Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation.

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and ß-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.

Relative quantitation of multi-antennary N-glycan classes: combining PGC-LC-ESI-MS with exoglycosidase digestion.

In the search for N-glycan disease biomarkers current glycoanalytical methods may not be revealing a complete picture of precious samples, and we may be missing valuable structural information that fall outside analysis windows. We report a targeted strategy combining PGC-LC-ESI-MS with exoglycosidases to improve the relative quantitation of tri and tetra-antennary glycan classes.

FKRP-dependent glycosylation of fibronectin regulates muscle pathology in muscular dystrophy.

The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG's glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.

GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources.

GlycoSuiteDB is a relational database that curates information from the scientific literature on glyco-protein derived glycan structures, their biological sources, the references in which the glycan was described and the methods used to determine the glycan structure. To date, the database includes most published O:-linked oligosaccharides from the last 50 years and most N:-linked oligosaccharides that were published in the 1990s. For each structure, information is available concerning the glycan type, linkage and anomeric configuration, mass and composition. Detailed information is also provided on native and recombinant sources, including tissue and/or cell type, cell line, strain and disease state. Where known, the proteins to which the glycan structures are attached are reported, and cross-references to the SWISS-PROT/TrEMBL protein sequence databases are given if applicable. The GlycoSuiteDB annotations include literature references which are linked to PubMed, and detailed information on the methods used to determine each glycan structure are noted to help the user assess the quality of the structural assignment. GlycoSuiteDB has a user-friendly web interface which allows the researcher to query the database using mono-isotopic or average mass, monosaccharide composition, glycosylation linkages (e.g. N:- or O:-linked), reducing terminal sugar, attached protein, taxonomy, tissue or cell type and GlycoSuiteDB accession number. Advanced queries using combinations of these parameters are also possible. GlycoSuiteDB can be accessed on the web at http://www.glycosuite.com.

Molecular analysis of the phospholipids of Escherichia coli k12.

Phospholipids from Escherichia coli K12 were converted to 1,2-diacylglycerols with phospholipase C from Bacillus cereus. High-pressure liquid chromatography of 1,2-diacylglycerol p-methoxybenzoates on LiChrosorb RP-18 using 2-propanol/acetonitrile (35:65) as eluant permitted separation of 14 molecular species. The main combinations of fatty acids were 1-16:0-2-16:1, 1-16:0-2-cyclo-17:0 and 1-16:0-2-18:1. Positional isomers were not present. The 1,2-di-16:0 compound was present at a significant level (7-10 mol%). Proportions of molecular species varied between phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Phospholipid from the outer membrane of E. coli K12 contained a lower level of molecules with two unsaturated chains than was present in the cytoplasmic membrane. The method is sensitive, has good resolving power and employs readily available equipment.

What place for polyacrylamide in proteomics?

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to deliver high quality protein resolution and dynamic range for the proteomics researcher. To remain as the preferred method for protein separation and characterization, several key steps need to be implemented to ensure quality sample preparation and speed of analysis. Here, we describe the progress made towards establishing 2D-PAGE as the optimal separation tool for proteomics research.

Desialylated LDL uptake in human and mouse macrophages can be mediated by a lectin receptor.

We have compared the uptake of desialylated low density lipoprotein (LDL) with other modified forms of LDL in mouse peritoneal macrophages and PMA-activated human U937 monocytes. Neuraminidase-treated LDL (NT-LDL) caused significant cholesterol ester accumulation in both cell types, although the efficiency relative to loading with acetylated LDL (AcLDL) was markedly different, suggesting a very different complement of receptors in the cells. We therefore determined the effect of PMA-activation on lipoprotein receptor expression in U937 cells and found that while scavenger receptor concentration was elevated after PMA-activation, there was no significant change in the expression of the LDL receptor. Receptor specificity of NT-LDL uptake was examined by competition experiments using the degradation assay. This showed that 125I-labelled NT-LDL uptake in U937 cells could largely be accounted for by the persistent expression of the LDL receptor in these cells. In contrast, in mouse peritoneal macrophages where LDL receptor expression is very low, 125I-labelled NT-LDL degradation was also effectively competed by asialofetuin. Surprisingly, 125I-labelled NT-LDL degradation was also effectively competed by AcLDL. Measurement of sialic acid content of AcLDL showed that approximately 14% of the LDL sialic acid, equivalent to 2 to 3 residues per particle, was lost during acetylation of LDL with acetic anhydride. Thus competition between 125I-labelled NT-LDL and AcLDL could be due to lectin receptor binding rather than competition for scavenger receptor binding.

