The Role of Autoimmunity-Related Gene CLEC16A in the B Cell Receptor-Mediated HLA Class II Pathway.

C-type lectin CLEC16A is located next to CIITA, the master transcription factor of HLA class II (HLA-II), at a susceptibility locus for several autoimmune diseases, including multiple sclerosis (MS). We previously found that CLEC16A promotes the biogenesis of HLA-II peptide-loading compartments (MIICs) in myeloid cells. Given the emerging role of B cells as APCs in these diseases, in this study, we addressed whether and how CLEC16A is involved in the BCR-dependent HLA-II pathway. CLEC16A was coexpressed with surface class II-associated invariant chain peptides (CLIP) in human EBV-positive and not EBV-negative B cell lines. Stable knockdown of CLEC16A in EBV-positive Raji B cells resulted in an upregulation of surface HLA-DR and CD74 (invariant chain), whereas CLIP was slightly but significantly reduced. In addition, IgM-mediated Salmonella uptake was decreased, and MIICs were less clustered in CLEC16A-silenced Raji cells, implying that CLEC16A controls both HLA-DR/CD74 and BCR/Ag processing in MIICs. In primary B cells, CLEC16A was only induced under CLIP-stimulating conditions in vitro and was predominantly expressed in CLIPhigh naive populations. Finally, CLIP-loaded HLA-DR molecules were abnormally enriched, and coregulation with CLEC16A was abolished in blood B cells of patients who rapidly develop MS. These findings demonstrate that CLEC16A participates in the BCR-dependent HLA-II pathway in human B cells and that this regulation is impaired during MS disease onset. The abundance of CLIP already on naive B cells of MS patients may point to a chronically induced stage and a new mechanism underlying B cell-mediated autoimmune diseases such as MS.

Cryptococcus neoformans Evades Pulmonary Immunity by Modulating Xylose Precursor Transport.

Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice yet surprisingly is not cleared. We found that this strain failed to induce the nonprotective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained interleukin 12p40 (IL-12p40) induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of proinflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus-associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, which is in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation.

CRISPR/Cas9-mediated mutation of OsSWEET14 in rice cv. Zhonghua11 confers resistance to Xanthomonas oryzae pv. oryzae without yield penalty.

BACKGROUND: Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa. OsSWEET14, encoding a sugar transporter, is known to be a major susceptible gene of bacterial blight targeted by four different transcription activator-like (TAL) effectors from either Asian or African Xoo strains. However, the OsSWEET14 single knockout or promoter mutants in the Kitaake background are moderately resistant or even susceptible to African Xoo strains. Therefore, in this study, we knocked out OsSWEET14 in rice cv. Zhonghua 11 background for disease assessment. RESULTS: In this study, CRISPR/Cas9 was utilized to disrupt the function of OsSWEET14 by modifying its corresponding coding region in the genome of rice cv. Zhonghua 11 (CR-S14). In total, we obtained nine different OsSWEET14-mutant alleles. Besides conferring broad-spectrum resistance to Asian Xoo strains, tested mutant alleles also showed strong resistance to African Xoo strain AXO1947. Moreover, the expression of OsSWEET14 was detected in vascular tissues, including the stem, leaf sheath, leaf blade and root. The disruption of OsSWEET14 led to increased plant height without a reduction in yield. CONCLUSIONS: Disruption of OsSWEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian Xoo strain PXO86. CR-S14 has normal reproductive growth and enhanced plant height under normal growth conditions. These results imply that CR-S14 may serve as a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance. The genetic background and increased plant height need to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.

Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation.

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside-H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.

Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNAc transporter.

Cryptococcus neoformans, an opportunistic fungal pathogen, produces a glycan capsule to evade the immune system during infection. This definitive virulence factor is composed mainly of complex polysaccharides, which are made in the secretory pathway by reactions that utilize activated nucleotide sugar precursors. Although the pathways that synthesize these precursors are known, the identity and the regulation of the nucleotide sugar transporters (NSTs) responsible for importing them into luminal organelles remain elusive. The UDP-galactose transporter, Ugt1, was initially identified by homology to known UGTs and glycan composition analysis of ugt1Δ mutants. However, sequence is an unreliable predictor of NST substrate specificity, cells may express multiple NSTs with overlapping specificities, and NSTs may transport multiple substrates. Determining NST activity thus requires biochemical demonstration of function. We showed that Ugt1 transports both UDP-galactose and UDP-N-acetylgalactosamine in vitro. Deletion of UGT1 resulted in growth and mating defects along with altered capsule and cellular morphology. The mutant was also phagocytosed more readily by macrophages than wild-type cells and cleared more quickly in vivo and in vitro, suggesting a mechanism for the lack of virulence observed in mouse models of infection.

