Enzymatic Release of Glycoprotein N-Glycans and Fluorescent Labeling.

Glycosylation, one of the most frequent protein posttranslational modifications, is involved in the mechanisms of cell-cell interactions and immune reactions and is modulated in the course of diseases. In contrary to chemical glycan release, enzymatic cleavage of N-glycans can be performed in any laboratory with relative ease. We present here two robust protocols to achieve N-glycan release. The first one uses trypsin to disrupt protein structure whereas the other involves the use of detergents prior to PNGase F digestion. Thereafter, N-glycans are isolated from peptides using reverse-phase cartridges and are desalted with carbograph cartridges before finally being derivatized with the fluorescent label 2AB.

Enzymatic Sequence Analysis of N-Glycans by Exoglycosidase Cleavage and Mass Spectrometry: Detection of Lewis X Structures.

Enzymatic sequencing of oligosaccharides provides structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can as well be applied to oligosaccharide mixtures to evaluate the occurrence of distinct oligosaccharide motives of functional and/or structural interest.Here we describe the application of enzymatic sequence analysis to a mixture of N-glycans released from α1-acid glycoprotein. The experimental conditions are optimized for detection of possible Lewis X structures after stepwise exoglycosidase digestion by MALDI-TOF mass spectrometry. However, the described method is generally applicable to analyze other structural properties of N-glycans using (respective) specific exoglycosidases.

The effect of blood sampling and preanalytical processing on human N-glycome.

Glycome modulations have been described in the onset and progression of many diseases. Thus, many studies have proposed glycans from blood glycoproteins as disease markers. Astonishingly, little effort has been given unraveling preanalytical conditions potentially influencing glycan analysis prior to blood biomarker studies. In this work, we evaluate for the first time the effect of hemolysis, storage and blood collection, but also influence of various times and temperatures between individual processing steps on the total N-glycome and on a glycan-biomarker score. Venous blood was collected from 10 healthy donors in 11 blood collection tubes with different additives, processed variously to obtain 16 preanalytical variables and N-glycans released from serum or plasma were analyzed by MALDI-TOF-MS and capillary electrophoresis coupled with fluorescence detection (CE-LIF) for the first time. Long time storage of deep frozen samples at -20°C or -80°C exerted only a minor influence on the glycome as demonstrated by CE-LIF. The N-glycome was very stable evidenced by MALDI-TOF when stored at 4°C for at least 48 hours and blood collected in tubes devoid of additives. The glycome was stable upon storage after centrifugation and aliquoting, which is an important information considering future diagnostic applications. Hemolysis, however, negatively correlated with an established glycan score for ovarian cancer, when evaluated by MALDI-TOF-MS measurement by affecting relative intensities of certain glycans, which could lead to false negative / positive results in glycan biomarker studies.

Invasion of Trypanosoma cruzi into host cells is impaired by N-propionylmannosamine and other N-acylmannosamines.

The etiologic agent of Chagas' disease, Trypanosoma cruzi, is widely distributed in South America, affecting millions of people with thousands of deaths every year. Adherence of the infectious trypomastigote to host cells is mediated by sialic acid. T. cruzi cannot synthesize sialic acids on their own but cleave them from the host cells and link them to glycans on the surface of the parasites using the trans-sialidase, a GPI-anchored enzyme. The infectivity of the protozoan parasites strongly depends on the activity of this enzyme. In this report, we investigated whether the transfer of sialic acids from the host to the parasites can be attenuated using novel sialic acid precursors. The cell line 86-HG-39 was infected with T. cruzi and treated with defined N-acylmannosamine analogues bearing an elongated N-acyl side-chain. By treatment of these cells the number of T. cruzi infected cell was reduced up to 60%. We also showed that the activity of the bacterial sialidase C was reduced with N-glycan substrates with elongated N-acyl side chains of the terminal sialic acids. The affinity of this sialidase decreased with the length of the N-acyl side-chain. The data presented suggest that N-acyl modified sialic acid precursors can change the transfer of sialic acids leading to modification of infection. Since the chemotherapy of this disease is inefficient and afflicted by side effects, the need of effective drugs is lasting. These findings propose a new path to prevent the dissemination of T. cruzi in the human hosts. These compounds or further modified analogues might be a basis for the search of new agents against Chagas' disease.

Enzymatic sequence analysis of N-glycans by exoglycosidase cleavage and mass spectrometry--detection of Lewis X structures.

Enzymatic sequencing of oligosaccharides gives structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can be applied to oligosaccharide mixtures as well to evaluate the occurrence of distinct oligosac-charide motives of functional and/or structural interest. Here we describe the application of enzymatic sequence analysis to a mixture of N-glycans released from alpha1-acid glycoprotein. The experimental conditions are optimized for detection of possible Lewis X structures after stepwise exoglycosi-dase digestion by MALDI-TOF mass spectrometry. However, the described method is generally applicable to analyze other structural properties of N-glycans by use of the respective specific exoglycosidases.

