Higher-resolution structure of the human insulin receptor ectodomain: Multi-modal inclusion of the insert domain

Insulin receptor (IR) signaling is critical to controlling nutrient uptake and metabolism. However, only a low-resolution (3.8 Å) structure currently exists for the IR ectodomain, with some segments ill-defined or unmodeled due to disorder. Here, we revise this structure using new diffraction data to 3.3 Å resolution that allow improved modeling of the N-linked glycans, the first and third fibronectin type III domains, and the insert domain. A novel haptic interactive molecular dynamics strategy was used to aid fitting to low-resolution electron density maps. The resulting model provides a foundation for investigation of structural transitions in IR upon ligand binding.

Transcriptome and glycome profiling of human hematopoietic CD133+ and CD34+ cells

New blood cells are continuously provided by self-renewing multipotent hematopoietic stem cells (HSC). The capacity of HSCs to regenerate the hematopoietic system is utilized in the treatment of patients with hematological malignancies. HSCs can be enriched using an antibody-based recognition of CD34 or CD133 glycoproteins on the cell surface. The CD133+ and CD34+ cells may have partly different roles in hematopoiesis. Furthermore, each cell has a glycome typical for that cell type. Knowledge of HSC glycobiology can be used to design therapeutic cells with improved cell proliferation or homing properties. The present studies characterize the global gene expression profile of human cord blood-derived CD133+ and CD34+ cells, and demonstrate the differences between CD133+ and CD34+ cell populations that may have an impact in transplantation when CD133+ and CD34+ selected cells are used. In addition, these studies unravel the glycome profile of primitive hematopoietic cells and reveal the transcriptional regulation of N-glycan biosynthesis in CD133+ and CD34+ cells. The gene expression profile of CD133+ cells represents 690 differentially expressed transcripts between CD133+ cells and CD133- cells. CD34+ cells have 620 transcripts differentially expressed when compared to CD34- cells. The integrated CD133+/CD34+ cell gene expression profiles proffer novel transcripts to specify HSCs. Furthermore, the differences between the gene expression profiles of CD133+ and CD34+ cells indicate differences in the transcriptional regulation of CD133+ and CD34+ cells. CD133+ cells express a lower number of hematopoietic lineage differentiation marker genes than CD34+ cells. The expression profiles suggest a more primitive nature of CD133+ cells. Moreover, CD133+ cells have characteristic glycome that differ from the glycome of CD133- cells. High mannose-type and biantennary complex-type N-glycans are enriched in CD133+ cells. N-glycosylation-related gene expression pattern of CD133+ cells identify the key genes regulating the CD133+ cell-specific glycosylation including the overexpression of MGAT2 and underexpression of MGAT4. The putative role of MAN1C1 in the increase of unprocessed high mannose-type N-glycans in CD133+ cells is also discussed. These studies provide new information on the characteristics of HSCs. Improved understanding of HSC biology can be used to design therapeutic cells with improved cell proliferation and homing properties. As a result, HSC engineering could further their clinical use.

Glycodelin A triggers T cell apoptosis through a novel calcium-independent galactose-binding lectin activity

Glycodelin A (GdA) is one of the progesterone inducible endometrial factors that protect the fetal semiallograft from maternal immune rejection. The immumoregulatory effects of GdA are varied, with diverse effects on the fate and function of most immune cell types. Its effects on T cells are particularly relevant as it is capable of regulating T cell activation, differentiation, as well as apoptosis. We have previously reported that GdA triggers mitochondrial stress and apoptosis in activated T cells by a mechanism that is distinct and independent of its effects on T cell activation. In this study we describe the characterization of a cell surface receptor for GdA on T cells. Our results reveal a novel calcium-independent galactose-binding lectin activity of GdA, which is responsible for its apoptogenic function. This discovery adds GdA to a select group of soluble immunoregulatory lectins that operate within the feto-placental compartment, the only other members being the galectin family proteins. We also report for the first time that both CD4(+) and CD8(+) T cell subsets are equally susceptible to inhibition with GdA, mediated by its novel lectin activity. We demonstrate that GdA selectively recognizes complex-type N-linked glycans on T cell surface glycoproteins. and propose that the galectin-1 glycoprotein receptor CD7 maybe a novel target for GdA on T cells. This study, for the first time, links the lectin activity of GdA to its biological function.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Carbohydrate binding specificity of the B-cell maturation mitogen from Artocarpus integrifolia seeds

