Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.     

Carbohydrate structure of the concanavalin A molecular variants of alpha-fetoprotein

The structure of the carbohydrate units of alpha-fetoprotein from fetal calf serum has been studied. Glycopeptides and oligosaccharides were prepared from alpha-fetoprotein by protease treatment and hydrazinolysis, respectively, and subjected to carbohydrate and amino acid analysis. Two N-glycosidic glycans are present in each alpha-fetoprotein molecule. These were separated into concanavalin A (ConA)-reactive and nonreactive species on ConA-Sepharose. Methylation analysis, Smith degradation, sequential exoglycosidase treatments, and sizing suggested that the major, ConA-nonreactive fraction is composed of triantennary and the minor, ConA-reactive fraction, of biantennary complex-type ConA-reactive and -nonreactive fractions of intact alpha-fetoprotein, respectively, and refractionated on Con A-Sepharose. The results indicate that 75% of alpha-fetoprotein molecules contain two triantennary complex-type glycans, 20% contain one triantennary and one biantennary glycan, and 5% contain two biantennary glycans. The last two molecular variants are bound to ConA. These results explain, at least in part, the previously found heterogeneity of alpha-fetoprotein with respect to charge and molecular size, and provide a biochemical basis for the differing reactivities toward ConA of alpha-fetoprotein from the yolk sac, fetal liver, and various tumors.     

Purification and characterization of novel kininogens from spotted wolffish and Atlantic cod.

Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish(Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDI-TOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kininogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried alpha2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units.     

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.     

Characterization of the oligosaccharide component of microsomal beta-glucuronidase from rat liver

The oligosaccharides of microsomal beta-glucuronidase were analysed by gel permeation and weak anion exchange chromatography following hydrazine release. N-linked glycans, constituted 80% of the total glycan pool and were mainly of the tri- and biantennary complex type with or without core and arm fucose. The major oligosaccharide, that comprised 30.6% of all the species analysed, was structurally identified by reagent array analysis method and found to be a triantennary complex structure, Galbeta1,4GlcNAcbeta1,2Manalpha1,6(3)(Galbeta1,4GlcNAcbeta1,4(Galbeta1,4GlcNAcbeta1,2) Manalpha1,3(6))Manbeta1,4GlcNAcbeta1,4 GlcNAc. O-Linked glycans comprised 20% of the total glycan pool, the major species being Galbeta1,3GalNAc. All of the N- and O-linked glycans were charged. Most of the negative charge was due to sialic acid (85.0%) with the remainder being phosphate present as phosphomonoesters (7.3%) and phosphodiesters (5%). This is the first report of O-linked carbohydrate chains in microsomal beta-glucuronidase. The presence of O-linked glycans and branched N-linked glycans in a microsomal enzyme, in relation to the current view of glycosyltransferase compartmentalization in the Golgi is discussed.     

The major oligosaccharides in the large subunit of the hemagglutinin from fowl plague virus, strain Dutch. Structure elucidation by one-dimensional and two-dimensional 1H nuclear magnetic resonance and by methylation analysis

The N-glycosidically linked glycans in the large subunit (HA1) of the hemagglutinin from fowl plague virus, strain Dutch (containing about 15%, w/w, of carbohydrates), were liberated by alkaline hydrolysis, and were filtrated through Bio-Gel as the re-N-acetylated oligosaccharide alditols. One major fraction (90%, mol/mol) was obtained. It was subfractionated by concanavalin A affinity chromatography and was analyzed by methylation/capillary gas chromatography/mass fragmentography and especially by one-dimensional and two-dimensional 1H nuclear magnetic resonance. The major HA1 glycans, which are not sialylated, were thus found to comprise about 40%, 30% and 20% (mol/mol), respectively, of biantennary intersected, biantennary, and triantennary N-acetyllactosaminic ('complex') oligosaccharides. About two thirds of the internal GlcNAc residues in these glycans are substituted by Fuc(alpha 1----6), all the triantennary species carry the third Gal(beta 1----4)GlcNAc(beta 1----unit at the Man(alpha 1----6)-branch, and roughly one fourth of the N-acetyllactosamine units in the non-intersected biantennary oligosaccharides are incomplete.     

