Branching Of N-linked Glycans Research Articles

A practical synthesis of a novel DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) for in vivo studies

Immunotherapy that targets N-linked glycans has not yet been developed due in large part to the lack of specificity of N-linked glycans between normal and malignant cells. N-Glycan chains are synthesized by the sequential action of glycosyl transferases in the Golgi apparatus. It is an overwhelming task to discover drug-like inhibitors of glycosyl transferases that block the synthesis of specific branching processes in cancer cells, killing tumor cells selectively. It has long been known that N-glycan biosynthesis can be inhibited by disruption of the first committed enzyme, dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1). Selective DPAGT1 inhibitors have the promising therapeutic potential for certain solid cancers that require increased branching of N-linked glycans in their growth progressions. Recently, we discovered that an anti-Clostridium difficile molecule, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) showed DPAGT1 inhibitory activity with the IC50 value of 0.25 μM. It was confirmed that APPB inhibits N-glycosylation of β-catenin at 2.5 nM concentration. A sharp difference between APPB and tunicamycin was that the hemolytic activity of APPB is significantly attenuated (IC50 > 200 μM RBC). Water solubility of APPB is >350-times greater than that of tunicamycin (78.8 mg/mL for APPB, <0.2 mg/mL for tunicamycin). A novel DPAGT1 inhibitor, APPB selectively inhibits growth of the solid tumors (e.g. KB, LoVo, SK-OV-3, MDA-MB-432S, HCT116, Panc-1, and AsPC-1) at low μM concentrations, but does not inhibit growth of a leukemia cell (L1210) and the healthy cells (Vero and HPNE) at these concentrations. In vitro metabolic stability using rat liver microsomes indicated that a half-life (t1/2) of APPB is sufficiently long (>60 min) for in vivo studies (PK/PD, safety profiles, and in vivo efficacy) using animal models. We have refined all steps in the previously reported synthesis for APPB for larger-scale. This article summarizes protocols of gram-scale synthesis of APPB and its physicochemical data, and a convenient DPAGT1 assay.•Remember that the abstract is what readers see first in electronic abstracting & indexing services.•This is the advertisement of your article. Make it interesting, and easy to be understood.•Be accurate and specific, keep it as brief as possible.

Improved Pharmacokinetic Profile for BAY 81-8973 Due to Increased Branching and Sialylation of N-Linked Glycans of Recombinant Factor VIII

The circulatory half-life of recombinant factor VIII (rFVIII) products is affected by glycosylation of the FVIII protein, including N-linked glycan branching and terminal sialic acid occupancy, primarily through receptor-mediated hepatic clearance (eg, asialoglycoprotein receptor [ASGPR] and lipoprotein receptor-related protein [LRP]). BAY 81-8973 (Kovaltry®, Bayer, Berkeley, CA) is an unmodified full-length rFVIII for treatment of hemophilia A. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of branching and sialylation of N-linked glycans. This study evaluated whether a relationship exists between N-linked glycosylation patterns and pharmacokinetic (PK) characteristics of BAY 81-8973 and 2 other rFVIII products (sucrose-formulated rFVIII [rFVIII-FS; Kogenate® FS, Bayer] and antihemophilic factor (recombinant) plasma/albumin-free method [rAHF-PFM; Advate®, Shire, Westlake Village, CA]).N-linked glycans or terminal carbohydrates were enzymatically removed from immobilized BAY 81-8973, rFVIII-FS, and rAHF-PFM proteins and analyzed using high-performance liquid chromatography to determine the percentage of individual N-linked glycan structures and degree of sialylation of each structure. PK data were available from 2 separate phase 1 crossover studies in which the PK profile of BAY 81-8973 was compared with that of rFVIII-FS (n=26) and rAHF-PFM (n=18) in patients with severe hemophilia A who received a single 50-IU/kg dose of each product.BAY 81-8973 and rFVIII-FS had increased N-linked glycan branching with higher levels of sialylation compared with rAHF-PFM. Levels of trisialylated glycans were 29.0% for BAY 81-8973 versus 11.5% for rFVIII-FS and 4.8% to 5.5% for rAHF-PFM; tetrasialylated glycans were 12.0% versus 2.8% and 0.6%, respectively. Degree of sialylation was 96% for BAY 81-8973, 94% for rFVIII-FS, and 78% to 81% for rAHF-PFM. Based on chromogenic assay results from the single-dose phase 1 PK studies, BAY 81-8973 half-life was 15% longer than that for rFVIII-FS and 16% longer than rAHF-PFM.Increases in the percentage of sialylated tri-antennary and tetra-antennary N-glycans correlated well with longer half-life of rFVIII in humans (adjusted R2=0.978 and 0.892 for tri-antennary and tetra-antennary N-glycans, respectively). Higher percentages of sialylation (ie, sialic acid capping) correlated with a longer half-life (adjusted R2=0.697), but the relationship was not as strong as that between glycan branching and half-life. Improved PK for BAY 81-8973 relative to rFVIII-FS and rAHF-PFM as seen in single-dose crossover PK studies might be related to this greater level of branching and sialylation, which may prolong the time BAY 81-8973 remains in the circulation. DisclosuresTeare:Bayer: Employment. Kates:Bayer: Employment. Shah:Bayer: Employment. Garger:Bayer: Employment.