Artocarpin, a mannose-specific lectin, is a homotetrameric protein (M(r) 65,000) devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range 5-6.5. Investigations of its carbohydrate binding specificity reveal that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man alpha 1-3[Man alpha 1-6]Man) and mannopentaose are the strongest ligands followed by Man alpha 1-3Man. Extension of these ligands by GlcNAc at the reducing ends of mannooligosaccharides tested remarkably improves their inhibitory potencies, while substitution of both the alpha 1-3 and alpha 1-6 mannosyl residues of mannotriose and the core pentasaccharide of N-linked glycans (Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc) by GlcNAc or N-acetyllactosamine in beta 1-2 linkage diminishes their inhibitory potencies. Sialylated oligosaccharides are non-inhibitory. Moreover, the substitution of either alpha 1-3 or alpha 1-6 linked mannosyl residues of M5Gn or both by mannose in alpha 1-2 linkage leads to a considerable reduction of their inhibitory power. Addition of a xylose residue in beta 1-2 linkage to the core pentasaccharide improves the inhibitory activity. Considering the fact that artocarpin has the strongest affinity for the xylose containing hepasaccharide from horseradish peroxidase, which differs significantly from all the mannose/glucose-specific lectins, it should prove a useful tool for the isolation and characterization of glycoproteins displaying such structure.

Synthesis of neoglycopeptides and analyses of their biodistributionin vivo to identify tissue specific uptake and novel putative membrane lectins

Complex typeN-linked oligosaccharides derived from fetuin, fibrinogen and thyroglobulin were coupled to acetyltyrosine affording a series of neoglycopeptides with retention of terminal structures and the beta-anomeric configuration of their reducing endN-acetylglycosamine residue. The neoglycopeptides thus synthesized could be labelled to high specific activities with125I in the aromatic side chain of tyrosine. Analysis of the fate of these neoglycopeptides in conjunction with inhibition with asialofetuin and oligosaccharides of defined structure in micein vivo revealed the uptake of galactosylated biantennary compound by kidneys, in addition to the known itinerary of triantennary galactosylated complex oligosaccharide from fetuin to liver and the galactosylated biantennary chain with fucosylation in the core to bone marrows. On the other hand, the agalacto, aglucosamino biantennary chains with and without fucosylation in the core region are taken up by submaxillary glands while the conserved trimannosyl core with fucose is primarily concentrated in stomach tissue. These studies thus define new routes for the uptake of complexN-linked glycans and also subserve to identify lectins presumably involved in their recognition.

Oligosaccharides modulate the apoptotic activity of glycodelin

GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitorof sperm-zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insec cell line yielded an apoptotically active Gd; however, the same geneexpressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary(CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans.

Crystal structure of a beta-prism II lectin from Remusatia vivipara

The crystal structure of a beta-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4 A. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the ``monocot mannose-binding'' lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.

Mapping the apoptosis inducing domain of an immunomodulatory protein: glycodelin A

Glycodelin A (GdA) is a dimeric glycoprotein synthesized by the human endometrium under progesterone regulation. Based on the high sequence similarity with beta-lactoglobulin, it is placed under the lipocalin superfamily. The protein is one of the local immunomodulators present at the feto-maternal interface which affects both the innate as well as the acquired arms of the immune system, thereby bringing about successful establishment and progression of pregnancy. Our previous studies revealed that the domain responsible for the immunosuppressive activity of glycodelin lies on its protein backbone and the glycans modulate the same. This study attempts to further delineate the apoptosis inducing region of GdA. Our results demonstrate that the stretch of amino acid sequence between Met24 to Leu105 is necessary and sufficient to inhibit proliferation of T cells and induce apoptosis in them. Further, within this region the key residues involved in harboring the activity were shown to be present between Asp52 and Ser65.