The covalent structure of individual N-linked glycopeptides from ovomucoid and asialofetuin

In order to explore whether individual N-linked glycans in a given glycoprotein may be processed to different end products and at the same time prepare a number of well characterized glycopeptides as substrates for glycopeptide hydrolases, we have prepared the individual glycopeptides representing the four major glycosylation sites in ovomucoid and the three sites in asialofetuin. The individual glycopeptides were characterized by amino acid sequence determination before and after removal of the glycan by peptide:N-glycanase (amidase), and the liberated glycans were subjected to mass spectrometric analysis. As expected from available sugar analyses of the individual glycans in ovomucoid, no major differences were detected between the four glycosylation sites in this glycoprotein, but a definite trend toward less processed (less extensively branched) species was observed in going from site 1 to 4. In fetuin, for which the glycan pool is known to be made up of about two-thirds triantennary and one-third biantennary structures, the analysis of the three glycopeptides gave triantennary to biantennary ratios of 75/25, 67/33, and 70/30, respectively, demonstrating that the three sites are processed to a very similar, albeit perhaps not identical, extent. All the glycopeptides obtained in these studies, including the CNBr-produced glycopeptide from ovalbumin, were purified by a set series of steps, gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DE52 and/or reverse phase high performance liquid chromatography. Based on the results, these procedures appear to have general application for the preparation of glycopeptides.     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells.

Follistatin (FS), a glycoprotein, plays an important role in cell growth and differentiation through the neutralization of the biological activities of activins. In this study, we analyzed the glycosylation of recombinant human FS (rhFS) produced in Chinese hamster ovary cells. The results of SDS-PAGE and MALDI-TOF MS revealed the presence of both non-glycosylated and glycosylated forms. FS contains two potential N-glycosylation sites, Asn95 and Asn259. Using mass spectrometric peptide/glycopeptide mapping and precursor-ion scanning, we found that both N-glycosylation sites were partially glycosylated. Monosaccharide composition analyses suggested the linkages of fucosylated bi- and triantennary complex-type oligosaccharides on rhFS. This finding was supported by mass spectrometric oligosaccharide profiling, in which the m/z values and elution times of some of the oligosaccharides from rhFS were in good agreement with those of standard oligosaccharides. Site-specific glycosylation was deduced on the basis of the mass spectra of the glycopeptides. It was suggested that biantennary oligosaccharides are major oligosaccharides located at both Asn95 and Asn259, whereas the triantennary structures are present mainly at Asn95.     

Isoform composition of antithrombin in a covalent antithrombin-heparin complex

Antithrombin (AT) circulates in two isoforms, alpha- (90-95%) and beta-AT (5-10%). AT inhibits clotting factors such as thrombin and factor Xa, a reaction catalyzed by heparin. Heparin has been used in many clinical situations but suffers from limitations such as a short intravenous half-life, bleeding risk, and the inability to inhibit thrombin bound to fibrin clots. In order to overcome some of heparin's limitations, we prepared a covalent AT-heparin complex (ATH) that has increased intravenous half-life, reduced bleeding risk, and can directly inhibit clot-bound thrombin. However, structural analysis is required to further develop this promising antithrombotic agent. It was found that the proportion of isoforms in ATH (55% alpha-AT, and 45% beta-AT) was significantly different than that in the commercial AT starting material (80% alpha-AT and 20% beta-AT). Further analysis of the rate of heparin-catalyzed inhibition of thrombin by AT isoforms prepared from ATH revealed that the beta-variant reacted approximately 2-fold faster.     

Diverse lectin-binding specificity of four ZP3 glycoprotein isoforms with a discrete isoelectric point in chicken egg coat

The vertebrate egg coat corresponding to mammalian zona pellucida is a filamentous matrix composed of highly and heterogeneously glycosylated proteins designated ZP glycoproteins including ZP1 to 4, ZPD and ZPAX, and play important roles in species-specific egg-sperm interactions. Recent advance in structural biology of chicken ZP3 provided new insights into molecular mechanisms of the egg-coat function involving its carbohydrate moieties. In this study, chicken ZP3 was separated into four major and distinct isoforms with different pI in 2D-PAGE. To investigate the meanings of the ZP3 heterogeneity in egg-sperm interactions, we preliminary analyzed glycan diversity on the molecules by using lectin-staining assays. The four major ZP3 isoforms 4-7 (from acidic to basic) were recognized equally with PNA (Galβ1-3GalNAc), but the isoforms 5-7 were recognized dominantly with WGA ((β-GlcNAc)n, clustered Sia), PHA-E (bi- and triantennary N-glycan containing Galβ1-4GlcNAcβ1-2Manα1-6) and RCA I (terminal Galβ1-4GlcNAc), respectively. Despite such sugar chain diversity among the ZP3 isoforms, a partner in the egg coat, ZP1, showed specific binding to each isoform equally. Localization of ZP1 and ZP3 in the egg-coat matrix were also analyzed.     

