Silencing of Intestinal Glycoprotein CD98 by Orally Targeted Nanoparticles Enhances Chemosensitization of Colon Cancer.

Colon cancer is among the most widely occurring cancer types, leading to considerably high mortality rate. The current chemotherapy achieves only limited success, and more effective therapeutic strategies are urgently needed. Human colonic biopsy specimens indicate increased expression of CD98 in patients with colon cancer, suggesting that CD98 might be a potential therapeutic target and/or a receptor for targeted drug delivery in colon cancer treatment. Herein, we coloaded CD98 siRNA (siCD98) and camptothecin (CPT) into CD98 Fab'-functionalized nanoparticles (NPs). The resultant Fab'-siCD98/CPT-NPs showed good monodispersity with an average diameter of approximately 270 nm and a ζ-potential of around -24 mV. These NPs mediated efficient drug delivery to the target cancer cells and tumor tissues, producing much better anticancer and antimigration effects compared to other relevant NPs. Mouse models with orthotopic colon tumors were treated with NP-embedded hydrogel, which revealed that Fab'-siCD98/CPT-NPs exhibited a therapeutic efficacy significantly better than that of single drug-loaded NPs or nonfunctionalized siCD98/CPT-NPs. This study indicates that the Fab'-siCD98/CPT-NP/hydrogel system is able to realize specific release of NPs in the colonic lumen and enable drugs (siCD98 and CPT) to be internalized into target cells, demonstrating a notable potential for clinical applications in colon-cancer-targeted combination therapy.

Prognostic Significance of the Expression of CD98 (4F2hc) in Gastric Cancer.

BACKGROUND: CD98 expression is high in various human neoplasms. However, the relationship of CD98 expression with the clinicopathological factors of gastric cancer (GC) remains unclear. This study examined CD98 expression and its clinicopathological impact on GC. PATIENTS AND METHODS: Three hundred and thirty-one patients with surgically resected GC were evaluated. Tumor sections were stained and analyzed using immunohistochemistry to assess CD98 expression. RESULTS: CD98 was positively expressed in 19% (66/331) of our patient cohort. Increased CD98 expression was significantly associated with advanced GC stage, lymph node metastasis, non-signet histology, lymphatic permeation, and vascular invasion. Positive CD98 expression was also a significant prediction marker for unfavorable prognosis postoperatively. However, CD98 was not identified as GC's independent prognostic predictor. CONCLUSION: CD98 could be a novel prediction marker for worse prognosis in GC-affected patients. Our data suggests that increased CD98 expression plays an essential role in tumor aggressiveness and metastasis.

Cloning, large scale over-expression in E. coli and purification of the components of the human LAT 1 (SLC7A5) amino acid transporter.

The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni(2+)-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.

Involvement of intestinal intraepithelial lymphocytes in turnover of intestinal epithelial cells: morphological and functional alterations due to daily administration of FK506.

To elucidate the interaction of intestinal intraepithelial lymphocytes (IELs) with intestinal epithelial cells (IECs), we investigated alterations of IECs by activating or inactivating IELs. The stimulation of IELs with anti-mouse CD3 monoclonal antibody induced massive apoptosis of IECs. Changes in IECs and IELs from mice that received daily administration of FK506 for 14days were investigated. IELs, particularly TCR-γδ⁺ IELs, were reduced in cell number, and a decrease of cytotoxic activity was observed. Under this condition, loss of apoptotic cells at the tips of villi and delayed turnover of IECs were detected. The expressions of alkaline phosphatase and CD98 amino acid transporters on IECs were decreased. Furthermore, abnormal skeletal organization of villi and weakened binding of IECs to the basement membrane were shown. These results suggest that inactivated IECs, which should be led to apoptosis, remained. It was strongly suggested that IELs participated in IEC turnover.

High expression of CD98 alters epithelial barrier functions to promote induction of airway allergy.