RelAp43, a member of the NF-κB family involved in innate immune response against Lyssavirus infection.

NF-κB transcription factors are crucial for many cellular processes. NF-κB is activated by viral infections to induce expression of antiviral cytokines. Here, we identified a novel member of the human NF-κB family, denoted RelAp43, the nucleotide sequence of which contains several exons as well as an intron of the RelA gene. RelAp43 is expressed in all cell lines and tissues tested and exhibits all the properties of a NF-κB protein. Although its sequence does not include a transactivation domain, identifying it as a class I member of the NF-κB family, it is able to potentiate RelA-mediated transactivation and stabilize dimers comprising p50. Furthermore, RelAp43 stimulates the expression of HIAP1, IRF1, and IFN-ß - three genes involved in cell immunity against viral infection. It is also targeted by the matrix protein of lyssaviruses, the agents of rabies, resulting in an inhibition of the NF-κB pathway. Taken together, our data provide the description of a novel functional member of the NF-κB family, which plays a key role in the induction of anti-viral innate immune response.

Semi-continuous, label-free immunosensing approach for Ca2+-based conformation change of a calcium-binding protein.

A label-free immunosensing method based on the conformational change of calcium-binding protein (CBP) depending on analyte concentration was explored for semi-continuous analysis of free Ca(2+). Glucose-galactose-binding protein as a CBP and produced as a recombinant protein by Escherichia coli was used as the immunogen to produce monoclonal antibodies by hybridoma technology. We finally screened the 3-6F cell clone, which produced the desired antibody specific to a particular structural conformation of the protein that occurred only upon CBP-calcium complex formation. To construct an immunosensor, the antibody was immobilized via a secondary antibody on an Octet Red optical fiber-based label-free sensor. Calcium analysis was conducted on the sensor in combination with CBP previously added to the aqueous sample, which distinguished the sensor signal according to the analyte concentration. The immunosensor produced a signal in real time with a response time of approximately 15 min and could be reused for analyses of different samples in a semi-continuous manner. The minimum detection limit of the analyte under optimal conditions was 0.09 mM and the upper limit was about 5 mM (log-logit transformed standard curve linearity: R(2) > 98%). In sample tests with milk, the analytical performance of the sensor was highly correlated (R(2) > 99%) with that of the reference system based on the KMnO4 titration method (ISO 12081). Although the sensor showed cross-reactivity at high concentrations (>1 mM) of cations including zinc, iron, manganese, and copper, these ionic components were not traceable (<0.01 mM) in milk.

Glucose transporter type 1 deficiency syndrome associated with autoantibodies to glutamate receptors.

BACKGROUND: The clinical spectrum of glucose transporter type 1 deficiency syndrome (GLUT1DS) has broadened, with increasing recognition of a milder phenotype. Antibodies targeting the subunits of glutamate receptors (GluRs), including GluN1, GluN2B, and GluD2, have been detected in various neurological disorders. Anti-GluD2 antibodies in particular may be associated with cerebellar symptoms. CASE REPORT: A 3-year-5-month-old boy with normal development exhibited myoclonus refractory to antiepileptic drugs from one year ago. He developed tremor and ataxia. Cerebrospinal fluid (CSF) revealed fasting-state glucose 50 mg/dl (CSF/blood glucose ratio of 0.50). Single photon emission computed tomography with 123I-iodoamphetamine revealed hypoperfusion in the cerebellum. At age 4 years and 5 months, treatment with intravenous methylprednisolone (IVMP) relieved his symptoms and improved the cerebellar hypoperfusion. However, his symptoms reappeared at age 5 years and 1 month. Treatment with IVMP was repeated, resulting in transient disappearance of symptoms. At age 6 years and 9 months, he was diagnosed with GLUT1DS by genetic analysis, and treatment with modified Atkins diet was started with efficacy. Levels of anti-GluN1, -GluN2B, and -GluD2 antibodies in the serum and CSF were measured 4 times. All antibodies in the CSF were elevated over 2 standard deviations above controls, and the levels fluctuated along with the severity of his symptoms. The level of anti-GluD2 antibodies in CSF declined to the normal range only after starting the modified Atkins diet. CONCLUSION: Treatment with IVMP transiently improved this patient's symptoms. Levels of anti-GluR antibodies may be associated with symptom severity.

