Asparagine Amidase Research Articles

Mild Chemical Deglycosylation of Horseradish Peroxidase Yields a Fully Active, Homogeneous Enzyme

Horseradish peroxidase isoenzyme C (HRP) contains eight N-linked glycans composed of Man, Xyl, Fuc, and GlcNAc. These glycans were resistant to enzymatic hydrolysis by endoglycosidases peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F, endo-β-N-acetylglucosaminidase H, and endo-b-N-acetylglucosaminidase F under conditions where ovalbumin was deglycosylated. However, using anhydrous trifluoromethanesulfonic acid (TFMS) in the presence of 90 mM phenol for 5 min at −10°C, all carbohydrate except GlcNAc was removed. Sixty percent of deglycosylated HRP was active after this TFMS treatment. Benzhydroxamic acid affinity chromatography separated active and inactive deglycosylated HRP. TFMS treatment, however, introduced negative charges in all inactive HRP and in about 90% of the active deglycosylated HRP. The nature of this modification has not been identified. After ion-exchange chromatography, homogeneous and fully active deglycosylated HRP, showing the original pI of 9, electronic absorption spectrum, and enzyme kinetics, was obtained. In this purified product no amino acid modifications were detected by amino acid analysis, partial sequencing, and mass spectrometry of tryptic peptides. The deglycosylated product showed greatly reduced solubility in salt solution compared to that of authentic HRP.

Structural Analysis of the Sialylated N- and O-Linked Carbohydrate Chains of Recombinant Human Erythropoietin Expressed in Chinese Hamster Ovary Cells. Sialylation Patterns and Branch Location of Dimeric N-acetyllactosamine Units

The N-linked carbohydrate chains of recombinant human erythropoietin expressed in CHO cells were quantitatively released with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F, separated from the remaining O-glycoprotein by gel-permeation chromatography, and subsequently fractionated via FPLC on Mono Q, HPLC on Lichrosorb-NH2 and high-pH anion-exchange chromatography on CarboPac PA1. The purified sialylated oligosaccharides were analyzed by one-dimensional and two-dimensional 500-MHz 1H-NMR spectroscopy. When necessary, oligosaccharides were treated with endo-β-galactosidase (and N-acetyl-β-glucosaminidase) followed by 1H-NMR analysis of the incubation products, to obtain additional structural information. Di-, tri-, tri′- and tetraantennary, N-acetyllactosamine-type oligosaccharides occur which can be completely (major) or partially (minor) sialylated. Three different types of α2-3-linked sialic acids are present, namely, N-acetylneuraminic acid (95%), N-glycolylneuraminic acid (2%) and N-acetyl-9-O-acetylneuraminic acid (3%). In the case of partial sialylation, a non-random distribution of the sialic acids over the branches is observed. One or two extra N-acetyllactosamine units, being exclusively located in the branches attached to the α1-6-linked Man residue, can be present in completely or partially sialylated di-, tri′-, and tetraantennary oligosaccharides. Tetraantennary oligosaccharides with, N-acetyllactosamine repeats could be digested quantitatively with endo-β-galactosidase from Bacteroides fragilis, whereas under the same conditions tri′antennary oligosaccharides Hardly reacted ( 75%). The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment, and purified via FPLC on Mono Q and HPLC on Lichrosorb-NH2. Two O-glycans were found, namely, Neu5Acα2-3Galβ1-3GalNAc-ol and Neu5Acα2-3Galβ1-3(Neu5 Acα2-6)GalNAc-ol.

The major N-linked carbohydrate chains from human urokinase. The occurrence of 4-O-sulfated, (alpha 2-6)-sialylated or (alpha 1-3)-fucosylated N-acetylgalactosamine(beta 1-4)-N-acetylglucosamine elements.

The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri'-antennary structures. The glycans contain predominantly GalNAc beta 1-4GlcNAc beta instead of Gal beta 1-4GlcNAc beta elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (alpha 1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4-)-4GalNAc beta 1-4GlcNAc beta 1-2 antennae.

Structural analysis of the sialylated N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Sialylation patterns and branch location of dimeric N-acetyllactosamine units.