BACKGROUND: Epithelial barrier dysfunction is critical in the induction of allergy; the aetiology is to be further understood. A recent report indicates that CD98 plays a role in the intestinal epithelial barrier dysfunction. OBJECTIVES: This study aimed to investigate the role of overexpression of CD98 in the induction of nasal allergy. METHODS: The nasal epithelium samples were collected from 30 patients with allergic rhinitis and 30 healthy subjects. The contents of CD98 and Staphylococcal enterotoxin B (SEB) in the nasal epithelium samples were evaluated by using Western blotting. The effect of SEB of inducing the expression of CD98 was evaluated with an airway epithelial cell line, the 16HBE14o cells. The epithelial barrier function was assessed with the indicators of transepithelial resistance (TER) and permeability to horseradish peroxidase (HRP). A mouse model was employed to evaluate the role of CD98 in the induction of nasal allergy. RESULTS: High levels of CD98 and SEB were detected in the nasal epithelium of patients with allergic rhinitis. A positive correlation was identified between CD98 and SEB in nasal epithelium samples. Exposure to SEB could induce the overexpression of CD98 in RPMI 2650 and 16HBE14o cells. The overexpression of CD98 down-regulated TER and increased the permeability to HRP in 16HBE14o monolayers. Concurrent exposure to SEB and OVA induced nasal allergies in a mouse model that could be blocked by pre-treatment with anti-CD98 antibody. CONCLUSIONS AND CLINICAL RELEVANCE: CD98 plays a critical role in compromising the airway epithelial barrier function that contributes to the induction of airway allergy.

Intestinal epithelial cell-specific CD98 expression regulates tumorigenesis in Apc(Min/+) mice.

The transmembrane glycoprotein CD98 regulates integrin signaling that in turn controls cell proliferation and survival. CD98 expression is upregulated in various carcinomas, including colorectal cancer. Recently, by generating gain- and loss-of-function mouse models featuring genetic manipulation of CD98 expression specifically in intestinal epithelial cells (IECs), we have explored the crucial role of CD98 in the regulation of intestinal homeostasis and inflammation-associated tumorigenesis. In the present study, we investigated the contribution of CD98 to intestinal tumorigenesis in Apc(Min/+) mice and the underlying mechanism of action. Mice featuring IEC-specific CD98 overexpression (Tg animals) were crossed with Apc(Min/+) mice, and the characteristics of intestinal adenoma formation were assessed. Compared with Apc(Min/+) mice, Tg/Apc(Min/+) animals exhibited increases in both intestinal tumor incidence and tumor size; these parameters correlated with enhanced proliferation and decreased apoptosis of IECs. IEC-specific CD98 overexpression resulted in increased synthesis of the oncogenic proteins c-myc and cyclin-D1 in Apc(Min/+) mice, independently of the Wnt-APC-ß-catenin pathway, suggesting the implication of CD98 overexpression-mediated Erk activation. IEC-specific CD98 overexpression enhanced the production of proinflammatory cytokines and chemokines that are crucial for tumorigenesis. We validated our results in mice exhibiting IEC-specific CD98 downregulation (CD98(flox/+)VillinCre animals). IEC-specific CD98 downregulation efficiently attenuated tumor incidence and growth in Apc(Min/+) mice. The reduction of intestinal tumorigenesis upon IEC-specific CD98 downregulation was caused by the attenuation of IEC proliferation and cytokine/chemokine production. In conclusion, we show that CD98 exerts an oncogenic activity in terms of intestinal tumorigenesis, via an ability to regulate tumor growth and survival.

Intestinal epithelial CD98 synthesis specifically modulates expression of colonic microRNAs during colitis.

