R. Reid Townsend

Physicochemical characterization of recombinant human nerve growth factor produced in insect cells with a baculovirus vector.

Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion‐exchange and reversed‐phase chromatography to near homogeneity. The Nterminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (<0.08 mol of N‐acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF frominsect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimers disease.

Chapter 5 Analysis of Glycoconjugates Using High-pH Anion-Exchange Chromatography

Publisher Summary This chapter discusses the analysis of monosaccharide and oligosaccharide of glycoproteins, glycolipids, proteoglycans, and glycosylphosphoinositol (GPI) anchors using high-pH anion-exchange chromatography (HPAEC). HPAEC with pulsed amperometric detection (PAD) does not require the derivatization and associated sample preparation steps that are needed for gas chromatography and other high-performance liquid chromatographic (HPLC) approaches. This relative simplicity of HPAEC with PAD for the composition analysis of glycoconjugates has made this fundamental analysis more generally accessible. The analysis of monosaccharides commonly found in the acid hydrolyzates of glycoproteins using isocratic elution (16 mM NaOH) on a Dionex AS-6 ion exchange column is reported in this chapter. Because PAD detects compounds other than carbohydrates, interfering and extraneous peaks can be observed. Quantitative monosaccharide composition is often the initial analysis toward the structural elucidation of glycoprotein glycans. An accurate molar ratio of covalently linked sugars relative to protein (1) provides direct proof that the polypeptide is glycosylated, (2) provides the global basis for strategies for identifying key structural features, (3) suggests classes of oligosaccharide chains, (4) can indicate changes in biosynthetic pathways, and (5) can be used as a quality control measure for therapeutic glycoproteins and glycoconjugates.

Higher levels of cystatin C are associated with worse cognitive function in older adults with chronic kidney disease: the chronic renal insufficiency cohort cognitive study.

To determine the association between cognition and levels of cystatin C in persons with chronic kidney disease (CKD).

Separation of high-mannose isomers from yeast and mammalian sources using high-pH anion-exchange chromatography

The biosynthesis of oligosaccharides N-linked at AsnXxxThr(Ser) sequons of glycoproteins is initiated by the co-translational transfer of a Glc,Man,GlcNAc, from dolichyl pyrophosphate’. The three glucose residues are rapidly removed by two glucosidases, one a( I+ 2)and the other a( 1 +3)-specific’. These first steps are identical in yeast and in man, and likely to be the same in plants’? however, the subsequent removal and addition of the peripheral mannoses are very different. The array of oligo-mannosyl structures which eventually occupy each glycosylation site are a function, in part, of the concerted action of mannosidases and mannosyl transferases on the archetypal Man,GlcNAc, structure’. These enzymes often display specificity for not only the type of residue and anomeric linkage, but also for its branch location. For example, a yeast mannosidase removes a single (l-+2)-x-linked mannose from a specific branch of the Man,GlcNAcz oligosaccharide4,‘. A yeast (1+6)-a-mannosyl transferase places a single mannose on one specific branch which is essential for elongation reaction?. Trimming by at least three mammalian mannosidases accounts for the heterogeneity of high-mannose structures at individual glycosylation sites (discussed in ref. 7). Thus, chromatographic methods which can separate not only by size and ring substitutions (linkage), but also according to branch isomerism, are required to understand the structural glycobiology of iv-linked oligosaccharide biosynthesis. High-pH anion-exchange chromatography (h.p.a.e.c.) has been shown to separate many oligosaccharide isomers (both neutral and charged) which differ only in a single linkagex~‘4~‘7). H owever, certain isomers were difficult to separate using

High-energy collision-induced dissociation mass spectrometry of synthetic mannose-6-phosphate oligosaccharides

The high-energy collision-induced dissociation spectra of a series of linear and branched synthetic mannosyl oligosaccharides that contain 6-phosphate substituents on either or both non-reducing terminal or penultimate residues have been studied. These phosphorylated structures were designed to mimic those of naturally derived N-glycans (Man-6-PO4) on lysosomal enzymes and to probe the minimally required binding motif for the Man-6-PO4 receptors. When a phosphate group was present, the spectra were dominated by ions that arise from cleavages at the glycosidic bonds (single and double) with charge retention on the phosphate-containing fragments. The spectra of linear structures that bear the nonreducing terminal Man-6-phosphate residues were devoid of Y-type ions, unlike those with similar phosphorylation at the penultimate residue. The location of the phosphorylated residue was deduced from the presence or absence of unique B and Y ions. In neutral branched structures, the ions were formed by cleavage at the glycosidic bond at either one or both of the branch points and the aglycon, which was attached to the disubstituted mannosyl residue. Branched oligosaccharides that contained one or two terminal Man-6-PO4 residues also showed double cleavages with charge retention on the phosphate-containing fragment. Our investigation shows that positive mode high energy collision-induced dissociation mass spectrometry can determine the location—terminal or penultimate—of Man-6-PO4 residues in N-linked type oligosaccharides.

