Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO® penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex N-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.

• MeSH
• amidy chemie   MeSH
• chromatografie kapalinová přístrojové vybavení   metody   MeSH
• glykopeptidy chemie   izolace a purifikace   metabolismus   MeSH
• glykosylace MeSH
• hemopexin chemie   izolace a purifikace   MeSH
• hydrofobní a hydrofilní interakce MeSH
• imunoglobulin G chemie   izolace a purifikace   MeSH
• isomerie MeSH
• lidé MeSH
• polysacharidy chemie   MeSH
• teplota MeSH
• trypsin chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

A sensitive and rapid high-performance liquid chromatography (HPLC) method was developed to enantioseparation of N-acetyl-dl-cysteine after precolumn derivatization using o-phthaldialdehyde and primary aliphatic amines. Seven polysaccharide-based chiral columns were tested in a reversed phase mode. Under the optimal chromatographic conditions, N-acetyl-dl-cysteine derivatives were completely enantioseparated on Chiralcel OZ-3R column with the resolution more than 2.5. The impact of various primary aliphatic amine additives as co-reagents (ethyl-, 1-propyl-, 1-butyl-, 1-pentylamine, (R)-sec-butylamine, tert-butylamine, isobutylamine, cyclopropyl-, cyclobutyl-, cyclopentyl and cyclohexylamine) used in precolumn derivatization step on the retention behavior (retention factor, selectivity and column efficiency) of N-acetyl-dl-cysteine derivatives was investigated. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH, salt concentration in the mobile phase and column temperature on the retention and selectivity was investigated. The developed method was properly validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy, precision, intermediate precision and selectivity according to International Council for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use guidelines using internal normalization procedure. Proposed HPLC method was successfully applied to the determination of optical purity in commercially available N-acetyl-L-cysteine samples.

• MeSH
• acetylcystein chemie   MeSH
• aminy chemie   MeSH
• o-ftalaldehyd chemie   MeSH
• polysacharidy chemie   MeSH
• stereoizomerie MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH

The labeling by amino acids and peptides was investigated for sensitive and fast analyses of oligosaccharides and N-linked glycans by capillary electrophoresis-mass spectrometry (CE-MS). Peptide tags with a various number of histidine residues were tested for maltooligosaccharide labeling in order to investigate the effect of the size of labels and a number of charges on CE-MS analysis. Nevertheless, the reductive amination labeling of N-linked glycans by a hexahistidine tag resulted in a multiple products formation, therefore a peptide tag was modified by hydrazine functionality in order to perform labeling by hydrazone formation chemistry. This labeling approach significantly improved sensitivity with LOD of labeled maltopentaose determined to be 40 nmol/L and also significantly reduced separation time of neutral maltooligosaccharides and N-linked glycans released from bovine ribonuclease B. Furthermore, the labeling by this multi-cationic peptide hydrazine tag also allowed performing analysis of acidic glycans by CE-MS in a positive ion mode as demonstrated by separation of sialylated N-linked glycans released from bovine fetuin.

• MeSH
• elektroforéza kapilární metody   MeSH
• fetuin A chemie   MeSH
• histidin chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• oligopeptidy chemie   MeSH
• oligosacharidy analýza   chemie   izolace a purifikace   MeSH
• polysacharidy analýza   chemie   izolace a purifikace   MeSH
• ribonukleasy chemie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Glycan metabolism balance is critical for cell prosperity, and macromolecule glycosylation is essential for cell communication, signaling and survival. Thus, glycotherapy may be a potential cancer treatment. The aim of the present study was to determine whether combined synthetic glycoconjugates (GCs) induce changes in gene expression that alter the survival of colon cancer cells. The current study evaluated the effect of the GCs N‑acetyl‑D‑glucosamine modified polyamidoamine dendrimer and calix[4]arene scaffold on cancer cell proliferation, apoptosis, invasion and sensitivity to immune cell‑mediated killing. Using reverse transcription‑quantitative polymerase chain reaction, the expression of genes involved in the aforementioned processes was measured. It was determined that GCs reduce the expression of the glucosaminyltransferases Mgat3 and Mgat5 responsible for surface glycosylation and employed components of the Wnt signaling pathway Wnt2B and Wnt9B. In addition, the calix[4]arene‑based GC reduced cell colony formation; this was accompanied by the downregulation of the metalloproteinase Mmp3. By contrast, the dendrimer‑based GC affected the expression of the glucose transporter components Sglt1 and Egfr1. Therefore, to the best of our knowledge, the present study is the first to reveal that N‑acetyl‑D‑glucosamine‑dendrimer/calix[4]arene GCs alter mRNA expression in a comprehensive way, resulting in the reduced malignant phenotype of the colon cancer cell line HT‑29.