Diversity in kinetics correlated with structure in nano body-stabilized LacY.

The structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY.

Natural self-assembly of allergen-S-layer fusion proteins is no prerequisite for reduced allergenicity and T cell stimulatory capacity.

BACKGROUND: Recombinant allergen-S-layer fusion proteins display a strongly reduced IgE-binding activity and promote the induction of allergen-specific Th0/1 cells and regulatory T cells. Such fusion proteins show a natural capacity to self-assemble into mono- or double-layer sheets reaching particle-like dimensions of 0.5-2 microm. We were interested in finding out whether self-assembly was crucial for the immunological characteristics of allergen-S-layer fusion proteins. METHODS: The IgE-binding and mediator-releasing capacities of nonassembled and self-assembled rSbpA-Bet v 1, consisting of the major birch pollen allergen Bet v 1 and the S-layer protein SbpA, were compared in inhibition ELISA and basophil activation assays using sera from patients allergic to birch pollen. T cell stimulation was evaluated using Bet v 1-specific T cell clones reactive to distinct epitopes of Bet v 1. Autologous B lymphocytes, monocytes and monocyte-derived dendritic cells were employed to evaluate potential differences in uptake and processing by different antigen-presenting cells. RESULTS: Both rSbpA-Bet v 1 variants showed significantly less IgE-binding and mediator-releasing activity than Bet v 1. However, self-assembly further minimized the reduced allergenicity of nonassembled rSbpA-Bet v 1. Both rSbpA-Bet v 1 variants induced comparable proliferation in Bet v 1-specific T cell clones. B cells inappropriately presented either variant of rSbpA-Bet v 1. Self-assembly amplified the T cell stimulatory capacity of monocytes and dendritic cells. CONCLUSIONS: The promising characteristics of allergen-S-layer fusion proteins regarding their potential use for allergy treatment do not depend on the formation of particle-like structures.

Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.

Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal- and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99% of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells approximately 40% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.

Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4.

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin-sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.

Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain.

Leishmania are parasitic protozoa with two major stages in their life cycle: flagellated promastigotes that live in the gut of the insect vector and nonflagellated amastigotes that live inside the lysosomes of the vertebrate host macrophages. The Pro-1 glucose transporter of L. enriettii exists as two isoforms, iso-1 and iso-2, which are both expressed primarily in the promastigote stage of the life cycle. These two isoforms constitute modular structures: they differ exclusively and extensively in their NH2-terminal hydrophilic domains, but the remainder of each isoform sequence is identical to that of the other. We have localized these glucose transporters within promastigotes by two approaches. In the first method, we have raised a polyclonal antibody against the COOH-terminal hydrophilic domain shared by both iso-1 and iso-2, and we have used this antibody to detect the transporters by confocal immunofluorescence microscopy and immunoelectron microscopy. The staining observed with this antibody occurs primarily on the plasma membrane and the membrane of the flagellar pocket, but there is also light staining on the flagellum. We have also localized each isoform separately by introducing an epitope tag into each protein sequence. These experiments demonstrate that iso-1, the minor isoform, resides primarily on the flagellar membrane, while iso-2, the major isoform, is located on the plasma membrane and the flagellar pocket. Hence, each isoform is differentially sorted, and the structural information for targeting each transporter isoform to its correct membrane address resides within the NH2-terminal hydrophilic domain.

The Autoimmune Disorder Susceptibility Gene CLEC16A Restrains NK Cell Function in YTS NK Cell Line and Clec16a Knockout Mice.