Post-translational modifications of human thrombin-activatable fibrinolysis inhibitor (TAFI): evidence for a large shift in the isoelectric point and reduced solubility upon activation

Thrombin-activable fibrinolysis inhibitor (TAFI) is distinct from pancreatic procarboxypeptidase B in several ways. The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes. During this study, we observed that (i) the isoelectric point (pI) of TAFI shifts dramatically from pH 5 toward pH 8 upon activation and (ii) TAFIa is significantly less soluble than TAFI. The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity. The analyses revealed that all five potential N-glycosylation sites were utilized including Asn22, Asn51, Asn63, Asn86 (located in the activation peptide), and Asn219 (located in the catalytic domain). Asn219 was also found in an unglycosylated variant. Four of the glycans, Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous. In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver. Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation. TAFI contains eight cysteine residues, of which two, Cys69 and Cys383, are not involved in disulfides and contain free sulfhydryl groups. The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257. This pattern is homologous to pancreatic procarboxypeptidase B, and it is therefore unlikely that permutations in the cysteine connectivity are responsible for the enzymatic instability. LC-MS/MS analyses covering more than 90% of the TAFI amino acid sequence revealed no additional modifications. When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications. However, after activation, TAFIa loses 80% of the attached glycans, generating a large shift in pI and a propensity to precipitate. These changes are likely to significantly affect the properties of TAFIa as compared to TAFI.     

Bi- and tri-antennary human transferrin glycopeptides and their affinities for the hepatic lectin specific for asialo-glycoproteins

Glycopeptides were isolated from a proteolytic digest of human transferrin. After mild acid hydrolysis the desialylated glycopeptides were labelled by the galactose oxidase/NaB(3)H(4) procedure and then fractionated by Sephadex-gel filtration or by anion-exchange chromatography. Either technique allowed separation of the two heterosaccharide chains (designated glycan I and glycan II) previously described for this protein by Spik, Vandersyppe, Fournet, Bayard, Charet, Bouquelet, Strecker & Montreuil (1974) (in Actes du Colloque Internationale No. 221 vol. 1, pp. 483-499). Subsequent chromatography on Sepharose-concanavalin A separated fractions containing different quantities of carbohydrates for each glycan, as indicated by analyses. The isolated glycan fractions were then tested for their abilities to bind to the immobilized rabbit hepatic lectin. Our studies suggest that either glycan can have a bi- or tri-antennary structure. Desialylated biantennary glycans I and II did not bind to the hepatic lectin. Desialylated triantennary glycan I was slightly retarded by the hepatic lectin, whereas the triantennary glycan II consisted of equal quantities of a retarded and a bound type. Desialylated triantennary glycan II was totally displaced from the hepatic lectin by using a buffer containing 0.05m-EDTA. The results suggest that greater structural heterogeneity exists in the carbohydrate moiety of human transferrin than was previously envisaged. Such heterogeneity could be reflected in several molecular forms of human transferrin, which, after desialylation, differ significantly in their affinities for the hepatic lectin.     

Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry

Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc.     

Glycopeptide profiling of beta-2-glycoprotein I by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome

β2-glycoprotein I (β2GPI) is a five-domain protein associated with the antiphospholipid syndrome (APS), however, its normal biological function is yet to be defined. β2GPI is N-glycosylated at several asparagine residues and the glycan moiety conjugated to residue 143 has been proposed to interact with the Gly40–Arg43 motif of β2GPI. The Gly40–Arg43 motif has also been proposed to serve as the epitope for the anti-β2GPI autoantibody associated with APS. We hypothesized that the structure or composition of the glycan at Asn-143 might be associated with the APS symptom by shielding or exposing the Gly40–Arg43 motif towards the anti-β2GPI autoantibody. To test this hypothesis we used mass spectrometry (MS) for comparative glycopeptide profiling of human β2GPI obtained from blood serum from four healthy test subjects and six APS patients. It revealed significant differences in the extent of sialylation and branching of glycans at Asn-143. Biantennary glycans were more abundant than triantennary glycans at Asn-143 in both healthy subjects and patients. In APS patient samples we observed a decrease in sialylated triantennary glycans and an increase in sialylated biantennary glycan structures, as compared to controls. These data indicate that some APS patients have β2GPI molecules with a reduced number of negatively charged sialic acid units in the glycan structure at Asn-143. This alteration of the electrostatic properties of the glycan moiety may attenuate the intramolecular interactions with the positively charged Gly40–Arg43 motif of β2GPI and, in turn, leads to conformational instability and exposure of the disease-related linear epitope Gly40–Arg43 to the circulating autoantibody. Thus, our study suggests a link between site-specific glycan profiles of β2GPI and the pathology of antiphospholipid syndrome.     

Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo

The structural diversity of glycoprotein N-linked oligosaccharides is determined by the expression and regulation of glycosyltransferase activities and by the availability of the appropriate acceptor/donor substrates. Cells in different tissues and in different developmental stages utilize these control points to manifest unique glycan expression patterns in response to their surroundings. The activity of a Toll-like receptor, called Tollo/Toll-8, induces a pattern of incompletely defined, but neural specific, glycan expression in the Drosophila embryo. Understanding the full extent of the changes in glycan expression that result from altered Tollo/Toll-8 signaling requires characterization of the complete N-linked glycan profile of both wild-type and mutant embryos. N-Linked glycans harvested from wild-type or mutant embryos were subjected to direct structural analysis by analytic and preparative high pressure liquid chromatography, by multidimensional mass spectrometry, and by exoglycosidase digestion, revealing a predominance of high mannose and paucimannose glycans. Di-, mono-, and nonfucosylated forms of hybrid, complex biantennary, and triantennary glycans account for 12% of the total wild-type glycan profile. Two sialylated glycans bearing N-acetylneuraminic acid were detected, the first direct demonstration of this modification in Drosophila. Glycan profiles change during normal development consistent with increasing alpha-mannosidase II and core fucosyl-transferase enzyme activities, and with decreasing activity of the Fused lobes processing hexosaminidase. In tollo/toll-8 mutants, a dramatic, expected loss of difucosylated glycans is accompanied by unexpected decreases in monofucosylated and nonfucosylated hybrid glycans and increases in some nonfucosylated paucimannose and biantennary glycans. Therefore, tollo/toll-8 signaling influences flux through several processing steps that affect the maturation of N-linked glycans.     

Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5

The uterine sarcoma human cell line MES-SA/Dx5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 150 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recognized by the lectins in the digoxigenin (DIG) glycan differentiation kit from Roche Allied Science, all of which were N-glycans. Pgp was isolated using both slab and preparative gel elution. The monoclonal antibody C219 was used to identify the presence of Pgp and Pgp treated with PNGase F on our blots. Pgp isolated from MES-SA/Dx5 cells contains at least two different complex N-glycans--one high mannose tree, detected by GNA, and one branched hybrid oligosaccharide-capped with terminal sialic acids, detected by SNA and MAA. DSA, specific for biantennary oligosaccharides possessing beta(1-4)-N-acetyl-D-glucosamine residues, also recognized the blotted Pgp and is probably detecting the core Galbeta(1-4)-GlcNAc(x) component found in other Pgps.     

N-glycosylation and biological activity of recombinant human alpha1-antitrypsin expressed in a novel human neuronal cell line

Human alpha‐1‐antitrypsin (A1AT) is a protease inhibitor that is involved in the protection of lungs from neutrophil elastase enzyme that drastically modifies tissue functioning. The glycoprotein consists of 394 amino acids and is N‐glycosylated at Asn‐46, Asn‐83, and Asn‐247. A1AT deficiency is currently treated with A1AT that is purified from human serum. In view of therapeutic applications, rA1AT was produced using a novel human neuronal cell line (AGE1.HN®) and we investigated the N‐glycosylation pattern as well as the in vitro anti‐inflammatory activity of the recombinant glycoprotein. rA1AT (300 mg/L) was biologically active as analyzed using elastase assay. The N‐glycan pool, released by PNGase F digestion, was characterized using 2D‐HPLC, MALDI‐TOF mass spectrometry, and by exoglycosidase digestions. A total of 28 N‐glycan structures were identified, ranging from diantennary to tetraantennary complex‐type N‐glycans. Most of the N‐glycans were found to be (α1–6) core‐fucosylated and part of them contain the Lewis X epitope. The two major compounds are a monosialylated diantennary difucosylated glycan and a disialylated diantennary core‐fucosylated glycan, representing 25% and 18% of the total N‐glycan pool, respectively. Analysis of the site‐specificity revealed that Asn‐247 was mainly occupied by diantennary N‐glycans whereas Asn‐46 was occupied by di‐, and triantennary N‐glycans. Asn‐83 was exclusively occupied by sialylated tri‐ and tetraantennary N‐glycans. Next, we evaluated the anti‐inflammatory activity of rA1AT using A1AT purified from human serum as a reference. rA1AT was found to inhibit the production of TNF‐α in neutrophils and monocytes as commercial A1AT does. Biotechnol. Bioeng. 2011;108:2118–2128. © 2011 Wiley Periodicals, Inc.     

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.     

Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator

Electrophoretic analysis of endoglycosidase-treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an N-glycosidically linked N-acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N-acetyllactosamine type of glycan is present in all molecules. Three glycopeptides were isolated and characterized by 1H-NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn-117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn-184 is the site where the presence or absence of a biantennary N-acetyllactosamine type of glycan causes the size heterogeneity. The third N-glycosylation site, Asn-448, was found to carry a triantennary or tetraantennary N-acetyllactosamine type of carbohydrate chain.