Unusual N-type glycosylation of salivary prolactin-inducible protein (PIP): multiple LewisY epitopes generate highly-fucosylated glycan structures.

Prolactin-inducible protein (PIP) is a glycoprotein found in body secretions from exocrine glands like saliva and seminal plasma. Important biological functions of PIP concentrations have been demonstrated, e.g. in tumor diagnosis and progression. PIP quantity has been also found useful to determine the success of chemotherapy of mammary carcinoma. Here, we present the analysis of the N-glycosylation of PIP isolated from different sources by LC-MS(/MS) and 1H-NMR. We found a very uncommon N-type glycosylation of PIP in healthy individuals from both, seminal fluid and saliva. PIP carries unusual highly fucosylated N-linked glycans with multiple Lewisy (Ley) epitopes on bi-, tri- and tetraantennary structures resulting in up to nine fucosyl residues on a tetraantennary glycan. In most organs, Ley epitopes are not present on N-glycans except in case of a tumor when it is highly up-regulated and important for prognosis. Here, for the first time on a specific glycoprotein Ley antigens are unambiguously characterized on an N-type glycan by NMR spectroscopy. So far, for specific glycoproteins Ley epitopes had only been reported on O-glycans. Furthermore, a correlation between a nonsynonymous single nucleotide polymorphism (SNP) and glycosylation pattern was detected: individuals heterozygous for the SNP causing the amino acid exchange 51Gln to 51His have glycan structures with a higher degree of sialylation compared to individuals lacking the SNP.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Glycan analysis of Prostate Specific Antigen (PSA) directly from the intact glycoprotein by HR-ESI/TOF-MS.

Glycans are important modulators of the biological function of proteins and are normally characterized from proteolytic glycopeptides or from (N-)glycans released enzymatically by glycosidase treatment or chemically by hydrazinolysis. We demonstrate that glycan compositions can easily be determined directly by LC-ESI/TOF-MS from intact glycoproteins even with a very complex glycosylation pattern. Interpretation of isotopically resolved mass spectra of prostate specific antigen (PSA) using bioinformatics tools gives within a few hours the glycan compositions of 38 glycoforms.

Tumor-associated Neu5Ac-Tn and Neu5Gc-Tn antigens bind to C-type lectin CLEC10A (CD301, MGL).

In human tumors, glycoproteins often exhibit abnormal glycosylation patterns, e.g. certain Lewis structures, TF antigen, Tn antigen and/or their sialylated forms, creating additional binding sites for glycoreceptors. In the present study, we have analyzed the carbohydrate specificity of the C-type lectin CLEC10A using glycan profiling by enzyme-linked immunosorbent assay (ELISA). In addition to the known ligands, we show binding to two tumor-associated antigens, namely Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn, with an affinity of CLEC10A in the micromolar range. Detailed analyses of the glycan-lectin interactions were carried out by surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR. CLEC10A binds Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn with dissociation constants of 297 and 80 µM, respectively, as determined by SPR. Comparison of the STD nuclear magnetic resonance (NMR) binding epitopes of Tn and Neu5Acα2,6-Tn revealed a constant binding mode of the N-acetylgalactosamine moiety. This finding is in good agreement with binding studies of CLEC10A transfectomas, which show a well-defined interaction of transmembrane CLEC10A with 6-sialylated-Tn structures. Since both Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn together with the previously known Tn antigen are expressed in human tumors such as mammary carcinoma, the interaction with CLEC10A expressed by macrophages and dendritic cells could be of major functional significance in tumor progression.

Glycan analysis: scope and limitations of different techniques--a case for integrated use of LC-MS(/MS) and NMR techniques.

The structure of glycans from glycoproteins is highly relevant for their function. We tightly integrate liquid chromatography-mass spectrometry (LC-MS), MS/MS, and nuclear magnetic resonance (NMR) data to achieve a complete characterization of even isobaric glycans differing in only one linkage position or in the substitution in one branch. As example, we analyzed ten desialylated underivatized glycans from bovine fibrinogen. The molecules were separated on a PGC column, and LC-MS data allowed an assignment of the compositions of the glycans. MS/MS data of the same glycans allowed elucidation of sequence and to some extent of branching and linkage. All MS/MS fragmentation methods led to multiple dissociations, resulting in several cases in ambiguous data. The MS/MS data were interpreted both by scientists and automatically by software, and the differential results are compared. Additional data from a tight integration of LC-MS and NMR data resulted in a complete structural characterization of the glycans. The acquisition of simple 1D (1)H NMR data led--in combination with LC-MS and MS/MS data--to an unambiguous assignment of the isobaric glycans. Compounds that were not separated in the chromatography could easily be assigned structurally by applying the 3D cross-correlation (3DCC) technology to arrive at NMR spectra of the pure components-without actually separating them. By applying LC-MS, MS/MS, 1D (1)H NMR, and 3DCC together, one can assign glycan structures from glycoconjugates with high confidence affording only 200 pmol of glycan material.