CLEC16A locus polymorphisms have been associated with several autoimmune diseases. We overexpressed CLEC16A in YTS natural killer (NK) cells and observed reduced NK cell cytotoxicity and IFN-γ release, delayed dendritic cell (DC) maturation, decreased conjugate formation, cell-surface receptor downregulation and increased autophagy. In contrast, siRNA mediated knockdown resulted in increased NK cell cytotoxicity, reversal of receptor expression and disrupted mitophagy. Subcellular localization studies demonstrated that CLEC16A is a cytosolic protein that associates with Vps16A, a subunit of class C Vps-HOPS complex, and modulates receptor expression via autophagy. Clec16a knockout (KO) in mice resulted in altered immune cell populations, increased splenic NK cell cytotoxicity, imbalance of dendritic cell subsets, altered receptor expression, upregulated cytokine and chemokine secretion. Taken together, our findings indicate that CLEC16A restrains secretory functions including cytokine release and cytotoxicity and that a delicate balance of CLEC16A is needed for NK cell function and homeostasis.

Long noncoding RNA Malat1 regulates differential activation of macrophages and response to lung injury.

Macrophage activation, i.e., classical M1 and the alternative M2, plays a critical role in many pathophysiological processes, such as inflammation and tissue injury and repair. Although the regulation of macrophage activation has been under extensive investigation, there is little knowledge about the role of long noncoding RNAs (lncRNAs) in this event. In this study, we found that lncRNA Malat1 expression is distinctly regulated in differentially activated macrophages in that it is upregulated in LPS-treated and downregulated in IL-4-treated cells. Malat1 knockdown attenuates LPS-induced M1 macrophage activation. In contrast, Malat1 knockdown enhanced IL-4-activated M2 differentiation as well as a macrophage profibrotic phenotype. Mechanistically, Malat1 knockdown led to decreased expression of Clec16a, silencing of which phenocopied the regulatory effect of Malat1 on M1 activation. Interestingly, Malat1 knockdown promoted IL-4 induction of mitochondrial pyruvate carriers (MPCs) and their mediation of glucose-derived oxidative phosphorylation (OxPhos), which was crucial to the Malat1 regulation of M2 differentiation and profibrotic phenotype. Furthermore, mice with either global or conditional myeloid knockout of Malat1 demonstrated diminished LPS-induced systemic and pulmonary inflammation and injury. In contrast, these mice developed more severe bleomycin-induced lung fibrosis, accompanied by alveolar macrophages displaying augmented M2 and profibrotic phenotypes. In summary, we have identified what we believe is a previously unrecognized role of Malat1 in the regulation of macrophage polarization. Our data demonstrate that Malat1 is involved in pulmonary pathogeneses in association with aberrant macrophage activation.

Islet cell autoantigens in insulin-dependent diabetes.

A burgeoning number of antigenic targets of the islet cell autoimmunity in IDD have been identified, and more can be anticipated through improved methods for their identification. The challenge for those investigating the pathogenesis of IDD will be to assign the relative importance of these antigens to the development of the disease, and to resolve whether there is a dominant primary immunologic event that is followed by a series of secondary immunizations to a variety of normally sequestered islet cell antigens in the sequence of pathogenic events that culminate in IDD. One interesting observation that may have potential pathogenic implications is the observation that of all islet cell autoantigens described, only two (i.e., 64 kD/GAD, 38 kD) are reactive in their native configurations, implying that recognition of conformational epitopes is most important. This property argues for primary immunizing agents rather than secondary ones after release of denatured antigens and antigenic recognition through their epitopes. Given the complex and multiple physiological functions of islet cells and the continuous variation in their activity, it is reasonable to speculate that the speed of the progression to IDD could vary between individuals with respect to their insulin needs and the relative activities of their islets. Activated islets may express autoantigens that have only limited expression in quiescent islets. The often times striking variation in the severity of insulitis seen in different islets of a single pancreas may be explained by the level of activity of individual islets. Furthermore, disparity in HLA-DR/DQ associations with disease may involve differences in the immunological recognition of autoantigens. Whereas there is still much to learn, it is clear that disease predictability and disease intervention studies have been enhanced through the identification of the islet cell autoantigens in IDD.