Topology of membrane proteins in native membranes using matrix-assisted laser desorption ionization/mass spectrometry

Publisher Summary Information on topology is important for understanding the structural basis underlying the translocation function of cation pumps like the Na,K-ATPase, Ca-ATPase or H,K-ATPase. Previous efforts to define topology have used approaches like hydropathy plots, proteolysis of vesicles, binding of regio-specific antibodies, or labeling with group-specific, membrane-sided reagents followed by identifying the modified sites. Proteolysis of sided vesicles followed by analysis of peptide products has been one of the most common approaches to determine exposed peptide sequences. Conversely, remaining membrane-associated peptides can be analyzed after exhaustive protease digestion. In the study in this chapter, matrix-assisted laser desorption ionization/mass spectrometry (MALDI/MS) is used to identify the peptides released from gastric parietal cell microsomes. MALDI, because of its sensitivity and relative tolerance to the presence of salts and buffers is examined for the analysis of unfractionated proteolytic digests. MALDI with postsource decay (PSD) analysis has been used to obtain sequence information on peptides even in crude digestion mixtures. The strategy in this chapter consists of proteolysis of intact vesicles, centrifugation at high speeds to separate membrane bound and soluble fractions and analysis of the mixture of released peptides by MALDI/MS. In addition, to increase the sensitivity and breadth of analysis, supernatant peptides are separated by reverse-phase high-pressure liquid chromatography (HPLC) and individual fractions are analyzed by MALDI/MS. PSD-analysis is also performed to obtain partial sequence information and identify peptides. On the basis of the released peptide products, a topological map for a major portion of the H,K-ATPase in gastric parietal cell tubulovesicles is proposed. The chapter focuses on the gastric H,K-ATPase as a test protein because purified gastric microsomal vesicles are well-enriched in the enzyme, the vesicles are oriented with a common asymmetry—that is, cytoplasmic side out, the vesicles are sealed allowing selective cytoplasmic digestion, and there is a pool of existing topological data from other methods.

Monosaccharide and Oligosaccharide Analysis of Recombinant Erythropoietin Electro-transferred onto Polyvinylidene Fluoride Membranes

Publisher Summary This chapter presents monosaccharide and oligosaccharide analysis of recombinant eiythropoietin electro-transferred onto polyvinylidene fluoride membranes. In a study described in the chapter, glass-distilled deionized water was used for the preparation of all buffers, eluents, and electrophoresis solutions. The storage flask and spigot were glass throughout to prevent contact with tubings that might support microbial growth and yield artifactual sugars during monosaccharide analysis. SDS-PAGE (12.5%) was performed using mini-gels run in a Tall Mighty Small unit at constant amperage (20 mA) using an LKB Multidrive 3.5 kV power supply. The kinetics of monosaccharide release from soluble rEPO and rEPO electro-transferred onto PVDF was compared. The time-dependent release of neutral, amino, and the 6-deoxy sugars (Fuc) from rEPO in solution (1A) was similar to that observed for electro-transferred rEPO (1B). The percent recovery of monosaccharides upon comparing electrotransferred and soluble rEPO hydrolysates was Gal, 80%; GlcN, 91%; Man, 70%; Fuc, 83%; and GalN, 89%.

S9.15 Structural elucidation ofO-Glycosylation on Glycoproteins

Electrospray mass spectrometry (ES-MS) has recently emerged as a new powerful technique for analysis of high molecular weight biopolymers (>100.000 Dal) at a subnanomolar level. The multiple charged molecules can be structurally attributed according to their state of charge. We used ES-MS for structural studies on glycopeptides. A superiority over well established FAB-MS methods was found in higher sensitivity as well as in ability to induce specific fragmentation of the carbohydrate portion. This allowed assignment of carbohydrate sequence and the binding site to the peptide portion. Using this approach, it is possible to avoid chemical derivatizations, which may influence alkali-labile groups, found frequently on carbohydrate chains. The positive and negative ion ES-MS was applied to analyse sialic acid containing O-glycopeptides from urine of patients suffering from inherited deficiency of lysosomal a-N-acetylgalactosaminidase. Beside the major species, Neu5Aca2-3Gal/31-(NeuAca2-6)3GalNAc-Ser(Thr), a number of mono-, diand trisialylglycans were structurally characterized in fractions, obtained after several chromatographic steps, including gel permeation and anion exchange chromatography. After appropriate Nand/or O-derivatizations, the samples were also analysed by FAB-MS for sequencing of sugar chains and by methylation analysis for linkage sites.