Glycosylation assists binding of HIV protein gp120 to human CD4 receptor.

The role of glycosylation of proteins on its binding affinity is not well understood. Even a monosaccharide (magenta) placed at a glycosylation site can significantly enhance binding of peptides to their receptor. If glycosylated, an HIV protein binds stronger and faster to its primary receptors on human cells.

Resolving the problem of chromatographic overlap by 3D cross correlation (3DCC) processing of LC, MS and NMR data for characterization of complex glycan mixtures.

Chromatographic overlap is a common problem in the analysis of complex mixtures. As a result, it is not possible to identify the components because each resulting NMR or MS spectrum contains multiple components. We introduce three-dimensional cross correlation (3DCC) that dissects NMR spectra of a mixture into spectra of the individual components without actually separating them. Correlation of peaks from MS and NMR profiles along a common LC time domain yields 3DCC NMR spectra of pure components correlated with a mass and a retention time. The method requires an LC run followed by fractionation and recording of MS and NMR spectra. The method is applicable to mixtures of any classes of molecules. Here, we demonstrate its application to a mixture of complex glycans obtained from a glycoprotein. Fourteen glycans eluting within only 3 min showed heavy overlap in the chromatographic run. 3DCC allowed their direct characterization without separation. Some of these structures from the glycoprotein bovine fibrinogen had not previously been described. The 3DCC procedure has been implemented in standard software. Actually, 3DCC can be used for any combination of separation techniques, like LC or GC, combined with two characterization methods like UV, IR, Raman, NMR or MS.

Biotransformation of Dimethenamid-P by the basidiomycete Irpex consors.

Twenty-nine basidiomycetes were screened in surface and liquid cultures for their capability to biotransform the chloroacetamide herbicide Dimethenamid-P (DMTA-P). The basidiomycete Irpex consors converted 70% of the herbicide (0.5 g L-1 DMTA-P) in liquid cultures within 6 days, applying a minimal medium under non-ligninolytic conditions. Nine transformation products of DMTA-P were identified by liquid chromatography-mass spectrometry analysis of the culture supernatants. The four main metabolites were isolated and subjected to GC-MS analysis and NMR spectroscopy. The analyses revealed that the thiophene ring was oxidized at three different positions. Metabolite M1 was identified as the S-oxide, which was isolable and relatively stable at room temperature. In metabolite M2, one methyl substituent of the thiophene ring was hydroxylated. The two metabolites M3A and M3B were diastereomers, but fully separated by HPLC. Here, oxidation of the aromatic CH carbon resulted in prototropic rearrangement to an αß-unsaturated thiolactone. None of the three major metabolites of DMTA-P has been described before.

Elapid snake venom analyses show the specificity of the peptide composition at the level of genera Naja and Notechis.

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms-that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes.

Formation of the immunogenic α1,3-fucose epitope: elucidation of substrate specificity and of enzyme mechanism of core fucosyltransferase A.

Glycans of glycoproteins are often associated with IgE mediated allergic immune responses. Hymenoptera venoms, e.g., carry α1,3-fucosyl residues linked to the proximal GlcNAc of glycoproteins. This epitope, formed selectively by α1,3-fucosyltransferase (FucTA), is xenobiotic and as such highly immunogenic and it also shows cross-reactivity if present on different proteins. Production of post-translationally modified proteins in insect cells is however commonly used and, thus, resulting glycoproteins can carry this highly immunogenic epitope with potentially significant side effects on mammals. To analyze mechanism, specificity and reaction kinetics of the key enzyme, we chose FucTA from Apis mellifera (honeybee) and characterized it by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR) experiments. Specifically, we show here that the donor substrate, GDP-Fucose, binds mostly via its guanine and less so via pyrophosphate and fucosyl fragments and has a K(D) = 37 µM. Affinity and kinetic studies with both the core α1,6-fucosylated and the unfucosylated octa- or heptasaccharides, respectively, as acceptor substrate revealed that honeybee FucTA prefers the latter structure with affinities of K(D) âˆ¼ 10 mM. Establishment of progress curve analysis using an explicit solution of the integrated Michaelis-Menten equation allowed for determination of key constants of the transfer reaction of the glycosyl residue. The dominant minimum acceptor substrate is an unfucosylated heptasaccharide with K(m) = 420 µM and k(cat) = 6 min(-1). Time-resolved NMR spectra as well as STD NMR allow molecular insights into specificity, activity and interaction of the enzyme with substrates and acceptors.