Extraction Of Membrane Proteins Research Articles

Synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d39-DDM) for solubilization of membrane proteins in TROSY NMR experiments.

This work reports the first synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d39-DDM). DDM is a mild non-ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d39-DDM for solubilizing large α-helical membrane proteins in samples for [(15)N-(1)H]TROSY (transverse relaxation-optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d39-DDM used d7-D-glucose and d25-n-dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α-configuration, were coupled together with an α(1 → 4) glycosidic bond to give maltose, which was then coupled with n-dodecanol by its substitution of a C1 bromine in the α-configuration to give DDM. (1)H NMR spectra were used to confirm a high level of deuteration in the synthesized d39-DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52-kDa sugar transport protein solubilized in DDM.

Abstract 15813: Loss-of-Function Mutation in Toll-Like Receptor 4 Partially Protects Against Peripheral and Cardiac Insulin Resistance During a Long-Term High-Fat Diet

Diabetes mellitus is an epidemic disease characterized by alterations in glucose transport, which is tightly regulated by a family of specialized proteins called the glucose transporters (GLUTs). Although diabetic cardiomyopathy is a common complication in diabetic patients, its pathogenesis is still not well understood. Toll-like receptor (TLR) 4, which plays a central role in pathogen recognition by the innate immune system, may also play a critical role in linking inflammation and metabolic disease. We hypothesized that TLR4 activation triggers cardiac insulin resistance. We used mice with a loss-of function mutation in TLR4 (C3H/HeJ) and age-matched wild-type (WT, C57BL/6N) mice (n=8/group) to investigate how feeding a high-fat diet (HFD, 60% kcal from fat) for 16 weeks affected whole-body and cardiac glucose metabolism. After 16 weeks, WT mice fed a HFD were obese and developed hyperglycemia and insulin resistance compared to WT mice on a control diet (10% kcal from fat). The C3H/HeJ mice were partially protected against HFD-induced obesity and insulin resistance. In the heart, WT mice fed a HFD had a 30% decrease (P<0.05) in GLUT4 protein content as measured by Western Blot of cardiac crude membrane protein extracts. In contrast, the loss-of-function point mutation in TLR4 partially rescued cardiac GLUT4 content in the face of a HFD. Interestingly, there was a 40% increase (P<0.05) in the novel GLUT isoform, GLUT8, in the heart when mice of either genotype were fed a HFD. Additionally, GLUT4 protein content was negatively (P<0.05) correlated with GLUT8 content in the myocardium, suggesting that GLUT8 may act as a compensatory mechanism in the face of HFD-induced GLUT4 downregulation. Phosphorylated Akt, a key protein of the insulin signaling pathway, was positively (P<0.05) correlated with GLUT4 content, while the basal/inactive form was negatively correlated. In conclusion, these data suggest that activation of TLR4 activation during diabetes and obesity alters glucose transport by an Akt mechanism, and as such is a pathogenic factor during peripheral and cardiac insulin resistance. Overall, TLR4 appears to be a key modulator in the cross-talk between inflammatory and metabolic pathways, as well as a potential therapeutic target for diabetes.

Rapid Analysis of Cell Surface N-Glycosylation from Living Cells Using Mass Spectrometry

Cell surfaces are covered with a dense carbohydrate layer referred to as the glycocalyx. Because different cell types express different glycan signatures, it is of paramount importance to have robust methods to analyze the glycome of living cells. To achieve this, a common procedure involves cell lysis and extraction of membrane (glyco)proteins and yields a major proportion of high-mannose N-glycans that most likely stem from intracellular proteins derived from the ER. Using HEK 293 cells as a model system, we developed a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells. We directly released glycopeptides from cell surfaces through tryptic digestion of freshly harvested and vital cells, thereby improving the detection and quantification of complex-type N-glycans by increasing their relative amount from 14 to 85%. It was also possible to detect 25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1, and 51 in Hep G2 cells. The additional signals provided deeper insight into cell-type-specific N-glycan features such as antennarity, fucosylation, and sialylation. Thus, this protocol, which can potentially be applied to any cells, will be useful in the fields of glycobiotechnology and biomarker discovery.

Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins.

In the past few years there has been a growth in the use of nano-particles for stabilizing lipid membranes with embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have described the use of an amphipathic polymer (Poly(styrene-co-maleic acid); SMA) to produce discoidal nanoparticles that contain a lipid bilayer with embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unecapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA Lipid Particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer "bracelet" encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of structure of the SMALP forms the foundation of future development and applications of SMALPs in membrane protein production and analysis.

A simple technique to improve the resolution of membrane acidic proteins of the haloarchaeon Haloferax volcanii by 2D electrophoresis.

Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the "omics" level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9-5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods.

An amphipathic polypeptide derived from poly-γ-glutamic acid for the stabilization of membrane proteins.

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-γ-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4-5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ETA ) in their active conformations. Furthermore, ETA in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.

Potential risk of blood transfusion‐transmitted brucellosis in an endemic area of China

Brucellosis is a severe zoonotic disease that is increasingly prevalent in north China. A study evaluating Brucella infection in blood donors was conducted at Kashi central blood station, Xinjiang, China. Four serologic and two molecular methods of detection of Brucella infection were used in plasma samples from blood donations collected from Kashi in northwest China, considered a brucellosis-endemic area. Blood donor samples collected in Shenzhen, southern China, a brucellosis-nonendemic area, were tested as a negative control group. In 3896 plasma samples collected from Kashi central blood station, 135 (3.5%) plasma samples were reactive by the Rose Bengal plate test (RBPT), and 120 (3.1%) of the 135 RBPT-reactive sample were also reactive with the standard tube agglutination test (SAT), respectively. All samples of the control group of 399 blood samples from Shenzhen blood center tested negative with RBPT and SAT. Of 135 seroreactive plasma samples, 39 (1.0%) reacted with B. melitensis membrane protein extracts by enzyme-linked immunosorbent assay and 25 were reactive to either rBP26 or rOMP31 by Western blot. Thirteen plasma samples and two follow-up blood samples were identified as carrying Brucella DNA by quantitative polymerase chain reaction (PCR) and nested PCR. Overall 15 (1:300) Kashi blood donations were found positive by nucleic acid testing, confirmed specific by DNA sequencing. The data indicate a probable high rate of Brucella bacteremia, suggesting a potential risk of transfusion-transmitted brucellosis. Blood donation screening for Brucella infection may be considered in the high Brucella-endemic areas of China.

Polar interactions trump hydrophobicity in stabilizing the self-inserting membrane protein Mistic.

Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and β-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins.

Erratum to: Molecular Characterization of a Lysophosphatidylcholine Acyltransferase Gene Belonging to the MBOAT Family in Ricinus communis L.

In the original publication of the article, figure 7 results were incorrectly published. Because of the discrepancies between our results published in Lipids 48(7):663–674, and others in the field with regards to Membrane Bound O-Acyltranferase (MBOAT) activity with 18:2n-6 and 18:3n-3 as substrates, we measured the activity of the castor lysophosphatidylcholine acyltransferase (RcLPCAT) using freshly prepared substrates. The 18:3-CoA and 18:2-CoA were prepared by chemical synthesis [1] and kindly provided by Dr. Stymne (Swedish University of Agricultural Sciences, Alnarp, Sweden). As indicated by our results using the new substrates, LPCAT activity was similar for 18:2 and 18:3 thioesters as it was for 18:1 and 16:1 thioesters, in agreement with the specificity recorded for Brassica LPCAT. These new results do not alter the major conclusion stated in our paper that MBOAT has limited activity with ricinoleoyl-CoA as a substrate, but does indicate it has similar activity with 18:2-CoA and 18:3-CoA as it does with 18:1-CoA and 16:1-CoA. The correct version of figure 7 is given below: RcLPCAT specificity using diverse acyl-CoA donors. LPCAT activity was assayed using 50 μM lysoPtdCho as the acceptor and different acyl-CoA as substrates at two concentrations, 10 μM (grey bars) or 40 μM (black bars) as indicated. The assay was performed as described in “Materials and Methods” of the published paper, in the presence of 30 μg of membrane protein extract prepared from yeast ale1 cells expressing RcLPCAT. Background LPLAT activities were determined from cells transformed with the pYES2 empty vector and the appropriate background rates were subtracted from the measured rate. The specific activity is presented and is expressed as nmol of released CoA min−1 μg−1 of membrane protein