Mecanismos celulares e moleculares envolvidos com alterações na expressão de glicanos durante a progressão do câncer colorretal

O câncer colorretal representa uma das maiores causas de morbidade e mortalidade relacionadas ao câncer. No Brasil, é o terceiro tipo de câncer mais frequente em homens e mulheres. Muitos estudos estão sendo desenvolvidos no sentido de esclarecer os diversos aspectos moleculares que regulam as alterações fenotípicas exibidas pelas células que constituem o câncer colorretal, no entanto, comparativamente, ainda são poucos os que são dedicados a investigar o papel de modificações co- e pós-traducionais neste processo. Entre os vários tipos destas modificações que ocorrem em proteínas, a glicosilação é a mais comum. Cogita-se que aproximadamente cinquenta por cento de todas as proteínas são glicosiladas. Durante a transformação maligna, mudanças no perfil de expressão de glicanos (carboidratos covalentemente ligados a proteínas ou lipídios) estão envolvidas em uma variedade de mecanismos celulares, tais como: perda da adesão célula-célula e célula matriz, migração, invasão e evasão da apoptose. Neste estudo, foi investigada a atividade antitumoral de inibidores da biossíntese de N-glicanos, swainsonina e tunicamicina, em células derivadas de câncer colorretal (Caco-2, HCT-116 e HT-29). Os resultados obtidos mostram que o tratamento das células HCT-116 com tunicamicina inibe mecanismos celulares relacionados ao fenótipo maligno, como formação de colônia dependente e independente de ancoragem, migração e invasão. Estes resultados sugerem que modulação da biossíntese de N-glicanos parece ser uma potencial ferramenta terapêutica para o tratamento do câncer colorretal. Em outra etapa do trabalho, foram avaliados também o impacto da estimulação com insulina e IGF-1 na expressão N-glicanos bissectados em células tumorais MDA-MB-435. Os resultados obtidos confirmaram também a existência de uma relação entre a estimulação dos receptores de insulina e IGF-1 e a regulação da expressão de N-glicanos bissectados em células tumorais MDA-MB-435, fornecendo assim informações relevantes sobre o papel desempenhado pela sinalização de insulina e IGF-1 durante a progressão de carcinomas.

Characterization of saccharides and phenolic acids in the Chinese herb Tanshen by ESI-FT-ICR-MS and HPLC

This study sought to determine the main components (saccharides and phenolic acids) in crude extract of the Chinese herb Tanshen by electrospray ionization Fourier transform ion cyclotron resonant mass spectrometry (ESI-FT-ICR-MS) in negative-ion mode. Eleven compounds were identified as phenolic acids by exact mass measurement and further confirmed by sustained off-resonance irradiation (SORI) CID data. In addition, monosaccharicles and oligosaccharides (n = 2 similar to 5) and a serial of corresponding anionic adducts of saccharide were observed without adding any anions additionally to the extract solution, and the anionic components were unambiguously identified as H2O, HCl, HCOOH, HNO3, C3H6O2, H2SO4 and C5H7NO3 according to the exact mass measurement results.

Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K(D) for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC(50)s that decrease from 15000 nM (1500 microg mL(-1)) for the 25 mol % functionalized polymers to 11 nM (1 microg mL(-1)) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.

Down-regulation of beta1,4-galactosyltransferase V is a critical part of etoposide-induced apoptotic process and could be mediated by decreasing Sp1 levels in human glioma cells.

beta1,4-Galactosyltransferase V (beta1,4GalT V; EC 2.4.1.38) is considered to be very important in glioma for expressing transformation-related highly branched N-glycans. Recently, we have characterized beta1,4GalT V as a positive growth regulator in several glioma cell lines. However, the role of beta1,4GalT V in glioma therapy has not been clearly reported. In this study, interfering with the expression of beta1,4GalT V by its antisense cDNA in SHG44 human glioma cells markedly promoted apoptosis induced by etoposide and the activation of caspases as well as processing of Bid and expression of Bax and Bak. Conversely, the ectopic expression of beta1,4GalT V attenuated the apoptotic effect of etoposide on SHG44 cells. In addition, both the beta1,4GalT V transcription and the binding of total or membrane glycoprotein with Ricinus communis agglutinin-I (RCA-I) were partially reduced in etoposide-treated SHG44 cells, correlated well with a decreased level of Sp1 that has been identified as an activator of beta1,4GalT V transcription. Collectively, our results suggest that the down-regulation of beta1,4GalT V expression plays an important role in etoposide-induced apoptosis and could be mediated by a decreasing level of Sp1 in SHG44 cells, indicating that inhibitors of beta1,4GalT V may enhance the therapeutic efficiency of etoposide for malignant glioma.

Beta1,4-galactosyltransferase V functions as a positive growth regulator in glioma.

beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAcbeta1-->6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.

The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.