• MeSH
• apoptóza genetika   MeSH
• buňky HT-29 MeSH
• glukosa metabolismus   MeSH
• glykokonjugáty farmakologie   MeSH
• lidé MeSH
• molekuly buněčné adheze genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky účinky léků   metabolismus   MeSH
• nádory tračníku genetika   metabolismus   MeSH
• proliferace buněk MeSH
• proteiny usnadňující transport glukosy genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• testy nádorových kmenových buněk MeSH
• transkriptom MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.

• MeSH
• chemické techniky analytické metody   normy   MeSH
• chromatografie kapalinová * MeSH
• chromatografie s reverzní fází * MeSH
• glykopeptidy analýza   MeSH
• hemopexin analýza   MeSH
• hydrofobní a hydrofilní interakce MeSH
• polysacharidy chemie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Human milk oligosaccharides (HMOs) are one of the major glycan source of the infant gut microbiota. The two species that predominate the infant bifidobacteria community, Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, possess an arsenal of enzymes including α-fucosidases, sialidases, and β-galactosidases to metabolise HMOs. Recently bifidobacteria were obtained from the stool of six month old Kenyan infants including species such as Bifidobacterium kashiwanohense, and Bifidobacterium pseudolongum that are not frequently isolated from infant stool. The aim of this study was to characterize HMOs utilization by these isolates. Strains were grown in presence of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3'-sialyl-lactose (3'-SL), 6'-sialyl-lactose (6'-SL), and Lacto-N-neotetraose (LNnT). We further investigated metabolites formed during L-fucose and fucosyllactose utilization, and aimed to identify genes and pathways involved through genome comparison. RESULTS: Bifidobacterium longum subsp. infantis isolates, Bifidobacterium longum subsp. suis BSM11-5 and B. kashiwanohense strains grew in the presence of 2'-FL and 3'- FL. All B. longum isolates utilized the L-fucose moiety, while B. kashiwanohense accumulated L-fucose in the supernatant. 1,2-propanediol (1,2-PD) was the major metabolite from L-fucose fermentation, and was formed in equimolar amounts by B. longum isolates. Alpha-fucosidases were detected in all strains that degraded fucosyllactose. B. longum subsp. infantis TPY11-2 harboured four α-fucosidases with 95-99 % similarity to the type strain. B. kashiwanohense DSM 21854 and PV20-2 possessed three and one α-fucosidase, respectively. The two α-fucosidases of B. longum subsp. suis were 78-80 % similar to B. longum subsp. infantis and were highly similar to B. kashiwanohense α-fucosidases (95-99 %). The genomes of B. longum strains that were capable of utilizing L-fucose harboured two gene regions that encoded enzymes predicted to metabolize L-fucose to L-lactaldehyde, the precursor of 1,2-PD, via non-phosphorylated intermediates. CONCLUSION: Here we observed that the ability to utilize fucosyllactose is a trait of various bifidobacteria species. For the first time, strains of B. longum subsp. infantis and an isolate of B. longum subsp. suis were shown to use L-fucose to form 1,2-PD. As 1,2-PD is a precursor for intestinal propionate formation, bifidobacterial L-fucose utilization may impact intestinal short chain fatty acid balance. A L-fucose utilization pathway for bifidobacteria is suggested.

• MeSH
• alfa-L-fukosidasa klasifikace   genetika   metabolismus   MeSH
• beta-galaktosidasa metabolismus   MeSH
• Bifidobacterium longum enzymologie   genetika   metabolismus   MeSH
• Bifidobacterium enzymologie   genetika   metabolismus   MeSH
• DNA bakterií genetika   MeSH
• feces mikrobiologie   MeSH
• fukosa metabolismus   MeSH
• genom bakteriální MeSH
• kojenec MeSH
• kyseliny mastné těkavé metabolismus   MeSH
• kyseliny sialové metabolismus   MeSH
• laktosa analogy a deriváty   metabolismus   MeSH
• lidé MeSH
• mateřské mléko metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• oligosacharidy metabolismus   MeSH
• propylenglykol metabolismus   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvence nukleotidů MeSH
• střeva mikrobiologie   MeSH
• trisacharidy metabolismus   MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