Interacción de FABP4 con proteínas de membrana de células endoteliales

IntroductionFatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. MethodologyHUVEC cells were incubated with and without FABP4 (100ng/ml) for 5minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker.A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. ResultsThe results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33kDa containing FAPB4 and its putative receptor. DiscussionThe existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular.

Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC–MS/MS

Glycosylation of membrane proteins plays an important role in cellular behaviors such as cell-cell interaction, immunologic recognition and cell signaling. However, the effective extraction of membrane proteins, the selective isolation of glycopeptides and the mass spectrometric characterization of glycosylation are challenging with current analytical techniques. In this study, a systematic approach was developed which combined: an integrated hydrophilic interaction chromatography solid phase interaction (HILIC SPE) for simultaneous detergent removal and glycopeptide enrichment, and mass spectrometric identification of both protein N-glycosylation sites and site-specific glycan composition. The HILIC SPE conditions were optimized to enable the use of a high concentration of strong detergents, such as SDS and Triton X-100 and to dissolve highly hydrophobic membrane proteins, thus increasing the yield of membrane protein extraction. We illustrated the performance of this approach for the study of membrane protein glycosylation from human embryonic kidney cell lines (HEK 293T). 200μg total protein digest was processed using this approach, leading to the identification of 811 N-glycosylation sites from 567 proteins within two experimental replicates. Furthermore, 177 glycopeptides representing 82 N-glycosites with both glycan composition and peptide sequence were identified by high energy collision dissociation. A method for systematic characterizing of cell membrane glycosylation has been developed in this manuscript. It is comprised of an integrated hydrophilic interaction chromatography solid phase extraction for the simultaneous detergent removal and intact glycopeptide enrichment. This HILIC SPE significantly increased the efficiency and sensitivity for glycosylation analysis and was combined with high energy collision dissociation to characterize site-specific N-glycosylation from HEK293 cell membrane. Totally 811 N-glycosylation sites from 567 proteins were identified and 177 intact glycopeptides with both glycan composition and peptides sequence were characterized, which provided a solution for site-specific N-glycosylation characterization of membrane.

Mycobacterium tuberculosispellicles express unique proteins recognized by the host humoral response

Mycobacterium tuberculosis (MTB) causes both acute and chronic infections in humans characterized by tolerance to antibiotics and reactivation to cause secondary tuberculosis. These characteristics have led to renewed interest in the in vitro pellicle, or biofilm mode of growth, where bacteria grow to produce a thick aggregate at the air-liquid interface and exhibit increased phenotypic resistance to antibiotics. We infected guinea pigs with the virulent H37Rv strain of MTB for 60 days at which point we collected blood. To identify antigenic proteins, membrane protein extracts of MTB H37Ra pellicles and shaken cultures grown for 3, 5, or 7 weeks were probed with the infected animals' sera after the proteins were separated by two-dimensional gel electrophoresis (2DGE). Antigenic proteins were then identified using MALDI-TOF/TOF mass spectrometry peptide mass fingerprinting. Antigenic pellicle proteins were compared across the three timepoints to identify those that were produced consistently during pellicle growth. They were also compared to those membrane proteins identified from harvested shaken cultures to determine pellicle-specific vs. universally expressed proteins. Using this technique, we identified 44 distinct antigenic proteins, nine of which were pellicle-specific. The sequence of antigenic pellicle-specific proteins was checked for sequence conservation across 15 sequenced MTB clinical isolates, three other members of the MTB complex, as well as M. avium and M. smegmatis. The antigenic pellicle-specific protein Rv0097 was found to have very high sequence conservation within the MTB complex but not with related mycobacteria, while FabG4 was highly conserved in all mycobacteria analyzed. These conserved pellicle-specific proteins represent targets for the development of future diagnostic tests and vaccines.

