Mass spectrometric approach for screening modifications of total serum N-glycome in human diseases: application to cirrhosis

Congenital and acquired modifications of glycosylation in diseases are a rapidly growing field that demonstrates the importance of glycosylation in human biology. Unfortunately, in clinical biochemistry, very few tests are available to explore oligosaccharide metabolism on a large scale. Such an assay needs to be of high throughput, rapid, and preferentially noninvasive. In the present study, we describe a method to analyze qualitative variations of N-glycosylation of human serum proteins. The method is based on direct release of N-linked oligosaccharides from patient serum samples, a single-step purification, and a matrix-assisted laser desorption ionization time of flight mass spectrometric analysis. A complementary structural study of the released oligosaccharides was achieved by enzymatic digestions, linkage analysis, and electrospray ionization ion trap mass spectrometry (ESI-IT-MS) of the permethylated N-glycome. A total of 26 oligosaccharide structures were individualized, their presence in human serum being the result of the combination of the biosynthesis and catabolic pathways. Application of the protocol to the serum of patients with cirrhosis demonstrates the ability of this assay to identify acquired modifications of glycosylation. Furthermore, we have analyzed the N-glycans and showed the increase in bisecting N-acetylglucosamine residue, core fucosylation, and the presence of an important population of neutral oligosaccharides. The study of total serum N-glycome modifications is a preliminary for the discovery of new noninvasive diagnostic or prognostic biomarkers resulting from the variations of the N-glycan metabolism during diseases.     

The sperm agglutination antigen-1 (SAGA-1) glycoforms of CD52 are O-glycosylated

CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Le(y) epitopes (NeuAc(0-1)Fuc(1-3)Hex(1-2) HexNAc(0-1)HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.     

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.     

Structural Analysis of N-Glycans of Storage Glycoproteins in Soybean (Glycine max. L) Seed

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz ¹H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man₃Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 3.4%).     

Site-specific N-glycan characterization of human complement factor H

Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). A 17.9-kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H (Endo H) deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. MS analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 and 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified. Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein.     

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.     

Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Le(x) in mice

The current interest in applying systems biology approaches to studying an organism's form or function promises to reveal further insights into the role of glycosylation in cells and whole organisms. This has prompted the development of a rapid, sensitive method of profiling the glycan component of both glycosphingolipids and glycoproteins from a single sample. Here we report a new mass spectrometric screening strategy for characterizing glycosphingolipid-derived oligosaccharides, which can be integrated into an existing highly sensitive glycoprotein glycomics strategy. Using ceramide glycanase to release the glycans from glycosphingolipids, this method provides a reliable profile of the glycosphingolipid-derived glycans present in a sample and has revealed new glycan structures. Glycoproteins are also efficiently recovered using this method, allowing the subsequent analysis of glycoprotein-derived glycans by mass spectrometry. The high sensitivity of this glycomic screening method allowed us to directly characterize the sialyl Le(x) epitope from mouse brain for the first time, where it was observed on an O-mannose structure. Thus, we present a mass spectrometric method that allows glycomic screening of N- and O-glycans as well as glycosphingolipid-derived glycans from a single tissue.     

Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap--Fourier transform mass spectrometry

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MS(n)) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.     

Glycosylation of sputum mucins is altered in cystic fibrosis patients

Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after sampling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.     

Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian [beta]-1,4-galactosyltransferase and [beta]-1,2-N-acetylglucosaminyltransferase II

A novel recombinant baculovirus expression vector was used to produce His-tagged human transferrin in a transformed insect cell line (Tn5beta4GalT) that constitutively expresses a mammalian beta-1,4-galactosyltransferase. This virus encoded the His-tagged human transferrin protein in conventional fashion under the control of the very late polyhedrin promoter. In addition, to enhance the synthesis of galactosylated biantennary N-glycans, this virus encoded human beta-1,2- N-acetylglucosaminyltransferase II under the control of an immediate-early (ie1) promoter. Detailed analyses by MALDI-TOF MS, exoglycosidase digestion, and two-dimensional HPLC revealed that the N-glycans on the purified recombinant human transferrin produced by this virus-host system included four different fully galactosylated, biantennary, complex-type glycans. Thus, this study describes a novel baculovirus-host system, which can be used to produce a recombinant glycoprotein with fully galactosylated, biantennary N-glycans.     

Identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms. The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries two N-glycans and four O-glycans. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples. Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), β1-4 Galactosidase, and β-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans. Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3,6 Galactosidase, and β1-3 Galactosidase), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans. SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 μg), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.     