In mammals, excitatory synapses contain two major types of ionotropic glutamate receptors: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptors (NMDARs). Both receptor types are comprised of several subunits that are post-translationally modified by N-glycosylation. However, the precise N-glycans that are attached to these receptor types are largely unknown. Here, we used biochemistry to confirm that native NMDARs are extensively N-glycosylated; moreover, we found that the NMDAR GluN2B subunit differs from GluN1 subunits with respect to endoglycosidase H sensitivity. Next, we used a complete panel of lectins to determine the glycan composition of NMDARs in both cerebellar tissue and cultured cerebellar granule cells. Our experiments identified 23 lectins that pulled down both the GluN1 and GluN2B NMDAR subunits. We then performed an electrophysiological analysis using representative lectins and found that pre-incubating cerebellar granule cells with the AAL, WGA, or ConA alters the receptor's biophysical properties; this lectin-mediated effect was eliminated when the cells were deglycosylated with peptide-N-glycosidase F. Similar lectin-mediated effects were observed using HEK293 cells that express recombinant GluN1/GluN2B receptors. Finally, using mutant recombinant GluN subunits expressed in HEK293 cells, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data provide new insight into the role that N-glycosylation plays in regulating the function of NMDA receptors in the central nervous system. All animal experiments were performed in accordance with relevant institutional ethics guidelines and regulations with respect to protecting animal welfare. We examined the N-glycan composition of NMDA receptors (NMDARs) using deglycosylating enzymes, lectin-based biochemistry, and electrophysiology. Our results revealed that cerebellar NMDARs associate with 23 different lectins that have unique specificities for glycan structures. Furthermore, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data shed light on the glycan composition of NMDARs, revealing potential targets for the development of novel therapeutic approaches.

• MeSH
• elektrofyziologické jevy fyziologie   MeSH
• HEK293 buňky MeSH
• krysa rodu rattus MeSH
• kyselina glutamová metabolismus   MeSH
• lidé MeSH
• neurony metabolismus   MeSH
• polysacharidy metabolismus   MeSH
• receptory N-methyl-D-aspartátu metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• synapse metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Controlled feeding of glucose has been employed previously to enhance the productivity of recombinant glycoproteins but there is a concern that low concentrations of glucose could limit the synthesis of precursors of glycosylation. Here we investigate the effect of glucose depletion on the metabolism, productivity and glycosylation of a chimeric human-llama monoclonal antibody secreted by CHO cells. The cells were inoculated into media containing varying concentrations of glucose. Glucose depletion occurred in cultures with an initial glucose ≤5.5 mM and seeded at low density (2.5 × 10(5) cells/mL) or at high cell inoculum (≥2.5 × 10(6) cells/mL) at higher glucose concentration (up to 25 mM). Glucose-depleted cultures produced non-glycosylated Mabs (up to 51%), lower galactosylation index (GI <0.43) and decreased sialylation (by 85%) as measured by mass spectrometry and HPLC. At low glucose a reduced intracellular pool of nucleotides (0.03-0.23 fmoles/cell) was measured as well as a low adenylate energy charge (<0.57). Low glucose also reduced GDP-sugars (by 77%) and UDP-hexosamines (by 90%). The data indicate that under glucose deprivation, low levels of intracellular nucleotides and nucleotide sugars reduced the availability of the immediate precursors of glycosylation. These results are important when applied to the design of fed-batch cultures.

• MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• glukosa metabolismus   MeSH
• glykoproteiny biosyntéza   metabolismus   MeSH
• glykosylace MeSH
• křečci praví MeSH
• lidé MeSH
• monoklonální protilátky biosyntéza   metabolismus   MeSH
• rekombinantní proteiny biosyntéza   genetika   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• avidin diagnostické užití   MeSH
• biosenzitivní techniky metody   přístrojové vybavení   využití   MeSH
• biotin diagnostické užití   MeSH
• bromkyan diagnostické užití   MeSH
• epichlorhydrin diagnostické užití   MeSH
• glutaraldehyd diagnostické užití   MeSH
• karbodiimidy diagnostické užití   MeSH
• klinické laboratorní techniky trendy   využití   MeSH
• kyselina jodistá diagnostické užití   MeSH
• makromolekulární látky izolace a purifikace   MeSH
• merkaptamin diagnostické užití   MeSH
• nanočástice diagnostické užití   MeSH
• polysacharidy diagnostické užití   MeSH
• streptavidin diagnostické užití   MeSH
• sukcinimidy diagnostické užití   MeSH

• MeSH
• diabetes mellitus 2. typu farmakoterapie   MeSH
• dospělí MeSH
• glukosa metabolismus   MeSH
• hypoglykemika MeSH
• inzulin sekrece   MeSH
• inzulinová rezistence účinky léků   MeSH
• Langerhansovy buňky účinky léků   MeSH
• lidé MeSH
• polysacharidy metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH