High Blocking Capacity of Fuzzy-Ball Fluid to Further Enhance Oil Recovery after Polymer Flooding in Heterogeneous Sandstone Reservoirs.

Even after a long time of polymer flooding, over half of the crude oil is still trapped in the reservoir due to the poor plugging capacity. It has been demonstrated that fuzzy-ball fluid can be utilized as an effective plugging fluid. The idea of further increasing oil recovery by fuzzy-ball fluid following polymer flooding drew us to investigate it due to its high performance and effect. In this paper, seepage behavior experiments and parallel core displacement experiments were carried out to evaluate the plugging ability and oil displacement effect of fuzzy-ball fluid. Also, the microscopic blocking mechanism of fuzzy-ball fluid was observed. The results showed that fuzzy-ball fluid has a good plugging capability thanks to the pressure consumption and accumulation plugging mechanisms. The resistance coefficient and residual resistance coefficient of fuzzy-ball fluid are also substantially greater than those of the polymer, at 76.25-239.96 and 13.95-49.91, respectively. Due to its outstanding plugging capability, fuzzy-ball fluid can achieve complete fluid diversion, with the flow fraction of the high-permeability core reduced to nearly 0% and that of the low-permeability core increased to nearly 100%. As a result, low-permeability core oil recovery and total oil recovery both can be enhanced by 46.12-49.24 and 22.81-24.40%, respectively. A field test of fuzzy-ball fluid flooding was carried out in wells TX1 and TX2 which have been flooding with polymers. After the fuzzy-ball fluid was introduced, total daily oil production increased by 64.15%. Fuzzy-ball fluid can significantly boost oil recovery after polymer flooding, according to laboratory and field trials, providing a technical solution for heterogeneous sandstone reservoirs to further enhance oil recovery.

Structures of HIV-1 Neutralizing Antibody 10E8 Delineate the Mechanistic Basis of Its Multi-Peak Behavior on Size-Exclusion Chromatography.

Antibody 10E8 is capable of effectively neutralizing HIV through its recognition of the membrane-proximal external region (MPER), and a suitably optimized version of 10E8 might have utility in HIV therapy and prophylaxis. However, 10E8 displays a three-peak profile on size-exclusion chromatography (SEC), complicating its manufacture. Here we show cis-trans conformational isomerization of the Tyr-Pro-Pro (YPP) motif in the heavy chain 3rd complementarity-determining region (CDR H3) of antibody 10E8 to be the mechanistic basis of its multipeak behavior. We observed 10E8 to undergo slow conformational isomerization and delineate a mechanistic explanation for effective comodifiers that were able to resolve its SEC heterogeneity and to allow an evaluation of the critical quality attribute of aggregation. We determined crystal structures of single and double alanine mutants of a key di-proline motif and of a light chain variant, revealing alternative conformations of the CDR H3. We also replicated both multi-peak and delayed SEC behavior with MPER-antibodies 4E10 and VRC42, by introducing a Tyr-Pro (YP) motif into their CDR H3s. Our results show how a conformationally dynamic CDR H3 can provide the requisite structural plasticity needed for a highly hydrophobic paratope to recognize its membrane-proximal epitope.

Development of a RPLC-UV method for monitoring uncleaved HIV-1 envelope glycoprotein.

One of the HIV-1 vaccine design efforts has focused on developing a recombinant HIV-1 trimeric envelope glycoprotein (Env) as an immunogen to induce broadly neutralizing antibodies. A native-like immunogen, the BG505.DS.SOSIP.664 gp140 (Env) construct has been well-characterized as a vaccine candidate. This vaccine candidate comprises of three identical gp120 and truncated gp41 subunits that form into a trimer of heterodimers. During production, recombinant Env is expressed as a gp140 precursor polypeptide in which a furin cleavable site is engineered to generate a heterodimer of gp120 and gp41 subunits. Each heterodimer is connected by an intermolecular disulfide bond, and three heterodimers form into a trimer. Furin cleavage is an important factor to mimic native-like HIV-1 Env conformations and is needed to help induce an immune response. Therefore, it is critical to monitor cleavage for ensuring functionality of the Env vaccine product. In this paper, a new RPLC-UV method coupled with reduction was developed to routinely determine the percentage of uncleaved gp140 relative to the cleaved gp120 and gp41 subunits. Baseline separation was achieved among the gp120, gp41 and uncleaved gp140 peaks, thus enabling relative quantification of uncleaved gp140. Overall, this RPLC-UV approach has been successfully applied to support Env vaccine candidate developments.