Protein phosphorylation: technologies for the identification of phosphoamino acids.

Protein phosphorylation plays a central role in many biological and biomedical phenomena. In this review, while a brief overview of the occurrence and function of protein phosphorylation is given, the primary focus is on studies related to the detection and analysis of phosphorylation both in vivo and in vitro. We focus on phosphorylation of serine, threonine and tyrosine, the most commonly phosphorylated amino acids in eukaryotes. Technologies such as radiolabelling, antibody recognition, chromatographic methods (HPLC, TLC), electrophoresis, Edman sequencing and mass spectrometry are reviewed. We consider the speed, simplicity and sensitivity of tools for detection and identification of protein phosphorylation, as well as quantitation and site characterisation. The limitations of currently available methods are summarised.

Identification and quantitation of cysteine in proteins separated by gel electrophoresis.

A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis of protein. The technique involves using 9-fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that recovers all of the amino acids (asparagine and glutamine are recovered in their acidic forms) except tryptophan. Cys adducts with acrylamide and iodoacetamide have been observed in hydrolysates of gel-separated proteins. To enable quantitation of Cys by amino acid analysis, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8.5, at 80 degrees C) and alkylation (0.25 M iodoacetamide, pH 8.5, at 37 degrees C) of proteins which have been separated by gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.

The relationship between the structures of the O polysaccharides from Escherichia coli O17 and O16.

The chemical structure of the O16 antigen from the lipopolysaccharide of Escherichia coli strain P4 has been determined. Comparison with the structures of other O16 antigens and that of the O17 antigen explains the previously reported cross-reaction of O antigen from the O16 strain K-12 with anti-O17 antibody [D. Liu and P.R. Reeves, Microbiology, 140 (1994) 49-57].

Characterization of human plasma glycoproteins separated by two-dimensional gel electrophoresis.

Purification of protein isoforms for the characterization of post-translational modifications, such as glycosylation, can be laborious and demanding. We report a means of determining monosaccharide composition and the identity of glycoproteins from a single spot on a two-dimensional (2-D) gel. The sensitivity of the method depends on the degree of glycosylation of the protein. We show that bovine fetuin can be analyzed and identified at the level of 100 pmol. 2-D reference maps enable quick identification of glycoprotein isoforms, and the nature of glycosylation differences. Human sera glycoforms were isolated by micropreparative 2-D PAGE using a narrow-range immobilized pH gradient. Single spots excised from one polyvinylidene difluoride blot of a 2-D gel were used sequentially for sialic acid analysis, neutral and amino sugar analysis, and finally amino acid analysis. The glycosylation variations in isoforms of human fetuin and alpha-1-antitrypsin were determined. The amino acid composition, in conjunction with protein pI and MW, successfully identified the glycoproteins.

Glycobiology and proteomics: is mass spectrometry the Holy Grail?

One characteristic of glycoproteins is that they are separated by two-dimensional electrophoresis (2-D PAGE) into typical 'trains' of protein spots which separate on the basis of different isoelectric point (pI) and/or molecular mass. The pattern of these trains often varies in development and disease. While the isoforms differ both in the number of sites of glycosylation and the types of carbohydrate attached to the protein, classical methods of glycan analysis are insensitive at the levels typically separated by 2-D PAGE. Developments in mass spectrometry technologies have enabled the characterization of most of the oligosaccharide attributes to be determined on picomole amounts of protein. These techniques are beginning to allow the glycoform heterogeneity on 2-D separated glycoproteins to be analyzed.