The midcycle increase in ovarian glucose uptake is associated with enhanced expression of glucose transporter 3. Possible role for interleukin-1, a putative intermediary in the ovulatory process.

This study characterizes the rat ovary as a site of hormonally dependent glucose transporter (Glut) expression, and explores the potential role of interleukin (IL)-1, a putative intermediary in the ovulatory process, in this regard. Molecular probing throughout a simulated estrous cycle revealed a significant surge in ovarian Glut3 (but not Glut1) expression at the time of ovulation. Treatment of cultured whole ovarian dispersates from immature rats with IL-1beta resulted in upregulation of the relative abundance of the Glut1 (4.5-fold) and Glut3 (3.5-fold) proteins as determined by Western blot analysis. Other members of the Glut family (i.e., Gluts 2, 4, and 5) remained undetectable. The ability of IL-1 to upregulate Glut1 and Glut3 transcripts proved time-, dose-, nitric oxide-, and protein biosynthesis-dependent but glucose independent. Other ovarian agonists (i.e., TNF alpha, IGF-I, interferon-gamma, and insulin) were without effect. Taken together, our findings establish the mammalian ovary as a site of cyclically determined Glut1 and Glut3 expression, and disclose the ability of IL-1 to induce the ovarian expression as well as translation of Glut1 and Glut3 (but not of Gluts 2, 4, or 5). Our observations also establish IL-1 as the first known regulator of Glut3, the most efficient Glut known to date. In so doing, IL-1, a putative component of the ovulatory process, may be acting to meet the increased metabolic demands imposed on the growing follicle and the ovulated cumulus-enclosed oocyte.

The formation of an insulin-responsive vesicular cargo compartment is an early event in 3T3-L1 adipocyte differentiation.

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.

Synthesis of a model trisaccharide for studying the interplay between the anti α-Gal antibody and the trans-sialidase reactions in Trypanosoma cruzi.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Galp antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal ß-d-Galp unit in the host glycoconjugate to terminal ß-d-Galp units in the parasite mucins to construct the d-NeuNAc(α2→3)ß-d-Galp motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α-d-Galp is only present in the infective stage whereas ß-d-Galf is characteristic of the epimastigote stage of the less virulent strains. Neither α-d-Galp nor d-Galf is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal ß-d-Galp units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Galp antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α-d-Galp(1→3)-[ß-d-Galp(1→6)]-d-Galp, the smallest structure containing both, the antigenic d-Galp(α1→3)-d-Galp unit and the sialic acid-acceptor ß-d-Galp unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by ß-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3'-sialyllactose or fetuin as donors. The product, 6-aminohexyl α-d-NeuNAc(2→3)-ß-d-Galp(1→6)-[α-d-Galp(1→3)]-ß-d-Galp, was purified and characterized.

Immunodetection of the expression of microsomal proteins encoded by the glucose 6-phosphate transporter gene.

Glucose 6-phosphate transport has been well characterized in liver microsomes. The transport is required for the functioning of the glucose-6-phosphatase enzyme that is situated in the lumen of the hepatic endoplasmic reticulum. The genetic deficiency of the glucose 6-phosphate transport activity causes a severe metabolic disease termed type 1b glycogen storage disease. The cDNA encoding a liver transporter for glucose 6-phosphate was cloned and was found to be mutated in patients suffering from glycogen storage disease 1b. While related mRNAs have been described in liver and other tissues, the encoded protein(s) has not been immunologically characterized yet. In the present study, we report (using antibodies against three different peptides of the predicted amino acid sequence) that a major protein encoded by the glucose 6-phosphate transporter gene is expressed in the endoplasmic reticulum membranes of rat and human liver. The protein has an apparent molecular mass of approx. 33 kDa using SDS/PAGE, but several lines of evidence indicate that its real molecular mass is 46 kDa, as expected. The glucose 6-phosphate transporter protein was also immunodetected in kidney microsomes, but not in microsomes derived from human fibrocytes, rat spleen and lung, and a variety of cell lines. Moreover, little or no expression of the glucose 6-phosphate transporter protein was found in liver microsomes obtained from three glycogen storage disease 1b patients, even bearing mutations that do not directly interfere with protein translation, which can be explained by a (proteasome-mediated) degradation of the mutated transporter.