Quantitation of residual valproic acid in flu vaccine drug substance.

Quantitative measurement of process-related impurities is a critical safety requirement for the production of drug substances of vaccine and therapeutic biologics. A simple and sensitive HPLC method has been developed for separation and quantitation of residual valproic acid (VPA) used in the cell transfection procedure for the manufacturing of an influenza vaccine. The method is comprised of a modified Dole liquid phase extraction followed by a quick pre-column derivatization using 2-bromoacetophenone. Nonanoic acid (NNA) is used as the internal standard (IS) and the quantification is performed by reversed-phase liquid chromatography. This new method can accurately measure as low as 6.8 µg/mL (LOQ) residual VPA in the vaccine drug substance.

Research on Progress in Combined Remediation Technologies of Heavy Metal Polluted Sediment.

Heavy metals contaminated sediment has become a worldwide environmental issue due to its great harm to human and aquatic organisms. Thus, economical, effective, and environmentally-friendly remediation technologies are urgently needed. Among which, combined remediation technologies have attracted widespread attention for their unique advantages. This paper introduces combined remediation technologies based on physical-, chemical-, and bio-remediation of heavy metal polluted sediments. Firstly, the research progress in physical-chemical, bio-chemical, and inter-organismal (including plants, animals, microorganisms) remediation of heavy metal polluted sediments are summarized. Additionally, the paper analyzes the problems of the process of combined remediation of heavy metals in river sediments and outlooks the future development trends of remediation technologies. Overall, this review provides useful technology references for the control and treatment of heavy metal pollution in river sediments.

Using LC-MS to Identify Clipping in Self-Assembled Nanoparticles During Vaccine Development.

A hemagglutinin stabilized stem nanoparticle (HA-SS-np) that is designed to provide broad protection against influenza is being developed as a potential vaccine. During an early formulation screening study, reducing gel (rCGE) analysis indicated product degradation in a few candidate buffers at the first-week accelerated stability point, whereas no change was shown in the size exclusion chromatography (SEC) measurement. A LC-MS workflow was therefore applied to investigate the integrity of this large HA-SS-np vaccine molecule (≈ 1 MDa). Application of LC-MS was critical to rationalize the conflicting results from the rCGE and SEC assays and led to the discovery that (1) an unexpected sequence clipping in the HA-SS-np subunits occurred, explaining the atypical reducing gel profile, and (2) an undisrupted disulfide bond held the two fragments together, explaining the unchanged SEC profile. This analytical case study led to a formulation buffer redesign, which mitigated the issue.

Overcoming the Multiple-Monomeric-Peak Profile of Broadly Neutralizing HIV-1 Antibody 10E8 with a Unique Size-Exclusion-Chromatography Method.

10E8 is a potent broadly neutralizing antibody (bNAb) that targets the membrane-proximal external region (MPER) of the HIV virus. During early analytical development of this bNAb directed towards clinical evaluation, 10E8 exhibited a multiple-monomeric-peak profile caused by secondary interactions in traditional size-exclusion chromatography (SEC), thereby rendering SEC unfit for the purpose of assessing aggregation, a target critical quality attribute. To overcome this challenge, an innovative and robust SEC method was successfully developed in which the mobile phase was tested for excipients capable of reducing the secondary interactions responsible for the multipeak profile, and an optimal mobile phase composed of 2× PBS and 100 mM arginine at pH 10.55 was established. Application of this optimized mobile phase was shown to allow quantification of the intrinsic level of aggregation of 10E8 without alteration to the SEC matrix itself. Furthermore, the newly developed method was linear, specific, accurate, and precise over an established range. Overall, an SEC method involving optimization of the mobile phase has been successfully developed, which allowed for assessment of antibody aggregation throughout process development, manufacturing, release, and stability testing.

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer.

Imaging of N-linked glycans from formalin-fixed paraffin-embedded tissue sections using MALDI mass spectrometry.