The transmembrane glycoprotein CD98 is known to be involved in intestinal inflammation. In the present study, we found that CD98 overexpression in intestinal epithelial cells does not normally affect the expression of colonic (epithelial and immune cell) microRNAs (miRNAs), small noncoding RNAs that posttranscriptionally regulate a wide variety of biological processes. However, upon dextran sulfate sodium (DSS) treatment, the expression of several colonic miRNAs, but not miRNAs from other tissues such as liver and spleen, were differentially regulated in mice overexpressing CD98 in epithelial cells compared with wild-type (WT) animals. For example, the level of colonic miRNA 132 was not affected by DSS treatment in WT animals but was upregulated in mice overexpressing CD98 in intestinal epithelial cells. Other colonic miRNAs, including colonic miRNA 23a and 23b, were downregulated in WT animals after DSS treatment but not in colonic epithelial cell CD98-overexpressing mice. Interestingly, the expression of potential miRNA target genes affected intestinal epithelial cells that overexpress CD98 and cell types that did not overexpress CD98 but were in close proximity to CD98-overexpressing intestinal epithelial cells. Taken together, these observations show that the combination of an inflammatory context and intestinal epithelial cell expression of CD98 affects the regulation of miRNA expression in colonic epithelial and immune cells. This is new evidence that protein expression modulates miRNA expression and suggests the existence of regulatory crosstalk between proteins and miRNAs in diseases such as colitis.

Correlation of L-type amino acid transporter 1 and CD98 expression with triple negative breast cancer prognosis.

Triple negative breast cancer (TNBC) is a heterogeneous, aggressive cancer for which there is no effective chemotherapy or targeted therapy. We aimed to evaluate L-type amino acid transporter (LAT) 1 and CD98 expression immunohistochemically in patients with breast cancer, especially TNBC. Out of 129 patients, LAT1 was positive in 56 patients (43.4%), and CD98 was positive in 41 patients (31.8%). The positive ratio of LAT1 expression in luminal A cases was 7.9%, 30.0% in luminal B cases, 71.4% in HER2 cases and 64.0% in TN cases. HER2 and TN subtypes expressed LAT1 and CD98 at higher levels than luminal A and B subtypes (both P < 0.001). LAT1 and CD98 expression correlated with tumor size (LAT1, P = 0.010; CD98, P = 0.007), nuclear grade (LAT1, P < 0.001; CD98, P < 0.001) and Ki67 labeling index (LAT1, P < 0.001; CD98, P = 0.001). LAT1 and CD98 expression was negatively associated with ER and PgR (both P < 0.001). In TNBC, the 5-year disease-free rate of CD98+ (63.6%) or LAT1+/CD98+ (61.9%) patients was significantly worse than that of CD98- (89.3%) patients or those with no co-expression of LAT1 and CD98 (89.7%), respectively (P = 0.014, P = 0.009). The 5-year survival rates of CD98 positive/negative patients were 77.3% and 100% (P = 0.050), respectively, whereas that of patients with LAT1+/CD98+ (76.2%) was significantly worse (100%) (P = 0.040). Multivariate analysis confirmed that CD98+ or LAT1+/CD98+ expression were risk factors for relapse in TNBC (P = 0.023, P = 0.019). Thus, in the present study we show that LAT1 and CD98 expression are prognostic factors. Inhibition of these proteins might provide a new therapeutic strategy in TNBC.

CD98 expression modulates intestinal homeostasis, inflammation, and colitis-associated cancer in mice.

Expression of the transmembrane glycoprotein CD98 (encoded by SLC3A2) is increased in intestinal inflammatory conditions, such as inflammatory bowel disease (IBD), and in various carcinomas, yet its pathogenetic role remains unknown. By generating gain- and loss-of-function mouse models with genetically manipulated CD98 expression specifically in intestinal epithelial cells (IECs), we explored the role of CD98 in intestinal homeostasis, inflammation, and colitis-associated tumorigenesis. IEC-specific CD98 overexpression induced gut homeostatic defects and increased inflammatory responses to DSS-induced colitis, promoting colitis-associated tumorigenesis in mice. Further analysis indicated that the ability of IEC-specific CD98 overexpression to induce tumorigenesis was linked to its capacity to induce barrier dysfunction and to stimulate cell proliferation and production of proinflammatory mediators. To validate these results, we constructed mice carrying conditional floxed Slc3a2 alleles and crossed them with Villin-Cre mice such that CD98 was downregulated only in IECs. These mice exhibited attenuated inflammatory responses and resistance to both DSS-induced colitis and colitis-associated tumorigenesis. Together, our data show that intestinal CD98 expression has a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in IECs therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as IBD and colitis-associated cancer.

Inhibition of L-type amino acid transporter 1 has antitumor activity in non-small cell lung cancer.

BACKGROUND: L-type amino acid transporter 1 (LAT1) is highly expressed in various human neoplasms. Antitumor activity of inhibiting LAT1 was analyzed in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Expression of LAT1 mRNA in 54 lung cancer cell lines was examined by RT-PCR. An inhibitor of LAT1, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), was administered to H1395 cell. LAT1 expression was examined in correlation with clinical features and outcome in 51 NSCLC patients. RESULTS: Inhibition of LAT1 by BCH reduced cell viability in H1395 cells. Furthermore, co-administration of gefitinib with BCH reduced the viability of the cells more than either agent alone. Inhibition of LAT1 reduced the level of phosphorylation of mTOR, p70S6K and 4EBP1. LAT1 protein expression was closely associated with wild type EGFR, and was an independent significant factor to predict a poor prognosis. CONCLUSION: Inhibition of LAT1 may be a new rationale to the effective therapy of NSCLC without EGFR mutation.

CD98 expression is associated with the grade of malignancy in thymic epithelial tumors.

CD98 has been associated with tumor growth and is highly expressed in human neoplasms. The aim of this study was to evaluate the clinicopathological significance of CD98 expression in thymic epithelial tumors. Forty-nine patients with thymic epithelial tumors were included in this study. Tumor sections were stained by immunohistochemistry for CD98; vascular endothelial growth factor (VEGF); micro-vessels (CD31 and CD34); cell cycle control marker (p53); and apoptosis marker (Bcl-2). CD98 expression for low-risk thymomas, high-risk thymomas, and thymic carcinomas were 1 (4%) of 27, 9 (82%) of 11, and 11 (100%) of 11, respectively. There was positive correlation between CD98 and VEGF (p<0.001), microvessel density (CD31 and CD34) (p<0.001) and p53 (p<0.001). However, Bcl-2 showed no positive correlation with CD98 expression. The expression of CD98 were also significantly associated with the grade of malignancy in thymic epithelial tumors. Multivariate analysis revealed that overexpression of CD98 was a significant independent factor predicting a poor outcome in thymic epithelial tumors. CD98 expression was associated with the grade of malignancy in thymic epithelial tumors. Moreover, CD98 expression was closely correlated with angiogenesis and cell cycle control, and was useful for predicting poor outcome.

MicroRNA-7 modulates CD98 expression during intestinal epithelial cell differentiation.

The transmembrane glycoprotein CD98 regulates multiple cellular functions, including extracellular signaling, epithelial cell adhesion/polarity, amino acid transport, and cell-cell interactions. MicroRNAs post-transcriptionally regulate gene expression, thereby functioning as modulators of numerous cellular processes, such as cell differentiation, proliferation, and apoptosis. Here, we investigated if microRNAs regulate CD98 expression during intestinal epithelial cell differentiation and inflammation. We found that microRNA-7 repressed CD98 expression in Caco2-BBE cells by directly targeting the 3'-untranslated region of human CD98 mRNA. Expression of CD98 was decreased, whereas that of microRNA-7 was increased in well-differentiated Caco2-BBE cells compared with undifferentiated cells. Undifferentiated crypt cells isolated from mouse jejunum showed higher CD98 levels and lower levels of mmu-microRNA-706, a murine original microRNA candidate for CD98, than well-differentiated villus cells. Importantly, microRNA-7 decreased Caco2-BBE cell attachment on laminin-1, and CD98 overexpression recovered this inhibition, suggesting that microRNA-7 modulates epithelial cell adhesion to extracellular matrix, which in turn could affect proliferation and differentiation during the migration of enterocytes across the crypt-villus axis, by regulating CD98 expression. In a pathological context, the pro-inflammatory cytokine interleukin 1-beta increased CD98 expression in Caco2-BBE cells by decreasing microRNA-7 levels. Consistent with the in vitro findings, microRNA-7 levels were decreased in actively inflamed Crohn disease colonic tissues, where CD98 expression was up-regulated, compared with normal tissues. Together, these results reveal a novel mechanism underlying regulation of CD98 expression during patho-physiological states. This study raises microRNAs as a promising target for therapeutic modulations of CD98 expression in intestinal inflammatory disorders.

Regulation of alternative macrophage activation by galectin-3.

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-gamma/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-beta-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.

Membrane trafficking of CD98 and its ligand galectin 3 in BeWo cells--implication for placental cell fusion.

CD98 heavy chain (CD98hc), expressed at high levels in developing human trophoblasts, is an integral membrane protein with multiple N-linked glycosylation sites and known to be important for cell fusion, adhesion, and amino acid transport. Western blotting and flow cytometry were used to study the effect of brefeldin A, an inhibitor of protein translocation through the Golgi, on CD98hc in the human placental trophoblast cell line BeWo. Although brefeldin A treatment caused increased cell surface expression of CD98hc, a novel partially glycosylated form of the protein was found and, concomitantly, cell fusion was reduced. Western blotting showed that CD98 and galectin 3, a proposed ligand for the glycosylated extracellular domain of CD98hc, co-immunoprecipitated, and double-label immuno-electron microscopy confirmed that CD98hc associated with galectin 3. Furthermore, cell fusion was reduced (specifically) by the disaccharide lactose, a known ligand for the carbohydrate recognition domain of galectin 3, suggesting that the association was functional. Taken together, the data suggest that N-glycosylation of CD98 and subsequent interaction with galectin 3 is critical for aspects of placental cell biology, and provides a rationale for the observation that, in the mouse, truncation of the CD98hc extracellular domain leads to early embryonic lethality [Tsumura H, Suzuki N, Saito H, Kawano M, Otake S, Kozuka Y, Komada H, Tsurudome M & Ito Y (2003) Biochem Biophys Res Commun 308, 847-851].

Activation of epithelial CD98 glycoprotein perpetuates colonic inflammation.

Anomalies in the regulation and function of integrins have been implicated in the etiology of various pathologic conditions, including inflammatory disorders such as irritable bowel disease. Several classes of cell surface glycoproteins such as CD98 have been shown to play roles in integrins-mediated events. Here, we investigated the role of CD98 in intestinal inflammation using both in vivo and in vitro approaches. We found that in Caco2-BBE monolayers and colonic tissues, expression of CD98 was upregulated by the proinflammatory cytokine, interferon gamma (INF gamma). Furthermore, CD98 was highly upregulated in colonic tissues from mice with active colitis induced by dextran sodium sulfate (DSS), but not in DSS-treated INF gamma -/- mice. Administration of an anti-CD98 antibody worsened DSS-induced colitis in mice but had no effect on untreated control mice. Finally, we used Caco2-BBE cell monolayers to model intestinal epithelial wound healing, and found that activation of epithelial CD98 in DSS-treated monolayers inhibited monolayer reconstitution, but had no affect on untreated control monolayers. Our data collectively indicate that (i) CD98 upregulation is mediated by INF gamma during intestinal inflammation and (ii) activation of epithelial CD98 protein aggravates intestinal inflammation by reducing intestinal epithelial reconstitution. Overall, our data suggest that epithelial CD98 plays an important role in the perpetuation of intestinal inflammation.

CD98 modulates integrin beta1 function in polarized epithelial cells.