Liver-specific increase of UTP and UDP-sugar concentrations in rats induced by dietary vitamin B6-deficiency and its relation to complex N-glycan structures of liver membrane-proteins.

This is the first known report on the influence of vitamin B6-deficiency on the concentration of UDP-sugars and other uracil nucleotides in rats. Animals aged 3 weeks or 2 months were fed a vitamin B6-free diet for periods varying from 3 days to 7 weeks. Nucleotides were quantified by enzymatic-photometry and by SAX-high precision liquid chromatography. In 3 week-old rats, vitamin B6-deficiency resulted in an up to 6.3-fold increase in the concentrations of UTP, UDP, UMP and UDP-sugars and less of CTP in rat liver, while no changes were observed in older rats. In young rats, the concentration of uracil nucleotides started to increase after 1 week diet, with a maximum after 2 weeks. After 5 weeks, the concentrations returned to normal values. In heart, lungs, kidney and brain, concentrations were measured after 2 weeks diet in young rats. In contrast to liver, the heart muscle uracil nucleotide concentrations were decreased by 40%. In kidney, the sum of UTP, UDP and UMP showed a decrease of 40%, whereas UDP-sugars were increased 1.4-fold. In the lungs, nucleotide concentrations were mostly unaffected by vitamin B6-deficiency, despite a 70% increase of UDP-GA. In brain, UDP-Glc, UDP-Gal and the sum of CTP and CDP showed an increase of 30-50%. We became surprised that the increased UDP-sugar concentrations did not influence the structure of liver plasma membrane-N-glycans. Despite the 4 to 6-fold increase of UTP and UDP-sugars, no changes in the complexity or sialylation of these N-glycans could be detected. This study demonstrates that, especially in liver, pyridoxal phosphate is closely involved in the control of uracil nucleotides during a defined period of development. In contrast to in vitro experiments, in vivo N-glycan biosynthesis in liver is regulated by a more complex or higher mechanism than substrate concentrations.

DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues.

Intercellular adhesion molecule-3 (ICAM-3) binds to the alpha(L)beta(2) integrin and mediates the contact between T cells and antigen-presenting cells. It has been suggested that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a C-type lectin of macrophages and DCs, is an additional ligand of ICAM-3. So far, the glycan structure mediating the interaction of native ICAM-3 with DC-SIGN is undefined. Here, we demonstrate that native ICAM-3 from human peripheral leukocytes binds recombinant DC-SIGN, is recognized by monoclonal Lewis x antibodies, and specifically interacts with DC-SIGN on immature DCs. The presence of Lewis x residues on ICAM-3 was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Investigations on different peripheral blood cell populations revealed that only ICAM-3 from granulocytes bound DC-SIGN. Cotransfection studies demonstrated that fucosyltransferase (FUT) IX and, to a significantly lesser extent, FUT IV, but not FUTs III and VII, mediate the synthesis of Lewis x residues on ICAM-3. These findings indicate that FUT IX is the main FUT mediating the synthesis of Lewis x residues of ICAM-3 in cells of the myeloid lineage, and that these residues bind DC-SIGN. The results suggest that ICAM-3 assists in the interaction of granulocytes with DC-SIGN of DCs.

Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on epithelia, blood vessel endothelia, and leukocytes. A variety of physiological functions have been assigned to CEACAM1. It is involved in the formation of glands and blood vessels, in immune reactions, and in the regulation of tumor growth. As a homophilic and heterophilic adhesion receptor, it signals through different cellular pathways. The existence of special oligosaccharide structures such as Lewis x or sialyl-Lewis x glycans within this highly glycosylated protein has been postulated, but chemical proof is missing so far. Because such structures are known to be essential for different cell-cell recognition and adhesion processes, characterizing the CEACAM1 glycan structure is of pivotal importance in revealing the biological function of CEACAM1. We examine the terminal glycosylation pattern of CEACAM1 from human granulocytes, focusing on Lewis x epitopes. Lewis x-specific antibodies react with immunoaffinity-purified native CEACAM1. Antibody binding was completely abolished by treatment with fucosidase III, confirming a terminal alpha(1-3,4) fucose linkage to the N-acetylglucosamine of lactosamine residues, a key feature of Lewis epitopes. To verify these data, MALDI-TOF MS analysis after stepwise exoglycosidase digestion of the CEACAM1 N-glycan mixture was performed. A complex mixture of CEACAM1-bound oligosaccharides could be characterized with an unusually high amount of fucose. The sequential digestions clearly identified several different Lewis x glycan epitopes, which may modulate the cell adhesive functions of CEACAM1.