The N-linked carbohydrate chains of recombinant human erythropoietin expressed in CHO cells were quantitatively released with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, separated from the remaining O-glycoprotein by gel-permeation chromatography, and subsequently fractionated via FPLC on Mono Q, HPLC on Lichrosorb-NH2 and high-pH anion-exchange chromatography on CarboPac PA1. The purified sialylated oligosaccharides were analyzed by one-dimensional and two-dimensional 500-MHz 1H-NMR spectroscopy. When necessary, oligosaccharides were treated with endo-beta-galactosidase (and N-acetyl-beta-glucosaminidase) followed by 1H-NMR analysis of the incubation products, to obtain additional structural information. Di-, tri-, tri'- and tetraantennary N-acetyllactosamine-type oligosaccharides occur which can be completely (major) or partially (minor) sialylated. Three different types of alpha 2-3-linked sialic acids are present, namely, N-acetylneuraminic acid (95%), N-glycolylneuraminic acid (2%) and N-acetyl-9-O-acetylneuraminic acid (3%). In the case of partial sialylation, a non-random distribution of the sialic acids over the branches is observed. One or two extra N-acetyllactosamine units, being exclusively located in the branches attached to the alpha 1-6-linked Man residue, can be present in completely or partially sialylated di-, tri'-, and tetraantennary oligosaccharides. Tetraantennary oligosaccharides with N-acetyllactosamine repeats could be digested quantitatively with endo-beta-galactosidase from Bacteroides fragilis, whereas under the same conditions tri' antennary oligosaccharides hardly reacted (< 15%). Using endo-beta-galactosidase from Escherichia freundii, these tri'antennary oligosaccharides could be digested more extensively (> 75%). The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment, and purified via FPLC on Mono Q and HPLC on Lichrosorb-NH2. Two O-glycans were found, namely, Neu5Ac alpha 2-3Gal beta 1-3GalNAc-ol and Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol.

Carbohydrate Structure Analysis of Batroxobin, a Thrombin-Like Serine Protease from Bothrops moojeni Venom

The carbohydrate side chains of batroxobin were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, pyridylaminated and separated by two-dimensional HPLC. Neutral oligosaccharide derivatives obtained after desialylation were characterized by methylation analysis, liquid secondary-ion mass spectrometry, digestion with exoglycosidases and endoglycosidases and, in part, by acetolysis, whereas sialic acid constituents were identified by reverse-phase HPLC after conjugation with 1,2-diamino-4,5-methylene-dioxybenzene. The overall glycosylation status of the protein was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results revealed that batroxobin is heterogeneously glycosylated carrying predominantly diantennary, partially incomplete complex-type glycans in addition to hybrid-type species. Most glycans were core-fucosylated at C6 of the innermost GlcNAc. As a characteristic feature, galactose was completely replaced by GalNAc beta 4-substituents in complex-type antennae, the GlcNAc-residues of which were, in part, fucosylated at C3. Furthermore, evidence was obtained that suggested the presence of a novel type of glycoprotein-N-glycan comprising two GalNAc beta 4GlcNAc beta 4GlcNAc beta 2Man-antennae. Sialic acid residues represented a mixture of N-acetylneuraminic acid (Neu5Ac) and N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2), which were exclusively linked to C3 of subterminal GalNAc. A precise assignment of these sialic acid derivatives to distinct oligosaccharide structures or antennae, however, was not carried out. Finally, MALDI-TOF-MS demonstrated that both potential N-glycosylation sites of batroxobin are substituted by carbohydrate chains. In conclusion, our studies revealed that this snake venom glycoprotein is characterized by a unique oligosaccharide pattern partly comprising novel structural elements.

The Major N-Linked Carbohydrate Chains from Human Urokinase. The Occurrence of 4-O-sulfated, (alpha2-6)-sialylated or (alpha1-3)-fucosylated N-acetylgalactosamine(beta1-4)-N-acetylglucosamine Elements

The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-β-glucosaminy)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri′-antennary structures. The glycans contain predominantly GalNAcβ1-4GlcNAcβ instead of Galβ1-4GlcNAcβ elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (α1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4−)-4GalNAcβ1-4GlcNAcβ1-2 antennae.

Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) from Silene alba cells.

The peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992) Glycoconjugate J 9:191-97] was partially purified from cultured Silene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of 'unconjugated N-glycans' in a suspension medium of cultured Silene alba cells.

Characterization of asparagine-linked oligosaccharides on a mouse submandibular mucin.