N-Linked Glycan Branching and Fucosylation Are Increased Directly in Hcc Tissue As Determined through in Situ Glycan Imaging.

Hepatocellular carcinoma (HCC) remains as the fifth most common cancer in the world and accounts for more than 700,000 deaths annually. Changes in serum glycosylation have long been associated with this cancer but the source of that material is unknown and direct glycan analysis of HCC tissues has been limited. Our laboratory previously developed a method of in situ tissue based N-linked glycan imaging that bypasses the need for microdissection and solubilization of tissue prior to analysis. We used this methodology in the analysis of 138 HCC tissue samples and compared the N-linked glycans in cancer tissue with either adjacent untransformed or tissue from patients with liver cirrhosis but no cancer. Ten glycans were found significantly elevated in HCC tissues as compared to cirrhotic or adjacent tissue. These glycans fell into two major classes, those with increased levels of fucosylation and those with increased levels of branching with or without any fucose modifications. In addition, increased levels of fucosylated glycoforms were associated with a reduction in survival time. This work supports the hypothesis that the increased levels of fucosylated N-linked glycans in HCC serum are produced directly from the cancer tissue.

Effects of altered sialic acid biosynthesis on N-linked glycan branching and cell surface interactions

GNE (UDP-GlcNAc 2-epimerase/ManNAc kinase) myopathy is a rare muscle disorder associated with aging and is related to sporadic inclusion body myositis, the most common acquired muscle disease of aging. Although the cause of sporadic inclusion body myositis is unknown, GNE myopathy is associated with mutations in GNE. GNE harbors two enzymatic activities required for biosynthesis of sialic acid in mammalian cells. Mutations to both GNE domains are linked to GNE myopathy. However, correlation between mutation-associated reductions in sialic acid production and disease severity is imperfect. To investigate other potential effects of GNE mutations, we compared sialic acid production in cell lines expressing wild type or mutant forms of GNE. Although we did not detect any differences attributable to disease-associated mutations, lectin binding and mass spectrometry analysis revealed that GNE deficiency is associated with unanticipated effects on the structure of cell-surface glycans. In addition to exhibiting low levels of sialylation, GNE-deficient cells produced distinct N-linked glycan structures with increased branching and extended poly-N-acetyllactosamine. GNE deficiency may affect levels of UDP-GlcNAc, a key metabolite in the nutrient-sensing hexosamine biosynthetic pathway, but this modest effect did not fully account for the change in N-linked glycan structure. Furthermore, GNE deficiency and glucose supplementation acted independently and additively to increase N-linked glycan branching. Notably, N-linked glycans produced by GNE-deficient cells displayed enhanced binding to galectin-1, indicating that changes in GNE activity can alter affinity of cell-surface glycoproteins for the galectin lattice. These findings suggest an unanticipated mechanism by which GNE activity might affect signaling through cell-surface receptors.

Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing.

Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the experimental options. Here we present a method to obtain information about protein glycosylation using a minimal amount of protein. We treat proteins that were captured or directly spotted in small microarrays (2.2 mm × 2.2 mm) with exoglycosidases to successively expose underlying features, and then we probe the native or exposed features using a panel of lectins or glycan-binding reagents. We developed an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We demonstrated the efficacy of the method to characterize differences between glycoproteins in their sialic acid linkages and N-linked glycan branching, and we validated the assignments by comparing results from mass spectrometry and chromatography. The amount of protein used on-chip was about 11 ng. The method also proved effective for analyzing the glycosylation of a cancer biomarker in human plasma, MUC5AC, using only 20 μL of the plasma. A glycan on MUC5AC that is associated with cancer had mostly 2,3-linked sialic acid, whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could complement the existing toolkit for studying glycosylation in disease.