Aberrant glycosylation is associated with most of the diseases. Direct imaging and profiling of N-glycans on tissue sections can reveal tissue-specific and/or disease-associated N-glycans, which not only could serve as molecular signatures for diagnosis but also shed light on the functional roles of these biomolecules. Mass spectrometry imaging (MSI) is a powerful tool that has been used to correlate peptides, proteins, lipids, and metabolites with their underlying histopathology in tissue sections. Here, we report an MSI technique for direct analysis of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissues. This technique consists of sectioning FFPE tissues, deparaffinization, and rehydration of the sections, denaturing tissue proteins, releasing N-linked glycans from proteins by printing peptide-N-glycosidase F over the sections, spray-coating the tissue with matrix, and analyzing N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Brain sections from a C57BL/6 mouse were imaged using this technique at a resolution of 100 µm. Forty-two N-glycans were analyzed from the mouse brain section. The mass spectrometry images were used to study the relative abundance of oligomannose, nonfucosylated, and fucosylated complex N-glycans in different brain areas including isocortex, hippocampal formation, and brainstem and specific glycans associated with different areas of the brain were identified. Furthermore, glioblastoma tumor xenografts in a NOD/SCID mouse were imaged. Several glycans with differential expression in tumor versus normal brain tissues were identified. The MSI technique allows for imaging of N-glycans directly from FFPE sections. This method can potentially identify tissue-specific and/or disease-associated glycans coexpressed with other molecular signatures or within certain histological structures.

Overexpression of α (1,6) fucosyltransferase associated with aggressive prostate cancer.

Aberrant protein glycosylation is known to be associated with the development of cancers. The aberrant glycans are produced by the combined actions of changed glycosylation enzymes, substrates and transporters in glycosylation synthesis pathways in cancer cells. To identify glycosylation enzymes associated with aggressive prostate cancer (PCa), we analyzed the difference in the expression of glycosyltransferase genes between aggressive and non-aggressive PCa. Three candidate genes encoding glycosyltransferases that were elevated in aggressive PCa were subsequently selected. The expression of the three candidates was then further evaluated in androgen-dependent (LNCaP) and androgen-independent (PC3) PCa cell lines. We found that the protein expression of one of the glycosyltransferases, α (1,6) fucosyltransferase (FUT8), was only detected in PC3 cells, but not in LNCaP cells. We further showed that FUT8 protein expression was elevated in metastatic PCa tissues compared to normal prostate tissues. In addition, using tissue microarrays, we found that FUT8 overexpression was statistically associated with PCa with a high Gleason score. Using PC3 and LNCaP cells as models, we found that FUT8 overexpression in LNCaP cells increased PCa cell migration, while loss of FUT8 in PC3 cells decreased cell motility. Our results suggest that FUT8 may be associated with aggressive PCa and thus is potentially useful for its prognosis.

Aberrant Mucin5B expression in lung adenocarcinomas detected by iTRAQ labeling quantitative proteomics and immunohistochemistry.

BACKGROUND: Lung cancer is the number one cause of cancer-related deaths in the United States and worldwide. The complex protein changes and/or signature of protein expression in lung cancer, particularly in non-small cell lung cancer (NSCLC) has not been well defined. Although several studies have investigated the protein profile in lung cancers, the knowledge is far from complete. Among early studies, mucin5B (MUC5B) has been suggested to play an important role in the tumor progression. MUC5B is the major gel-forming mucin in the airway. In this study, we investigated the overall protein profile and MUC5B expression in lung adenocarcinomas, the most common type of NSCLCs. METHODS: Lung adenocarcinoma tissue in formalin-fixed paraffin-embedded (FFPE) blocks was collected and microdissected. Peptides from 8 tumors and 8 tumor-matched normal lung tissue were extracted and labeled with 8-channel iTRAQ reagents. The labeled peptides were identified and quantified by LC-MS/MS using an LTQ Orbitrap Velos mass spectrometer. MUC5B expression identified by iTRAQ labeling was further validated using immunohistochemistry (IHC) on tumor tissue microarray (TMA). RESULTS: A total of 1288 peptides from 210 proteins were identified and quantified in tumor tissues. Twenty-two proteins showed a greater than 1.5-fold differences between tumor and tumor-matched normal lung tissues. Fifteen proteins, including MUC5B, showed significant changes in tumor tissues. The aberrant expression of MUC5B was further identified in 71.1% of lung adenocarcinomas in the TMA. DISCUSSIONS: A subset of tumor-associated proteins was differentially expressed in lung adenocarcinomas. The differential expression of MUC5B in lung adenocarcinomas suggests its role as a potential biomarker in the detection of adenocarcinomas.

Ubiquitination of tumor necrosis factor receptor-associated factor 4 (TRAF4) by Smad ubiquitination regulatory factor 1 (Smurf1) regulates motility of breast epithelial and cancer cells.

Smad ubiquitin regulatory factors (Smurfs) are HECT-domain ubiquitin E3 ligases that regulate diverse cellular processes, including normal and tumor cell migration. However, the underlying mechanism of the Smurfs' role in cell migration is not fully understood. Here we show that Smurf1 induces ubiquitination of tumor necrosis factor receptor-associated factor 4 (TRAF4) at K190. Using the K190R mutant of TRAF4, we demonstrate that Smurf1-induced ubiquitination is required for proper localization of TRAF4 to tight junctions in confluent epithelial cells. We further show that TRAF4 is essential for the migration of both normal mammary epithelial and breast cancer cells. The ability of TRAF4 to promote cell migration is also dependent on Smurf1-mediated ubiquitination, which is associated with Rac1 activation by TRAF4. These results reveal a new regulatory circuit for cell migration, consisting of Smurf1-mediated ubiquitination of TRAF4 and Rac1 activation.

Ablation of Smurf2 reveals an inhibition in TGF-ß signalling through multiple mono-ubiquitination of Smad3.

TGF-ß signalling is regulated by post-translational modifications of Smad proteins to translate quantitative difference in ligand concentration into proportional transcriptional output. Previous studies in cell culture systems suggested that Smad ubiquitination regulatory factors (Smurfs) act in this regulation by targeting Smads for proteasomal degradation, but whether this mechanism operates under physiological conditions is not clear. Here, we generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse embryonic fibroblasts and dermal fibroblasts, we show that TGF-ß-mediated, Smad-dependent transcriptional responses are elevated in the absence of Smurf2. Instead of promoting poly-ubiquitination and degradation, we show that Smurf2 actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3 interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3 ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data support a model in which Smurf2 negatively regulates TGF-ß signalling by attenuating the activity of Smad3 rather than promoting its degradation.

Smad ubiquitination regulatory factor 2 promotes metastasis of breast cancer cells by enhancing migration and invasiveness.

Controlled protein degradation mediated by ubiquitin/proteasome system (UPS) plays a crucial role in modulating a broad range of cellular responses. Dysregulation of the UPS often accompanies tumorigenesis and progression. Here, we report that Smad ubiquitination regulatory factor 2 (Smurf2), a HECT-domain containing E3 ubiquitin ligase, is up-regulated in certain breast cancer tissues and cells. We show that reduction of Smurf2 expression with specific short interfering RNA in metastatic breast cancer cells induces cell rounding and reorganization of the actin cytoskeleton, which are associated with a less motile and invasive phenotype. Overexpression of Smurf2 promotes metastasis in a nude mouse model and increases migration and invasion of breast cancer cells. Moreover, expression of Smurf2CG, an E3 ligase-defective mutant of Smurf2, suppresses the above metastatic behaviors. These results establish an important role for Smurf2 in breast cancer progression and indicate that Smurf2 is a novel regulator of breast cancer cell migration and invasion.

Requirement of Fut8 for the expression of vascular endothelial growth factor receptor-2: a new mechanism for the emphysema-like changes observed in Fut8-deficient mice.

alpha1,6-Fucosylation plays key roles in many biological functions, as evidenced by the study of alpha1,6-fucosyltransferase (Fut8) knockout (Fut8(-/-)) mice. Phenotypically, Fut8(-/-) mice exhibit emphysema-like changes in the lung, and severe growth retardation. Fut8(-/-) cells also show marked dysregulation of the TGF-beta1 receptor, EGF receptor, integrin activation and intracellular signalling, all of which can be rescued by reintroduction of Fut8. The results of the present study demonstrated that vascular endothelial growth factor receptor-2 (VEGFR-2) expression was significantly suppressed in Fut8(-/-) mice, suggesting that Fut8 was required for VEGFR-2 expression. The expression of VEGFR-2 mRNA and protein was consistently down-regulated by knockdown of the Fut8 gene with small interference RNA in A549 cells, as well as in TGP49 cells, suggesting that suppression occurs at the level of transcription. In contrast, the expression level of ceramide, an inducer of cell apoptosis, was increased in the lungs of Fut8(-/-) mice. The terminal transferase dUTP nick end-labelling (TUNEL) assay was used to identify apoptotic cells. The number of TUNEL-positive septal epithelia and endothelia cells was significantly increased in the alveolar septa of lungs from Fut8(-/-) mice when in comparison with lungs from wild-type mice. It is well known that, in emphysema, ceramide expression can be greatly enhanced by blockade of the VEGFR-2. Thus, suppression of VEGFR-2 expression may provide a novel explanation for the emphysema-like changes in Fut8(-/-) mice.