The elimination of O-linked glycans from glycoproteins under non-reducing conditions.

A simple procedure is described for the elimination of O-linked glycans from bovine submaxillary mucin under non-reducing conditions, using triethylamine in aqueous hydrazine. The glycans were isolated as the hydrazones, which were converted to the reducing glycans by exchange with acetone in neutral aqueous solution. The glycan alditols obtained after reduction corresponded to those obtained by the reductive beta-elimination of O-glycans.

GlycoMod--a software tool for determining glycosylation compositions from mass spectrometric data.

GlycoMod (http://www.expasy.ch/tools/glycomod/) is a software tool designed to find all possible compositions of a glycan structure from its experimentally determined mass. The program can be used to predict the composition of any glycoprotein-derived oligosaccharide comprised of either underivatised, methylated or acetylated monosaccharides, or with a derivatised reducing terminus. The composition of a glycan attached to a peptide can be computed if the sequence or mass of the peptide is known. In addition, if the protein is known and is contained in the SWISS-PROT or TrEMBL databases, the program will match the experimentally determined masses against all the predicted protease-produced peptides (including any post-translational modifications annotated in these databases) which have the potential to be glycosylated with either N- or O-linked oligosaccharides. Since many possible glycan compositions can be generated from the same mass, the program can apply compositional constraints to the output if the user supplies either known or suspected monosaccharide constituents. Furthermore, known oligosaccharide structural constraints on monosaccharide composition are also incorporated into the program to limit the output.

Configurations of glycosidic phosphates of lipopolysaccharide from Salmonella minnesota R595.

The anomeric configurations of the reducing terminal glucosamine and 4-amino-4-deoxy-L-arabinose phosphates in lipopolysaccharide from Salmonella minnesota R595 have been determined by nuclear magnetic resonance. Chemical shifts for the anomeric protons were obtained by selective decoupling of the phosphorus spectrum and proton-proton coupling constants by polarization transfer from protons to phosphorus. In both cases, the phosphate is attached to the sugar in an axial orientation.

Proteomic analysis of the genetic premature aging disease Hutchinson Gilford progeria syndrome reveals differential protein expression and glycosylation.

Proteomics has revealed differential protein expression and glycosylation in membrane proteins from premature aging Hutchinson-Gilford progeria syndrome fibroblasts (progeria). Progeria is a rare autosomal dominant genetic disorder of premature aging characterized by marked growth retardation and specific, progressive, premature senescent changes of the skin and other tissues. Affected children live to an average age of 13 years. The 1q20-24 region of chromosome 1 which codes for one of these proteins, lamin A/C, has previously been implicated by Brown et al. (1990) who described identical twins with progeria, where cytogenetic analysis showed an inverted insertion in the long arm of the chromosome in 70% of cells. Luengo et al. (2002) similarly reported an interstitial deletion of chromosome 1q23, in a 9-year-old patient with a classic clinical picture of progeria.

Characterization of O-linked glycosylation motifs in the glycopeptide domain of bovine kappa-casein.

kappa-Casein is the major glycoprotein in bovine milk. It has a proteinase-sensitive (chymosin) site which cleaves the glycoprotein into two segments: N-terminal para-kappa-casein domain and the C-terminal kappa-casein macroglycopeptide domain which is highly heterogeneous in oligosaccharide content. We have identified six sites of O-glycosylation on the macroglycopeptide by solid-phase Edman degradation: Thr121, Thr131, Thr133, Thr136 (A variant only), Thr142 and Thr165. No Ser residues are glycosylated. The glycosylation status of 15 of 17 potential O-glycosylation sites in the B variant was accurately predicted using the four peptide motifis previously proposed for the glycosylation of human glycophorin A (Pisano, A., Redmond, J.W., Williams, K.L. and Gooley, A.A., Glycobiology, 3, 429-435, 1993), provided one additional assumption is made concerning an inhibitory role for a nearby Ile.