An insight into the cells’ glycans and lectin-glycosensing sites

Glycosylation produces a diverse and abundant repertoire of glycans that help protein folding and cellular secretion [1]. Glycoproteins often sprout sialic acid (alias N-acetyl neuraminic acid, abbreviated as Neu5Ac) at the termini of their Nand O-glycans in a process called sialylation [2]. Alterations to the normal function of the glycosylation machinery, including increased branching of N-linked glycans and sialylation, are major hallmarks of cancer progression. Particularly, increased sialylation on the cell surface induces tumor proliferation by promoting cell detachment from primary tumors through charge repulsion [3]. Despite the potential of increased or altered sialylation as diagnostic and prognostic biomarkers, there is still no early glycosensing platform that can be ubiquitously applied to detect glycans sialylation in diverse bio-samples with high selectivity and sensitivity. Further analyses have been recently found that the metabolic control of glycan’s sialylation is regulated by two factors: (1) the rate of flux through the sialic acid pathway and (2) the expression of an appropriate complement of sialyltransferase. However, none of these factors have been exploited with sufficient precision to achieve specific endpoints. Metabolic glycoengineering has been established as a very useful technique for the modulation of glycan sialylation structures on cell surfaces, and various constituents of the target sialoglycans are studied by lectins, a famous class of glycan-binding proteins [4]. Structurally, lectins have small or shallow binding sites, which results in relatively weak and non-covalent glycan interactions, often with binding constants in the millimolar range. However, the weak binding affinity and non-covalent characteristics of the lectin glyco-interactive sites can be enhanced dramatically by multivalent binding approaches [5]. Among these, the most powerful one is sensing the structural motifs and intensity of the accessible cell surface sialylation constellations [6, 7]. Djansugurova group from Kazakh Institute of General Genetics and Cytology, in collaboration with Ahmed group in the University of Maryland, reported in a preliminary communication the restoration of surface sialylation sites in a T47D breast cancer line compared to HB4A normal cells after sialic acid supplementation under conditions of nutrient deprivation [8]. Looking forward, Ahmed’s group [9] focused further efforts on modulating cell surface glycan sialylation and found increases in cell surface binding for wheat germ agglutinin (WGA), a lectin that binds to both 2,3and 2,6-linked sialic acid with slightly greater enhancement in the cancer cells (T47D, MCF7 and MDA MB231) when compared to the normal lines (MCF10A and HB4A). However, upon using plant lectins that discriminate between 2,3and 2,6-linkages, the Sambucus nigra lectin (SNA) binding to 2,6-sialic acids revealed roughly proportional increases in all lines, whereas Maackia amurensis lectin I (MAL-I) binding to 2,3-linked sialic acid showed greater enhancement in the malignant lines, compared to the normal cells. These research results highlight, for the first time, a detailed differential lectin-glycosensing site approach for the application of cell surface sialylation detection, specifically the highly dense clustered patches of sialo-epitopes NeuAc(2–3)bGal(1–4)bGlcNAc/Glc, which are displayed on the mammal cell surface envelope, enabling the detection of targeted sialoglycans in a highly sensitive and cost-effective manner [10]. More work along this line will potentially be useful for both cytochemical C.-Z. Li (&) Nanobioengineering and Bioelectronics Lab, Department of Biomedical Engineering, Florida International University, Miami, FL 33174, USA e-mail: licz@fiu.edu

How does mild hypothermia affect monoclonal antibody glycosylation?

The application of mild hypothermic conditions to cell culture is a routine industrial practice used to improve recombinant protein production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimization. In this study, we investigated the impact of mild hypothermia on protein glycosylation. Chinese hamster ovary (CHO) cells expressing a monoclonal antibody (mAb) were cultured at 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32°C and was accompanied by a reduction in intracellular nucleotide sugar donor (NSD) concentrations and a decreased proportion of the more processed glycan structures on the mAb constant region. To better understand CHO cell metabolism at 32°C, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggested reduced fluxes of carbon species towards nucleotide and NSD synthesis and more energy was used for product formation. Expression of the glycosyltransferases that are responsible for N-linked glycan branching and elongation were significantly lower at 32°C. As a result of mild hypothermia, mAb glycosylation was shown to be affected by both NSD availability and glycosyltransferase expression. The combined experimental/FBA approach generated insight as to how product glycosylation can be impacted by changes in culture temperature. Better feeding strategies can be developed based on the understanding of the metabolic flux distribution.