TRAF6 mediates Smad-independent activation of JNK and p38 by TGF-beta.

In many physiological and disease processes, TGF-beta usurps branches of MAP kinase pathways in conjunction with Smads to induce apoptosis and epithelial-to-mesenchymal transition, but the detailed mechanism of how a MAP kinase cascade is activated by TGF-beta receptors is not clear. We report here that TRAF6 is specifically required for the Smad-independent activation of JNK and p38, and its carboxyl TRAF homology domain physically interacts with TGF-beta receptors. TGF-beta induces K63-linked ubiquitination of TRAF6 and promotes association between TRAF6 and TAK1. Our results indicate that TGF-beta activates JNK and p38 through a mechanism similar to that operating in the interleukin-1beta/Toll-like receptor pathway.

Reduced alpha4beta1 integrin/VCAM-1 interactions lead to impaired pre-B cell repopulation in alpha 1,6-fucosyltransferase deficient mice.

Mice with a targeted gene disruption of Fut8 (Fut8(-/-)) showed an abnormality in the transition from pro-B cell to pre-B cell, reduced peripheral B cells, and a decreased immunoglobulin production. Alpha 1,6-fucosyltransferase (FUT8) is responsible for the alpha 1,6 core fucosylation of N-glycans, which could modify the functions of glycoproteins. The loss of a core fucose in both very late antigen 4 (VLA-4, alpha4beta1 integrin) and vascular cell adhesion molecule 1 (VCAM-1) led to a decreased binding between pre-B cells and stromal cells, which impaired pre-B cells generation in Fut8(-/-) mice. Moreover, the B lineage genes, such as CD79a, CD79b, Ebf1, and Tcfe2a, were downregulated in Fut8(-/-) pre-B cells. Indeed, the frequency of preBCR(+)CD79b(low) cells in bone marrow pre-B cells in Fut8(-/-) was much lower than that in Fut8(+/+) cells. These results reveal a new role of core fucosylated N-glycans in mediating early B cell development and functions.

Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose.

The alpha1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the alpha1,6-fucosylated chain among alpha1,2-, alpha1,3-, alpha1,4-, and alpha1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the alpha1,2-fucosylated PA-sugar chain (H antigen) relative to the alpha1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with alpha1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the alpha1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from alpha1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA.

Crystal structure of mammalian alpha1,6-fucosyltransferase, FUT8.

Mammalian alpha1,6-fucosyltransferase (FUT8) catalyses the transfer of a fucose residue from a donor substrate, guanosine 5'-diphosphate-beta-L-fucose to the reducing terminal N-acetylglucosamine (GlcNAc) of the core structure of an asparagine-linked oligosaccharide. Alpha1,6-fucosylation, also referred to as core fucosylation, plays an essential role in various pathophysiological events. Our group reported that FUT8 null mice showed severe growth retardation and emphysema-like lung-destruction as a result of the dysfunction of epidermal growth factor and transforming growth factor-beta receptors. To elucidate the molecular basis of FUT8 with respect to pathophysiology, the crystal structure of human FUT8 was determined at 2.6 A resolution. The overall structure of FUT8 was found to consist of three domains: an N-terminal coiled-coil domain, a catalytic domain, and a C-terminal SH3 domain. The catalytic region appears to be similar to GT-B glycosyltransferases rather than GT-A. The C-terminal part of the catalytic domain of FUT8 includes a Rossmann fold with three regions that are conserved in alpha1,6-, alpha1,2-, and protein O-fucosyltransferases. The SH3 domain of FUT8 is similar to other SH3 domain-containing proteins, although the significance of this domain remains to be elucidated. The present findings of FUT8 suggest that the conserved residues in the three conserved regions participate in the Rossmann fold and act as the donor binding site, or in catalysis, thus playing key roles in the fucose-transferring reaction.

From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples.

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using alpha 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.