The type II transmembrane protein CD98, best known as the heavy chain of the heterodimeric amino acid transporters (HAT), is required for the surface expression and basolateral localization of this transporter complex in polarized epithelial cells. CD98 also interacts with beta1 integrins resulting in an increase in their affinity for ligand. In this study we explored the role of the transmembrane and cytoplasmic domains of CD98 on integrin-dependent cell adhesion and migration in polarized renal epithelial cells. We demonstrate that the transmembrane domain of CD98 was sufficient, whereas the five N-terminal amino acids of this domain were required for CD98 interactions with beta1 integrins. Overexpression of either full-length CD98 or CD98 lacking its cytoplasmic tail increased cell adhesion and migration, whereas deletion of the five N-terminal amino acids of the transmembrane domain of CD98 abrogated this effect. CD98 and mutants that interacted with beta1 integrins increased both focal adhesion formation and FAK and AKT phosphorylation. CD98-induced cell adhesion and migration was inhibited by addition of phosphoinositol 3-OH kinase (PI3-K) inhibitors suggesting these cell functions are PI3-K-dependent. Finally, CD98 and mutants that interacted with beta1, induced marked changes in polarized renal epithelial cell branching morphogenesis in collagen gels. Thus, in polarized renal epithelial cells, CD98 might be viewed as a scaffolding protein that interacts with basolaterally expressed amino acid transporters and beta1 integrins and can alter diverse cellular functions such as amino acid transport as well as cell adhesion, migration and branching morphogenesis.

Lysophosphatidylcholine enhances cytokine production of endothelial cells via induction of L-type amino acid transporter 1 and cell surface antigen 4F2.

OBJECTIVE: A diverse range of lipid oxidation products detected in oxidized low-density lipoprotein (oxLDL) and atherosclerotic lesions are capable of eliciting biological responses in vascular cells. We performed DNA microarray experiments to explore novel responses of human umbilical vein endothelial cells (HUVECs) to oxLDL and its components. METHODS AND RESULTS: cDNA microarray analysis showed that oxLDL, lysophosphatidylcholine (LysoPC), 4-hydroxy-2-nonenal, and oxysterols altered gene expression specifically, but some genes were commonly induced in HUVECs. Solute carrier family 3 member 2 and family 7 member 5, encoding the heavy chain of the cell surface antigen 4F2 (4F2hc) and the L-type amino acid transporter 1 (LAT1), respectively, were induced by oxLDL and many oxidation products. LAT1 requires 4F2hc to form a heterodimeric functional complex to transport neutral amino acids into the cell. LysoPC increased membrane protein levels of LAT1 confirmed by Western blot analysis and also uptake of L-[(14)C]leucine, which was inhibited by a competitive inhibitor for LAT1. The release of interleukin 6 (IL-6) and IL-8 was increased in LysoPC-treated cells and was attenuated by the LAT1 inhibitor. CONCLUSIONS: These findings suggest that an increase in uptake of neutral amino acids induced by LysoPC results in enhancement of inflammatory responses of endothelial cells.

Lateral membrane protein associations of CD4 in lymphoid cells detected by cross-linking and mass spectrometry.

Interactions of membrane proteins are important in various aspects of cell function. However, weak membrane protein-protein interactions are difficult to study using techniques such as co-immunoprecipitations. CD4 is a cell surface protein involved in T cell activation and the binding of the human immunodeficiency virus to HIV target cells. Here we report the use of cross-linking followed by affinity purification of CD4 in combination with mass spectrometry for identification of proteins that are in the proximity of CD4. Besides the components of the CD4 receptor complex, CD4 and lck, we have identified by tandem mass spectrometry 17 tryptic peptides from transferrin receptor CD71, three peptides from protein phosphatase CD45, and one peptide from 4F2 lymphocyte activation antigen CD98. The efficiency of the cross-linking did not correlate with the level of cell surface expression of the detected molecules, excluding a possible bias of the cross-linking toward the most abundant cell surface molecules. Whereas the association of CD4 with CD45 has been reported, the associations with CD71 and CD98 have not been previously described. We used small-scale immunoprecipitation after cross-linking in combination with fluorescence resonance energy transfer (FRET) measurements to investigate the association between CD4 and CD71. Our data show that CD71 self-associates on the cell surface, that a small fraction of CD4 can be detected by copurifying it with CD71 after cross-linking, and that the level of association between CD4 and CD71 significantly increases after phorbol 12-myristate 13-acetate-induced endocytosis of CD4. This suggests that a small fraction of CD4 associates with clusters of CD71. As both molecules undergo endocytic recycling, the association and cross-linking result from their clustering in the same pit and/or vesicle. The CD4-CD98 association probably results from nonspecific cross-linking.

Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function.

There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.