Transcriptional Regulation of the Protocadherin β Cluster during Her-2 Protein-induced Mammary Tumorigenesis Results from Altered N-Glycan Branching

Changes in the levels of N-acetylglucosaminyltransferase V (GnT-V) can alter the function of several types of cell surface receptors and adhesion molecules by causing altered N-linked glycan branching. Using a her-2 mammary tumor mouse model, her-2 receptor signaling was down-regulated by GnT-V knock-out, resulting in a significant delay in the onset of her-2-induced mammary tumors. To identify the genes that contributed to this GnT-V regulation of early events in tumorigenesis, microarray analysis was performed using her-2 induced mammary tumors from wild-type and GnT-V-null mice. We found that 142 genes were aberrantly expressed (>2.0-fold) with 64 genes up-regulated and 78 genes down-regulated after deletion of GnT-V. Among differentially expressed genes, the expression of a subgroup of the cadherin superfamily, the protocadherin β (Pcdhβ) cluster, was up-regulated in GnT-V-null tumors. Altered expression of the Pcdhβ cluster in GnT-V-null tumors was not due to changes in promoter methylation; instead, impaired her-2-mediated signaling pathways were implicated at least in part resulting from reduced microRNA-21 expression. Overexpression of Pcdhβ genes inhibited tumor cell growth, decreased the proportion of tumor-initiating cells, and decreased tumor formation in vivo, demonstrating that expression of the Pcdhβ gene cluster can serve as an inhibitor of the transformed phenotype. Our results suggest the up-regulation of the Pcdhβ gene cluster as a mechanism for reduced her-2-mediated tumorigenesis resulting from GnT-V deletion.

N-Acetylglucosamine Functions in Cell Signaling

The amino sugar N-acetylglucosamine (GlcNAc) is well known for the important structural roles that it plays at the cell surface. It is a key component of bacterial cell wall peptidoglycan, fungal cell wall chitin, and the extracellular matrix of animal cells. Interestingly, recent studies have also identified new roles for GlcNAc in cell signaling. For example, GlcNAc stimulates the human fungal pathogen Candida albicans to undergo changes in morphogenesis and expression of virulence genes. Pathogenic E. coli responds to GlcNAc by altering the expression of fimbriae and CURLI fibers that promote biofilm formation and GlcNAc stimulates soil bacteria to undergo changes in morphogenesis and production of antibiotics. Studies with animal cells have revealed that GlcNAc influences cell signaling through the posttranslational modification of proteins by glycosylation. O-linked attachment of GlcNAc to Ser and Thr residues regulates a variety of intracellular proteins, including transcription factors such as NFκB, c-myc, and p53. In addition, the specificity of Notch family receptors for different ligands is altered by GlcNAc attachment to fucose residues in the extracellular domain. GlcNAc also impacts signal transduction by altering the degree of branching of N-linked glycans, which influences cell surface signaling proteins. These emerging roles of GlcNAc as an activator and mediator of cellular signaling in fungi, animals, and bacteria will be the focus of this paper.

Prediction of N-linked glycan branching patterns using artificial neural networks

A model was developed for novel prediction of N-linked glycan branching pattern classification for CHO-derived N-linked glycoproteins. The model consists of 30 independent recurrent neural networks and uses predicted quantities of secondary structure elements and residue solvent accessibility as an input vector. The model was designed to predict the major component of a heterogeneous mixture of CHO-derived glycoforms of a recombinant protein under normal growth conditions. Resulting glycosylation prediction is classified as either complex-type or high mannose. The incorporation of predicted quantities in the input vector allowed for theoretical mutant N-linked glycan branching predictions without initial experimental analysis of protein structures. Primary amino acid sequence data were effectively eliminated from the input vector space based on neural network prediction analyses. This provided further evidence that localized protein secondary structure elements and conformational structure may play more important roles in determining glycan branching patterns than does the primary sequence of a polypeptide. A confidence interval parameter was incorporated into the model to enable identification of false predictions. The model was further tested using published experimental results for mutants of the tissue-type plasminogen activator protein [J. Wilhelm, S.G. Lee, N.K. Kalyan, S.M. Cheng, F. Wiener, W. Pierzchala, P.P. Hung, Alterations in the domain structure of tissue-type plasminogen activator change the nature of asparagine glycosylation. Biotechnology (N.Y.) 8 (1990) 321–325].