Enzymatic synthesis of amino sugar fatty acid esters

N‐Acetyl‐glucosamine fatty acid esters were synthesized by a lipase‐catalyzed transesterification with methyl hexanoate and N‐acetyl‐glucosamine (GlcNAc), which resulted in the formation of 2‐(acetylamino)‐2‐deoxy‐6‐O‐hexanoate‐D‐glucose, a novel glycolipid. Additionally N‐butyryl‐glucosamine (GlcNBu) was used for a similar synthesis, leading to the formation of 2‐(butyrylamino)‐2‐deoxy‐6‐O‐hexanoate‐D‐glucose. The higher hydrophobicity of GlcNBu led to an increase in the overall yield and the initial reaction rate when compared to the reaction with GlcNAc. By pre‐dissolving GlcNAc and GlcNBu in dimethyl sulfoxide (DMSO), it was possible to completely dissolve both sugars in the organic solvent, thus further enhancing the initial reaction rate and yield respectively.Practical applications: Glycolipids are used in a wide range of applications, ranging from food, cosmetic, and pharmaceutical formulations, where they can be used as emulsifiers or foaming agents to classic cleaning products, utilizing their good detergent properties. Further applications may include fields like membrane protein extraction, bioremediation, or tertiary oil recovery. Novel glycolipids with tailor‐made properties might be useful to improve any of the named applications and widen the diversity of available environmentally friendly surfactants, often termed “green surfactants.” Glycolipids are the most prominent example therefrom.

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device

Cell Surface Protein-protein Binding on COS-7 Cells.

Examination of interactions between a transmembrane protein and a soluble protein by pull-down or immunoprecipitation assays can be tricky and complicated due to the detergent extraction of membrane proteins during the lysate preparation step. The choice and concentration of detergents must be determined empirically and the procedure can be burdensome. Here, we describe a simplified binding assay by expressing the membrane protein of interest in COS-7 cells and applying detergent-free solutions containing an extracellular protein to be tested. The binding is then examined by immunocytochemistry.

Detergent-Free Extraction of Membrane Proteins into Native Nanodiscs. Application to the Reaction center of Rhodobacter Sphaeroides

Recently, it has been discovered that styrene-maleic acid (SMA) copolymers are able to solubilize membrane proteins in the form of nanodiscs (SMALPs), also called ‘native nanodiscs’, directly from their native membrane [1,2]. To understand what physical properties of membranes and SMA modulate this unique property of SMA, we first studied the solubilization of vesicles of synthetic phospholipids by SMA using transmission experiments. We found that SMA is an excellent membrane solubilizer, able to solubilize different types of membranes below, at and above the gel to crystalline-liquid phase temperature. Based on the kinetics of solubilization under different experimental conditions we developed a model for the mode of action of SMA that will also explain why SMA is an efficient solubilizer, whereas membrane scaffold proteins (MSPs) and amphipols are not.Next we used the SMA technology to purify and characterize reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides in the form of native nanodiscs. Monitoring of heat stability and recombination kinetics of P+QB- in photo-excited RCs in native nanodiscs showed that (1) RCs are much more stable in native nanodiscs than in detergent, and (2) the charge recombination kinetics display native-membrane like behavior.Our study contributes fundamental knowledge about the mode of action of SMA that is essential for optimizing methods to extract membrane proteins from different organisms and it promotes the general applicability of SMALPs as host for membrane proteins in studies on interactions of proteins with native lipids and in protein structure determination studies.1. Knowles et al., 2009, JACS, 131, 7484-7485.2. Long et al., 2013, BMC Biotechnology, 13:41.