Purification and structural characterization of de-N-acetylated form of GD3 ganglioside present in human melanoma tumors

The presence of gangliosides containing de-N-acetylated sialic acids in human tissues has been so far shown by using mouse monoclonal antibodies specific for the de-N-acetylated forms, but the isolation and chemical characterization of such compounds have not yet been performed. Since indirect evidence suggested that de-N-acetylGD3 ganglioside could be present in human melanoma tumors, we analyzed the gangliosides purified from a 500-g pool of those tumors. The de-N-acetylGD3 that was found to migrate just below GD2 in thin-layer chromatography was isolated from the disialogangliosides by high-pressure liquid chromatography using the specific antibody SGR37 to monitor the elution. The amount of antigen was found to be 320 ng per gram of fresh tumor or 0.1% of total gangliosides. Gas chromatography-mass spectrometry analysis of the antibody-positive ganglioside showed that sialic acids were formed of one molecule of N-acetylneuraminic acid and one molecule of neuraminic acid. Radioactive re-N-acetylation of the antigen yielded a GD3-like ganglioside with the radioactive label on the external sialic acid. The constitutive fatty acids were found to differ markedly from those of GD3 and 9-O-acetylGD3 isolated from the same pool of tumors. The major fatty acids were C16:0 and C18:0 in de-N-acetylGD3, whereas GD3 and its 9-O-acetylated derivative contained a large amount of C24:1. These data show that de-N-acetylGD3 ganglioside is indeed present in human melanoma tumors, and the fatty acid content suggests the existence of a de-N-acetylase mostly active on the molecular species of gangliosides with short-chain fatty acids.     

N-linked glycans of the human insulin receptor and their distribution over the crystal structure

The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility.     

Identification and characterization of glycosylation sites in human serum clusterin.

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.     

Combinations of SPR and MS for characterization of native and recombinant proteins in cell lysates

Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni²⁺ for SPR analysis of the specific Ni²⁺ binding response and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein and of cauliflower were investigated for specific binding to the phosphopeptide ligands immobilized on a sensorchip by SPR. Subsequently, the bound protein was eluted and analyzed by MS for characterization of intact mass and peptide mass mapping.     

Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies

Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MSⁿ experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified.     

Structural characterization of the N-glycan moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax quadricarinatus

Glycosylation is of importance for the structure and function of proteins. In the case of vitellin (Vt), a ubiquitous protein accumulated into granules as the main yolk protein constituent of oocytes during oogenesis, glycosylation could be of importantance for the folding, processing and transport of the protein to the yolk and also provides a source of carbohydrate during embryogenesis. Vt from the crayfish Cherax quadricarinatus is synthesized as a precursor protein, vitellogenin (Vg), in the hepatopancreas, transferred to the hemolymph, and mobilized into the growing oocyte via receptor-mediated endocytosis. The gene sequence of C. quadricarinatus shows a 2584-amino-acid protein with 10 putative glycosylation sites. In this study a combined approach of lectin immunoblotting, in-gel deglycosylation, and mass spectrometry was used to identify the glycosylation sites and probe the structure of the glycan moieties using C. quadricarinatus Vg as a model system. Three of the consensus sites for N-glycosylation-namely, Asn(152), Asn(160) and Asn(2493)-were glycosylated with the high-mannose glycans, Man(5-9)GlcNAc(2), and the glucose-capped oligosaccharide Glc(1)Man(9)GlcNAc(2).     

Expression and characterization of biologically active human Fas ligand produced in CHO cells

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh-FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 μg/L × 10⁶ cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays. S. Zappitelli and L. D’Alatri contributed equally to this work.     

Efficient expression, purification, and characterization of C-terminally tagged, recombinant human asparagine synthetase

Several lines of evidence suggest that up-regulation of asparagine synthetase (AS) in human T-cells results in metabolic changes that underpin the appearance of asparaginase-resistant forms of acute lymphoblastic leukemia (ALL). Inhibitors of human AS therefore have potential as agents for treating leukemia and tools for investigating the cellular basis of AS expression and drug-resistance. A critical problem in developing and characterizing potent inhibitors has been a lack of routine access to sufficient quantities of purified, reproducibly active human AS. We now report an efficient protocol for preparing multi-milligram quantities of C-terminally tagged, wild type human AS in a baculovirus-based expression system. The recombinant enzyme is correctly processed and exhibits high catalytic activity. Not only do these studies offer the possibility for investigating the kinetic behavior of biochemically interesting mammalian AS mutants, but such ready access to large amounts of enzyme also represents a major step in the development and characterization of inhibitors that might have clinical utility in treating asparaginase-resistant ALL.     

Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry.

A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol.