malignant or autoimmune B cells using monoclonal antibodies (mAbs), which can activate apoptotic signaling pathways and mediate complement-mediated cytoxicity potentially through mechanisms involving cholesterol-and sphingolipid-rich membrane microdomains known as lipid rafts (2-4). Rafts are thought to function in part as platforms for signaling from those receptors with properties that allow their access to the tightly packed lipid raft environment, which otherwise excludes most membrane proteins (5-7). The operational criteria for assigning raft association of a protein are insolubility in nonionic detergents and buoyancy on density gradients. The detergent best characterized for raft isolation is Triton X-100, and we showed previously that antibodies induce translocation of CD20 from the soluble fraction of Triton X-100 cell lysates into the buoyant insoluble fraction, consistent with its induced association with rafts (8). However, raft association of some proteins can only be demonstrated using very low concentrations of Triton X-100 or other nonionic detergents (9 -11), and we recently found that unligated CD20, although soluble in 1% Triton X-100, was insoluble in 1% Brij 58 (1). Brij 58-insoluble CD20 localized to cholesterol-dependent, buoyant fractions on sucrose density gradients. Importantly, deletion of a short membrane-proximal cytoplasmic sequence, previously shown to be essential for eff...

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Section: Discussionsupporting

confidence: 61%

The CD20 Calcium Channel Is Localized to Microvilli and Constitutively Associated with Membrane Rafts

Ayer

Polyak

et al. 2004

Journal of Biological Chemistry

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“…Studies suggest that anti-FMC7 antibody binds to a conformationally determined epitope on the CD20 antigen (17,18). It is interesting to note that the cluster with decreased CD20 expression (i.e., the high CD38 typical BCLL cluster) also had concurrently decreased FMC7 expression.…”

Section: Discussionmentioning

confidence: 99%

Unsupervised immunophenotypic profiling of chronic lymphocytic leukemia

Habib

Finn

2006

Cytometry Part B Clinical

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Background: Proteomics and functional genomics have revolutionized approaches to disease classification. Like proteomics, flow cytometry (FCM) assesses concurrent expression of many proteins, with the advantage of using intact cells that may be differentially selected during analysis. However, FCM has generally been used for incremental marker validation or construction of predictive models based on known patterns, rather than as a tool for unsupervised class discovery. We undertook a retrospective analysis of clinical FCM data to assess the feasibility of a cell-based proteomic approach to FCM by unsupervised cluster analysis.Methods: Multicolor FCM data on peripheral blood (PB) and bone marrow (BM) lymphocytes from 140 consecutive patients with B-cell chronic lymphoproliferative disorders (LPDs), including 81 chronic lymphocytic leukemia (CLLs), were studied. Expression was normalized for CD19 totals, and recorded for 10 additional B-cell markers. Data were subjected to hierarchical cluster analysis using complete linkage by Pearson's correlation. Analysis of CLL in PB samples (n = 63) discovered three major clusters. One cluster (14 patients) was skewed toward ''atypical'' CLL and was characterized by high CD20, CD22, FMC7, and light chain, and low CD23. The remaining two clusters consisted almost entirely (48/49) of cases recorded as typical BCLL. The smaller ''typical'' BCLL cluster differed from the larger cluster by high CD38 (P = 0.001), low CD20 (P = 0.001), and low CD23 (P = 0.016). These two typical BCLL clusters showed a trend toward a difference in survival (P = 0.1090). Statistically significant cluster stability was demonstrated by expanding the dataset to include BM samples, and by using a method of random sampling with replacement.Conclusions: This study supports the concept that unsupervised immunophenotypic profiling of FCM data can yield reproducible subtypes of lymphoma/chronic leukemia. Expanded studies are warranted in the use of FCM as an unsupervised class discovery tool, akin to other proteomic methods, rather than as a validation tool. q

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“…33,34 Interestingly, Polyak et al found that FMC7 may be an indicator of membrane cholesterol content as cholesterol depletion markedly diminishes the expression of FMC7. 17,35 Thus, samples with discordant CD20 may represent a form of DLBCL that has an altered membrane cholesterol metabolism.…”

Section: Discussionmentioning

confidence: 99%

“…This was also true for FMC7, an epitope of CD20 (data not shown). 16,17 Interestingly, one sample showed at least 3 populations of CD19 ϩ cells displaying different intensities of FMC7, suggesting that clonal populations with different CD20 expression can exist within the same tumor ( Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). All but one sample with "dim" CD20 expression also had "dim" expression for FMC7.…”

Section: Cd20 Expression By Fcm Is Heterogeneousmentioning

confidence: 99%