Abstract
We used shotgun proteomics to identify plasma membrane and lipid raft proteins purified from B cells obtained from mantle cell lymphoma (MCL) patients in leukemic phase. Bioinformatics identified 111 transmembrane proteins, some of which were profiled in primary MCL cases, MCL-derived cell lines, and normal B cells using RT-PCR and Western blotting. Several transmembrane proteins, including CD27, CD70, and CD31 (PECAM-1), were overexpressed when compared with normal B cells. CD70 was up-regulated (>10-fold) in three of five MCL patients along with its cognate receptor CD27, which was up-regulated (4-9-fold) in five of five patients, suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and protein kinase C betaII were also detected in the plasma membranes, suggesting that these proteins are constitutively active in MCL. Protein kinase C betaII has been associated with lipid rafts, and shotgun proteomics/protein profiling revealed that key lipid raft proteins, raftlin (four of five patients) and CSK (C-terminal Src kinase)-binding protein (Cbp)/phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) (four of four patients) were down-regulated in MCL. Levels of other known lipid raft proteins, such as Lyn kinase and flotillin 1, were similar to normal B cells. However, 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, was associated with lipid rafts and was up-regulated approximately 7-fold in MCL compared with normal B cells. Significantly inhibitors of 5-LO activity (AA861) and 5-LO-activating protein (FLAP) (MK886, its activating enzyme) induced apoptosis in MCL cell lines and primary chronic lymphocytic leukemia cells, indicating an important role for the leukotriene biosynthetic pathway in MCL and other B cell malignancies. Thus, using shotgun proteomics and mRNA and protein expression profiling we identified a subset of known and unknown transmembrane proteins with aberrant expression in MCL plasma membranes. These proteins may play a role in the pathology of the disease and are potential therapeutic targets in MCL.
Highlights
We used shotgun proteomics to identify plasma membrane and lipid raft proteins purified from B cells obtained from mantle cell lymphoma (MCL) patients in leukemic phase
Using shotgun proteomics we identified extrinsic and intrinsic proteins in plasma membranes purified from primary B cells obtained from two patients (MCL-P2 and MCL-P5) and from two MCL-derived cell lines (Z138 and JVM2)
These results indicated that CSK-binding protein (Cbp)/phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a known lipid raft protein, was underexpressed in primary MCL cells, and RTPCR showed that mRNA levels in MCL patients were markedly down-regulated in four of four MCL patient samples
Summary
Cell Purification and Cell Lysate Preparation—MCL peripheral blood samples were derived from patients attending the CLL clinic at Leicester Royal Infirmary after ethics committee review and after obtaining informed, written consent. Five patients with lymphocyte counts greater than 50 ϫ 106/ml were selected for this study to minimize possible contamination of tumor cells by normal hemopoietic cells Western Blot Analysis—Aliquots (10 g) of cell lysate or plasma proteins were separated by 4 –20% 1D SDS-PAGE, transferred onto nitrocellulose membranes, and probed with the appropriate antibodies before detection by enhanced chemiluminescence (ECL or ECL plus Western blotting Detection Reagent, GE Healthcare, Amersham Biosciences) as described previously [19]. The amplification efficiencies of the target and TATA box-binding protein genes were approximately equal
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