Insights into the TOR-S6K Signaling Pathway in Maize (Zea mays L.). Pathway Activation by Effector–Receptor Interaction

The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

Low Expression Of The FUCA1 Gene Is An Adverse Prognostic Factor In Myeloma and Combined With High Sialyltransferase Gene Expression Identifies Patients At Increased Risk Of Early Disease Progression and Death

BackgroundGlycosylation is a post translational modification which results in the addition of carbohydrate determinants to proteins and lipids. This has a major influence on the function of molecules known to be important in myeloma cell adhesion and trafficking such as integrins and selectins. We previously reported that ST3GAL6, a sialyltransferase involved in the synthesis of functional selectin ligands in humans, is over expressed in myeloma cells and associated with inferior survival in the MRC Myeloma IX study (MRC IX). We now extend our analysis of the MRC IX dataset to evaluate the potential prognostic significance of other glycosylation genes that are differentially regulated between normal and malignant plasma cells. MethodsAnalyzing publically available microarray transcriptomic datasets (Mayo Clinic GSE6477, University of Arkansas (UAMS) GSE24080, GSE2658) we first identified dysregulated glycosylation genes in MM. The prognostic significance of these candidate genes in MRCIX (singly and in combination) was analyzed using Kaplan Meier survival estimates for progression free survival and overall survival (PFS, OS). These results were validated in independent datasets (TT2 and TT3). Any genes significantly associated with survival were correlated with FISH abnormalities and ISS and multivariate analysis was performed to determine their independence as prognostic factors. QPCR was performed in cell lines to validate GEP findings. Matching SNP-based mapping array and methylation array data were analyzed. Membrane protein extracts from MM cell lines were applied directly to lectin microarrays following fluorescent labeling to generate cell surface glycan profiles. Immunohistochemistry for glycosylation enzymes and glycans was performed on patient bone marrow samples. ResultsHigh expression (top quartile as cut-off) of the sialyltransferase gene ST3GAL1 showed a trend towards inferior OS (median survival 35 mos .v. 45 mos, p=0.07) with significantly reduced PFS (19 .v. 14 mos, p=0.015). Increased expression of ST3GAL1 correlated with the presence of t(4;14) (p=0.009), del13q (p=0.001), +1q (p=0.01) and hypodiploidy (p=0.00001). Low expression (lower quartile by GEP) of the gene FUCA1, which encodes tissue alpha-L-fucosidase, was linked to inferior outcome (median OS 44 .v. 38 mos, p=0.025). On multivariate analysis low FUCA1 expression was independent of other important prognostic factors (HR=1.61, p=0.017). Patients with t(11;14) by FISH were more likely to have low FUCA1 than t(11;14) negative patients. There was no significant association between ISS and ST3GAL6, ST3GAL1 or FUCA1. We verified the effect of low FUCA1 in the GSE24080 dataset from TT2 and TT3 combined. Patients with low FUCA1 had significantly worse OS (p=0.0002). SNP mapping array and methylation array analysis did not show any evidence of correlation between copy number alterations or hypermethylation of FUCA1 and its low expression level. Since FUCA1 participates in N-glycan degradation with removal of fucose residues, this raises the possibility that a reduction in FUCA1 may lead to excessive fucosylation of cancer related glycans involved in adhesion and trafficking, such as sLex. Indeed, lectin arrays of MM cell lines revealed high levels of a-1,3/a-1,6 linked fucose, as determined by binding of Aleuria Aurantia Lectin (AAL). The HECA-452 antibody recognizes a functional trisaccharide domain shared by sialyl Lewis a and sLex and known to bind to E-selectin. Preliminary immunohistochemistry revealed increased HECA452 staining in bone marrow samples, which had both strong ST3GAL6 and low FUCA1 staining. Combining gene expression data showed that reduced expression of FUCA1 with increased expression of either ST3GAL6 or ST3GAL1 or both, identified a subgroup of patients (18%) in MRC Myeloma IX with particularly poor outcome (figure 1). Similar results were found in TT2 and TT3 with 17% of patients affected. [Display omitted] ConclusionsAltered glycosylation gene expression patterns may identify patients at high risk of disease progression and early death. Our data implicates sialyltransferases and selectin ligands as potential therapeutic targets in MM. Disclosures:Morgan:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Johnson and Johnson: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

7th International Symposium on Enabling Technologies for Life Sciences (ETP)

7^th International Symposium on Enabling Technologies for Life Sciences (ETP)