The asparagine-linked oligosaccharides from an adult female mouse submandibular gland mucin were released by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F or endo-beta-N-acetylglucosaminidase H. Endo-beta-N-acetylglucosaminidase H appeared to be more effective at releasing the asparagine-linked oligosaccharides from this mucin than was peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase F. After quantitative reductive labelling with the fluorophore, 8-aminonaphthalene-1,3,6-sulphonic acid, the oligosaccharides were separated by polyacrylamide gel electrophoresis and isolated. The individual oligosaccharides were sequenced by a battery of recombinant exoglycosidases. Approximately 50% of the oligosaccharides were of the high-mannose type. The five-mannose member of this family was the most prevalent. The second group of oligosaccharides were of the non-bisected hybrid type. No complex asparagine-linked oligosaccharides were detected. The hybrids exhibited both biantennary and triantennary branching patterns. The triantennary hybrid was the most common hybrid at > 30% of all oligosaccharides. With approximately 98% of the hybrid oligosaccharides sialylated and all lacking a bisecting N-acetylglucosamine, these oligosaccharides as a group have been only rarely observed in other glycoproteins. The fully sialylated triantennary hybrid may be unique.

Do de-N-glycosylation enzymes have an important role in plant cells?

In this review de-N-glycosylation was defined as the removal of the glycan(s) from a N-glycosylprotein, by means of enzymes acting on the di- N-acetylchitobiosyl part of the invariant pentasaccharide inner-core of N-glycosylproteins. Peptide- N 4-( N-acetyl-β- d-glucosaminyl) asparagine amidases (PNGase) and endo- N-acetyl-β- d-glucosaminidases (ENGase) were both considered as de-N-glycosylation enzymes. A detailed description of the characterization and the function of plant PNGases and ENGases is presented, together with a brief presentation on the occurrence and the current knowledge on the function of microbial and animal enzymes. De-N-glycosylation of plant glycoproteins was proposed as a possible mechanism for the release of oligosaccharides displaying biological activities and the removal of N-glycans could also explain the regulation of protein activity. Each enzyme seems to have a specific function during germination and post-germinative development. All the arguments concur that de-N-glycosylation enzymes have an important role in plant cells and confirm that the N-glycosylation/de-N-glycosylation system should occur more commonly than presently recognized in living organisms.

Glycosylation of Bombesin Receptors: Characterization, Effect on Binding, and G-Protein Coupling

In the present study, we investigated the nature and the importance of glycosylation of two mammalian bombesin receptors, the neuromedin B receptor (NMB-R) and the gastrin-releasing peptide receptor (GRP-R), using chemical cross-linking and enzymatic deglycosylation. [125I]-(D-Tyr0)NMB cross-linked to native NMB-R on rat C-6 glioblastoma cells or rat NMB-R transfected into BALB 3T3 cells revealed a single broad band, M(r) = 63,000, on both cell types that was not altered by DTT. NMB inhibited cross-linking specifically and saturably with an IC50 of 4.8 and 6.1 nM for C-6 and NMB-R transfected cells, respectively, and there was a close correlation between its ability to inhibit binding and its ability to inhibit cross-linking. A single broad band of M(r) = 82,000 was cross-linked with [125I]GRP on mouse GRP-R transfected BALB 3T3 cells. Peptide-N4-(N-acetyl-beta- glucosaminyl)asparagine amidase F (PNGase F) digestion increased the mobility of the original band in C-6, NMB-R, and GRP-R transfected cell membranes. Endoglycosidase H (Endo-H) and endoglycosidase F2 (Endo-F2) digestion had no effect on both transfected cells. Neuraminidase digestion slightly increased the mobility of the original band in NMB-R transfected cell membranes; however, it had no effect on GRP-R transfected cell membranes. Endo-alpha-N-acetylglucosaminidase (O-glycanase) digestion subsequent to neuraminidase treatment showed no additional effect on either receptor. Serial partial deglycosylation of cross-linked NMB-Rs with PNGase F treatment for different incubation periods revealed one band of partially glycosylated receptor (53 kDa) besides the fully glycosylated and fully deglycosylated ones, showing that NMB-R has two oligosaccharide chains. Similarly, three partially deglycosylated species (72, 62, and 52 kDa) are seen with the GRP-R, indicating that the GRP-R has four oligosaccharide chains. Treatment of unlabeled membranes with PNGase F followed by affinity labeling resulted in fully deglycosylated NMB-R or 75% deglycosylated GRP-R. Deglycosylation of the NMB-R did not alter its affinity for NMB or alter G-protein coupling; however, 75% deglycosylation of the GRP-R both decreased its affinity for GRP and altered its ability to couple to G-proteins. The present results demonstrate that NMB-R on native and transfected cells is an N-linked sialoglycoprotein with two triantenary and/or tetraantenary complex oligosaccharide chains. The apparent M(r) of this sialoglycoprotein is 63,000, and this protein does not contain disulfide-linked subunits or O-linked carbohydrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal Structure of Peptide-N4-(N-acetyl-.beta.-D-glucosaminyl)asparagine amidase F at 2.2-.ANG. Resolution

Peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F) is an amidase that cleaves the beta-aspartylglucosylamine bond of asparagine-linked glycans. The 34.8-kDa (314 amino acids) enzyme has a very broad substrate specificity and is extensively used for studies of the structure and function of glycoproteins. Enzymatic activity of PNGase F requires recognition of both the peptide and the carbohydrate components of the substrate. Only limited information regarding the mechanism of action of the enzyme is available. The three-dimensional structure of PNGase F has been determined by X-ray crystallography at 2.2-A resolution. The protein folds into two domains comprising residues 1-137 and 143-314, respectively. Both domains have eight-stranded antiparallel beta-sandwich motifs that are very similar in geometry. Both sandwiches have parallel principal axes and lie side by side. The covalent link between the domains is located at the top end of the molecule. Extensive hydrogen-bonding contacts occur along the full length of the interface between the two domains. Three different areas, all at the interface between the two domains, have been identified as possible locations for the active site of the enzyme. These include a hydrophobic bowl of about 20 A in diameter on one surface of the molecule, a long polar cleft on the opposite side, and a cleft at the bottom, which is lined with large aromatic residues including eight tryptophans.

Carbon Starvation Increases Endoglycosidase Activities and Production of "Unconjugated N-Glycans" in Silene alba Cell-Suspension Cultures

We previously reported the occurrence of oligomannosides and xylomannosides corresponding to unconjugated N-glycans (UNGs) in the medium of a white campion (Silene alba) cell suspension. Attention has been focused on these oligosaccharides since it was shown that they confer biological activities in plants. In an attempt to elucidate the origin of these oligosaccharides, we studied two endoglycosidase activities, putative enzymes involved in their formation. The previously described peptide-N4-(N-acetyl-glucosaminyl) asparagine amidase activity and the endo-N-acetyl-beta-D-glucosaminidase activity described in this paper were both quantified in white campion cells during the culture cycle with variable initial concentrations of sucrose. The lower the sucrose supply, the higher the two activities. Furthermore, endoglycosidase activities were greatly enhanced after the disappearance of sugar from the medium. The production of UNGs in the culture medium rose correlatively. These data strongly suggest that the production of UNGs in our white campion cell-suspension system is due to the increase of these endoglycosidase activities, which reach their highest levels of activity during conditions of carbon starvation.

A fluorescence high-performance liquid chromatography assay for enzymes acting on the di- N-acetylchitobiosyl part of asparagine-linked glycans

The glycoasparagine, Man 7GlcNAc 2Asn (‘Man 7’) was labelled with resorufin and used as a specific substrate for the detection and quantification of endo- β- N-acetyl glucosaminidases (Endos) acting on the di- N-acetylchitobiosyl part of asparagine-linked glycans. Peptide- N 4-( N-acetly-β-glucosaminyl) asparagine amidases (PNGases) cannot transform this substrate but they can be detected by the procedure described earlier using the resorufin-labelled N-glycopeptide [Glycoconjugate J., 9 (1992) 162–167]. These two substrates can be used in a simple, reproducible and very sensitive fluorescence HPLC assay in order to monitor Endo and PNGase activities during isolation and purification processes, or studies of the evolution of such activities during cultivation of the producing cells.

Intramolecular charge heterogeneity in purified major histocompatibility class II alpha and beta polypeptide chains

Major histocompatibility (MHC) class II antigens are heterodimeric cell surface glycoproteins consisting of an alpha and a beta chain. Although one-dimensional SDS-polyacrylamide gel electrophoresis analysis of purified MHC class II antigens shows a single diffuse band for each chain, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of this heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mature protein. To investigate the role of the three N-linked oligosaccharides of the alpha and beta chains in determining the isoelectric point of each chain, affinity-purified MHC class II antigens from human and rat sources were deglycosylated using asparagine amidase. The complete enzymatic removal of all three N-linked oligosaccharides was confirmed by SDS-polyacrylamide gel electrophoresis as well as by four different lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of the HLA DR2 chains which lack the transmembrane and cytoplasmically exposed regions in Escherichia coli. Two-dimensional electrophoresis of these single chains also reveal multiple banding patterns. The two-dimensional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimension, treatment of the proteins with alkaline phosphatase to remove any potential phosphorylation, or preincubation in the presence of iodoacetamide. Multiple forms of recombinant alpha and beta chains were also observed in Tris-glycine-urea gels which merged into a single band in the presence of SDS. In addition, partially fractionated bands from preparative isoelectric focusing gels, when refocused, showed an identical number of multiple spots spanning the same range of isoelectric points. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the observed charge heterogeneity is independent of glycosylation and phosphorylation of the proteins.

Functional role of N-glycosylation in alpha 5 beta 1 integrin receptor. De-N-glycosylation induces dissociation or altered association of alpha 5 and beta 1 subunits and concomitant loss of fibronectin binding activity.

Fibronectin (FN)-mediated cell adhesion is controlled mainly by alpha 5 beta 1 (recognizing the RGD sequence) and alpha 4 beta 1 (recognizing the CS-1 peptide sequence of FN) integrin receptors. Integrin-dependent cell adhesion to FN is greatly promoted by optimal GM3 concentration at the surface membrane (Zheng, M., Fang, H., Tsuruoka, T., Tsuji, T., Sasaki, T., and Hakomori, S. (1993) J. Biol. Chem. 268, 2217-2222), and cell adhesion mediated by alpha 4 beta 1 (to FN) or alpha 6 beta 1 (to laminin) is inhibited by modifying N-glycosylation processing of the integrin receptor (e.g. Akiyama, S. K., Yamada, S. S., and Yamada, K. M. (1989) J. Biol. Chem. 264, 18011-18018). We therefore studied the specific role of N-glycosylation in alpha 5 beta 1 function. Key findings of the present study were as follows. (i) Adhesion of K562 cells to FN-coated plates, which is mediated solely by alpha 5 beta 1, was inhibited when cells were treated with a mixture of endo-N-acetylglucosaminidase F and peptide -N4-(N-acetylglucosaminyl)asparagine amidase F (endo-F/PNGase-F). (ii) The alpha 5 beta 1 receptor at the K562 cell surface tended to dissociate into alpha 5 and beta 1 subunits when an extract of cells treated with endo-F/PNGase-F was precipitated by integrin subunit-specific antibodies, i.e. the alpha 5 subunit was preferentially precipitated by anti-alpha 5 monoclonal antibody ZH5, and the beta 1 subunit was preferentially precipitated by anti-beta 1 monoclonal antibody ZH1. When intact cells were extracted and treated with either ZH5 or ZH1, both alpha 5 and beta 1 were coprecipitated, indicating that the two subunits are normally tightly associated with each other. (iii) Adhesion of alpha 5 beta 1-containing liposomes (phosphatidylcholine:cholesterol liposomes incorporating purified alpha 5 beta 1) to FN-coated plates was abolished by treatment of liposomes with endo-F/PNGase-F. Liposomes incorporating alpha 5 beta 1 pretreated with endo-F/PNGase-F also did not bind to FN. When purified alpha 5 beta 1 receptor was treated with endo-F/PNGase-F followed by ZH5 or ZH1, the alpha 5 or beta 1 subunit was precipitated separately, respectively. In contrast, both subunits were always coprecipitated when intact purified alpha 5 beta 1 receptor was directly treated with ZH5 or ZH1. These findings indicate that N-glycosylation of both the alpha and beta subunits of the alpha 5 beta 1 integrin receptor is essential for association of these subunits and for optimal binding to FN.

Structure of the O‐linked carbohydrate chains of porcine zona pellucida glycoproteins

The N-linked carbohydrate chains of porcine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and subsequently separated from the O-glycoprotein by gel-permeation chromatography on Bio-Gel P-100. The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment. Fractionation of the extremely heterogeneous mixture of O-linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of 32 O-glycans were determined by one- and two-dimensional 1H-NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Gal beta 1-4GlcNAc beta 1-3Gal beta 1-3GalNAc-ol, Gal beta 1-4(6SO4-)GlcNAc, and alpha 2-3-linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n = 0 to > 6: [Neu5Gc/Ac alpha2-3]0-1[Gal beta 1-4(6SO4-)GlcNAc beta 1-3]nGal beta 1-4GlcNAc beta 1-3Gal Beta 1-3GalNAc-ol. In addition, smaller compounds were identified in which the Gal beta 1-3GalNAc-ol core is substituted by Neu5Gc/Ac alpha 2-6-linked to GalNAc-ol and/or Neu5Gc/Ac alpha 2-3-linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6-O-sulfated GlcNAc residues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAc beta 1-3GalNAc-ol core structure. Analysis of the endo-beta-galactosidase digests of pools of N- and O-glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N-acetyllactosamine) chains. In the case of the N-linked carbohydrate chains, multiple branching of the core structures occurs, resulting in an even larger heterogeneity than observed for the O-linked carbohydrate chains.

Improved fractionation of sialylated glycopeptides by pellicular anion-exchange chromatography

The glycoprotein bovine fetuin was treated with trypsin and the Asn-81 tryptic glycopeptide was purified (90% pure by Edman sequencing) by reversed-phase chromatography (RP-HPLC). The Asn-81 glycopeptide, which eluted as a single peak by RP-HPLC, was separable into five peaks on the NucleoPac PA100 column, a pellicular anion-exchange column. Each of the five Asn-81 glycopeptide peaks was shown to contain N-linked oligosaccharides by treatment of each peak with peptide N 4-(N-acetyl-β- d-glucosaminyl) asparagine amidase F (PNGase F) and subsequent oligosaccharide analysis by high-pH anion-exchange chromatography with pulsed amperometric detection. High-pH anion-exchange chromatography-pulsed amperometric detection oligosaccharide analysis revealed that each peak contained a different population of sialylated N-linked oligosaccharides. Hence each peak contained a different group of glycopeptide glycoforms. It was observed that the longer the retention time of the Asn-81 glycopeptide peak on the anion-exchange column, the greater the oligosaccharide sialylation. Two glycopeptide peaks which differed in their distribution of disialylated oligosaccharides demonstrated that the glycopeptide separation was a result of something more than gross differences in sialic acid content. The two other N-linked tryptic glycopeptides of fetuin were also separated into multiple peaks on the NueleoPac PA100 column and these separations were shown to be due to differences in oligosaccharide sialylation. The separations of the three fetuin N-linked glycopeptides demonstrate that pellicular anion-exchange chromatography offers improved separation speed and resolution for the separation of sialylated glycopeptides.

Murine Heparin Cofactor II: Purification, cDNA Sequence, Expression, and Gene Structure

Heparin cofactor II (HCII) is a glycoprotein in human plasma that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Unexpectedly, we found that HCII activity in murine plasma is present in two proteins of 68 and 72 kDa. The two proteins have the same N-terminal amino acid sequence, and both react with an antibody raised against the C-terminal nine amino acid residues of murine HCII predicted from the cDNA sequence. Treatment of the two proteins with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase yields a single 54-kDa band. Thus, murine plasma contains two forms of HCII that appear to have identical amino acid sequences but differ in the composition of their N-linked oligosaccharides. HCII cDNA clones isolated from a murine liver library include a 1434 bp open reading frame following the first Met codon, a TAA stop codon, and 580 bp of 3'-untranslated sequence terminating in a poly(A) tail. The amino acid sequence deduced from the cDNA contains the N-terminal sequence of purified murine plasma HCII preceded by a 23-residue hydrophobic sequence presumed to be the signal peptide. The amino acid sequence of murine HCII is 87% identical to that of human HCII, the greatest variability occurring in the N-terminal portion of the protein. Northern blot analysis reveals a 2.3-kb HCII mRNA in murine and human liver, but no HCII mRNA is detectable in heart, brain, spleen, lung, skeletal muscle, kidney, testis, placenta, pancreas, or intestine. Southern blot analysis of restriction fragment length polymorphisms in progeny on interspecific and intersubspecific crosses indicates that mice have a single HCII gene (designated Hcf2), which maps to chromosome 16 between Prm-1 and Igl. The murine HCII gene is approximately 7.1 kb in size and consists of at least four exons and three introns. The intron/exon organization is identical to that of the human HCII gene except at the 5' end, where the murine gene may lack a large intron in the 5'-untranslated region. Our results indicate that HCII is more highly conserved than the human and murine homologues of other serpins such as alpha 1-antitrypsin and alpha 1-antichymotrypsin.

Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain.

Recombinant human uterine tissue plasminogen activator (tPA) glycosylation mutants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain due to the replacement of either Tyr67 by Asn (YN-tPA) or Gly60 by Ser (GS-tPA) were expressed in mouse epithelial cells (C127) in the presence of [6-3H]glucosamine. Glycopeptides comprising individual glycosylation sites were isolated and oligosaccharides attached were liberated by treatment with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols obtained after reduction were either directly characterized by high-pH anion-exchange chromatography (high-mannose and hybrid-type glycans) or preparatively subfractionated after enzymic desialylation and separation from sulphated asialooligosaccharides (complex-type sugar chains). Individual (sub)fractions of glucans were studied by methylation analysis, liquid secondary-ion mass spectrometry and, in part, by exoglycosidase digestion, whereas corresponding deglycosylated peptides were identified by amino acid analysis and N-terminal amino acid sequencing. The results revealed that Asn117 of YN-tPA carried exclusively high-mannose-type glycans with five to nine mannose residues similar to wild-type tPA expressed in this cell line [Pfeiffer, G., Schmidt, M., Strube, K.-H. & Geyer, R. (1989) Eur. J. Biochem. 186, 273-286]. In contrast, Asn117 of GS-tPA carried only small amounts (about 25%) of high-mannose and hybrid-type species and predominantly complex-type sugar chains (about 75%) which were partially incomplete and mostly devoid of fucose. Newly introduced N-glycosylation sites at Asn67 (YN-tPA) or Asn58 (GS-tPA) as well as those at Asn184 and Asn448 were solely substituted by complex-type glycans. Each carbohydrate attachment site displayed a peculiar oligosaccharide pattern with regard to branching and substitution by Gal alpha 3-residues, sulphate groups, intersecting GlcNAc and lactosamine repeats. Our study clearly demonstrates that creation of a new glycosylation site at Asn58 influenced the oligosaccharide processing and, hence, the glycosylation pattern at Asn117, whereas introduction of a new site at Asn67 did not. The relative amounts of complex-type glycans at Asn117 of GS-tPA correlated with the degree of carbohydrate substitution of Asn58. Therefore, it can be concluded that the presence of a sugar chain at the position and not the Gly to Ser mutation itself is responsible for the observed alteration of GS-tPA glycosylation.

Enzymatic Modeling of the Oligosaccharide Chains of Glycoproteins Immobilized onto Polystyrene Surfaces

A method for the modification of the oligosaccharide moiety of even small amounts of purified glycoproteins by enzymatic glycosylation and deglycosylation is described. The method includes noncovalent immobilization of the glycoproteins onto the polystyrene surface of the wells of microtiter plates used as reaction tubes, deglycosylation or glycosylation by incubation either with exoglycosidases or endoglycosidases or with glycosyltransferases, and the characterization of the modified glycan structures by probing them with lectins. Placental transferrin receptor employed as a model glycoprotein was modified in amounts of as little as 100 ng removing sialic acid residues, hybrid-type glycans or all types of N-glycans with neuraminidase, endo-β-N-acetylglucosaminidase H or peptide-N4-(acetyl-β-glucosaminyl) asparagine amidase. Asialotransferrin receptor was α-2,6-sialylated with α-2,6-sialyltransferase from rat liver, but could not be α-2,3-sialylated with α-2,3-sialyltransferase from porcine liver. Changes in the structure and in the relative amount of the oligosaccharides could be monitored semiquantitatively with high sensitivity by the binding of digoxigenin-labeled lectins and anti-digoxigenin Fab fragments. The method is easy to use, does not require immobilization of the enzymes employed, offers simple separation of the enzymes and the product, and leaves the protein intact for further studies.