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Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N-glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N-glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex-type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high-mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.

An N-linked glycosylation in a novel C-lectin protein from snake venom was observed by Edman degradation and liquid chromatography-electrospray mass spectrometry. The peptides obtained by trypsin cleavage were analyzed to confirm the amino acid sequence and Asn5 was found to be the N-glycosylation site. The result was further confirmed by N-glycosidase digestion. In addition, the protein and tryptic peptides with and without glycan chain were characterized by mass spectrometry according to the mass difference. The glycopeptide obtained from proteolytic digestion was analyzed and the glycoforms were identified as high-mannose type by tandem MS coupled with alpha-mannosidase digestion. An oxidized Met residue was detected and located in the protein by mass spectrometry.

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays important roles in fertilization and consists of three glycoproteins; ZPA, ZPB and ZPC. In pig, neutral complex-type N-linked chains obtained from a ZPB/ZPC mixture possess sperm-binding activity. We have recently reported that among neutral N-linked chains triantennary and tetraantennary chains have a sperm-binding activity stronger than that of diantennary chains. Triantennary and tetraantennary chains are localized at the second of the three N-glycosylation sites of ZPB. In this study, we focused on the localization of neutral N-linked chains in ZPC. ZPB and ZPC can not be separated from each other unless the acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-beta-galactosidase digestion. A large part of the acidic N-linked chains becomes neutral by the digestion, but the main neutral N-linked chains are not susceptible to the enzyme. N-glycanase digestion indicated that ZPC has three N-glycosylation sites. Three glycopeptides each containing one of the N-glycosylation sites were obtained by tryptic digestion of ZPC and the N-glycosylation sites were revealed as Asn124, Asn146 and Asn271. The carbohydrate structures of the neutral N-linked chains from each glycopeptide were characterized by two-dimensional sugar mapping analysis taking into consideration the structures of the main, intact neutral N-linked chains of ZPB/ZPC mixture reported previously. Triantennary and tetraantennary chains were found mainly at Asn271 of ZPC, whereas diantennary chains were present at all three N-glycosylation sites. Thus, ZPC has tri-antennary and tetra-antennary chains as well as ZPB, but the localization of the chains is different from that in ZPB.

The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A (PNGase A) was purified from almonds (Prunus amygdalus var. dulcis). Contrary to previous results in the literature, the enzyme appeared to be a heterodimer with subunits of 55 and 27 kDa when analysed by SDS/PAGE and two-dimensional electrophoresis. Peaks corresponding to molecular masses of 54.2, 21.2 and 75.5 kDa were observed with matrix-assisted laser-desorption/ionization mass spectrometry. The N-terminal sequences of the larger and the smaller chain were determined to be LASGYHSWAD and EPTPLHDFPP, respectively. Both polypeptides reacted with concanavalin A, indicating their glycoprotein nature. Upon digestion of PNGase with pepsin, the N-linked oligosaccharides were released with active PNGase and analysed as their 2-aminopyridine derivatives by two-dimensional HPLC and by matrix-assisted laser-desorption mass spectrometry. The most abundant N-glycan of the four species found exhibited the well known vacuole type structure, i.e. the pentasaccharide core with xylose and alpha1,3-linked fucose. The other structures either had an additional mannose residue and/or lacked the fucose. PNGase A was largely but not absolutely resistant to self-deglycosylation. However, only at an extremely high enzyme/substrate ratio, N-glycans released from PNGase A itself caused a detectable contamination of a PNGase digest of a glycopeptide.

Major proteins synthesized in the hypopharyngeal gland of the worker honeybee change from bee-milk proteins to alpha-glucosidase in accordance with the age-dependent role change of the worker bee. Previously, we showed that the gene for alpha-glucosidase is expressed specifically in the forager-bee gland [Ohashi, K., Sawata, M., Takeuchi, H., Natori, S. & Kubo, T. (1996) Biochem. Biophys. Res. Commun. 221, 380-385]. Here, we describe the isolation and analysis of cDNAs for two bee-milk 56-kDa and 64-kDa proteins. The 56-kDa protein was a glycoprotein which shared 63.2% and 56.9% amino acid sequence identities with proteins encoded by cDNA for royal-jelly-related protein 57-1 (pRJP57-1) and pRJP57-2. The 64-kDa protein cDNA was identical to pRJP57-1. Thus, these bee-milk proteins seem to form a structurally related protein family. The gene for the 64-kDa protein/RJP57-1 was expressed specifically in the nurse-bee gland, whereas that for the 56-kDa protein was expressed in both the nurse-bee and forager-bee glands. mRNAs for the 56-kDa and 64-kDa proteins were detected by in situ hybridization in a whole acinus of the nurse-bee gland, whereas mRNAs for the 56-kDa protein and alpha-glucosidase were detected in that of the forager-bee gland. Therefore, the individual secretory cells of the acinus of the hypopharyngeal gland were shown to express these genes differently with the age-dependent role change of the worker bee.

The cDNA of soybean agglutinin (SBA), a glycoprotein lectin, obtained from the mRNA of soybean seeds at mid-maturation, was cloned in a lambda gt 10 phage and subcloned in a pUC-8 plasmid. Probing with a fragment of the lectin gene [Vodkin, L. O., Rhodes, P. R. & Goldberg, R. B. (1983) Cell 34, 1023-1031] afforded a clone of 1012 nucleotides containing the complete coding region of 858 nucleotides for the precursor to soybean agglutinin. The deduced amino acid sequence contains the 253 residues of the mature lectin and an hydrophobic N-terminal signal peptide of 32 amino acids. Expression in Escherichia coli of the cDNA coding for the precursor to the lectin or for the mature lectin led to the accumulation of large quantities of inclusion bodies, from which mature SBA was isolated in small yield (up to 1 mg/l). It was identical with the native lectin in the hemagglutinating activity and carbohydrate specificity, N-terminal sequence and oligomeric structure, but, because it was not glycosylated, its subunit mass was lower by 2 kDa. Our findings show that pre-SBA is processed into the mature form in the bacteria, and that, contrary to what has been suggested [Nagai, K. & Yamaguchi, H. (1993) J. Biochem. (Tokyo) 113, 123-125], glycosylation is not essential for the folding of the lectin, nor for its subunit assembly into a biologically active tetramer. To obtain recombinant SBA in secreted form, the pre-SBA cDNA was subcloned in pTM1 vector and the construct inserted into vaccinia virus. When monkey BS-C-1 cells were infected by the virus, using a double expression protocol, recombinant lectin was secreted into the growth medium, from which it was isolated by immunoaffinity chromatography at a yield similar to that from the bacteria. Except for its lower hemagglutinating activity, the product was indistinguishable from native SBA in all properties tested. It was also susceptible to digestion by endo-beta-N-acetylglucosaminidase H or N-glycanase which caused a decrease of 2 kDa in its subunit mass and gave the same results on lectin blot analysis, indicating that it too is a glycoprotein with a single oligomannose unit.

The primary structures of 21 novel monoantennary and diantennary N-glycans of the glycoprotein alphaD-hemocyanin (alphaD-Hc) of Helix pomatia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an alphaD-Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb-NH2, was characterized using 1H-NMR spectroscopy, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of alphaD-Hc were released with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F digestion, separated from the protein on Bio-Gel P-100, and subfractionated on Bio-Gel P-4. A portion of subfractions was reduced with sodium borodeuteride, and the non-reduced and reduced samples were further fractionated on CarboPac PA-1, Lichrosorb-NH2/Lichrosphere-NH2, and/or Lichrosphere-C18. Purified oligosaccharides and oligosaccharide-alditols were analyzed using 500/600-MHz 1H-NMR spectroscopy. In total, four novel types of antenna were identified, namely, [structures: see text] which are all attached to O-2 of alphaMan residues of the trimannosyl-N,N'-diacetylchitobiose core element, which is generally beta-1,2-xylosylated and alpha-1,6-fucosylated, Man(alpha1-6)[Man(alpha1-3)][+/-Xyl(beta1-2)]Man(beta1-4)GlcNAc(beta1-4) [+/-Fuc(alpha1-6)]GlcNAc.

Fetidins are 40-kDa and 45-kDa hemolytic and antibacterial glycoproteins present in the coelomic fluid of the earthworm Eisenia fetida andrei. By screening a cDNA library with a polyclonal antifetidin serum, we have cloned a cDNA that encoded the 40-kDa fetidin. The clone contains an insert of 1.44 kb encoding a protein of 34 kDa, which corresponds to the size of deglycosylated fetidins. The recombinant protein inhibits Bacillus megaterium growth. Restriction fragment polymorphisms were observed on Southern blots and correspond to a known protein polymorphism. The sequence of the cDNA contains a peroxidase signature and fetidins from earthworm coelomic fluid have peroxidase activity. The 40-kDa and 45-kDa fetidins therefore represent two related polymorphic defence factors in invertebrates.

Semenogelin I and semenogelin II constitute the major gel-forming proteins in human semen. The gel proteins were rapidly solubilized and separated from spermatozoa in ejaculates collected at pH 9.7 in buffer containing 4 mol/l urea and dithiothreitol. This protected the semenogelins from proteolytic degradation by prostate-specific antigen, and allowed their isolation by affinity chromatography on heparin-Sepharose. Semenogelins I and II were almost selectively retained and eluted partially separated in 0.25 mol/l NaCl. Further purification was achieved by chromatography on Superose. Approximately 10-20 mg semenogelin I and 2-5 mg semenogelin II were recovered from each sample with a purity exceeding 95% as judged by SDS/PAGE. The molecular mass of semenogelin I (49 958 Da) and the major form of semenogelin II (63 539 Da) measured by mass spectrometry was consistent with the reported cDNA data. The occurrence of a second, larger form of semenogelin II was due to asparagine-linked glycosylation. The amino-termini of the purified proteins were blocked, but digestion with pyroglutamate amino-peptidase enabled the identification of amino-terminal sequences consistent with the reported cDNA data. The amino acid compositions of the purified proteins were also consistent with those derived from cDNA data. The absorption coefficients (280 nm, 1%, 1 cm) for semenogelins I and II were 5.5 and 5.4, respectively, and the isoelectric point was above pH 9.5 for both proteins.

The soluble human interleukin-6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95% was achieved. A subsequent IL-6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute-phase protein 1-antichymotrypsin in HepG2 cells stably transfected with IL-6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglycosylation with peptide N-glycosidase F led to a shift in molecular mass from 65 kDa to 45 kDa. It has previously been shown that the shIL6R can be generated by shedding the membrane-bound form or by expression of an alternatively spliced mRNA. Here we show that the shIL6R isolated from human plasma is recognized by an affinity-purified peptide antibody raised against an amino acid sequence unique for the alternatively spliced isoform. Thus, the shIL6R isoform generated through alternative splicing which has been previously detected in supernatants of cultured cell lines is also an in vivo product circulating in human plasma.

The sperm receptor activity of pig zona pellucida has been previously shown to exist in one of the components, pig zona protein 3 alpha (PZP3 alpha), that can be purified after the removal of sialylated and/or sulfated N-acetylpoly(lactosamine) by digestion with endo-beta-galactosidase. In this study, we examined whether N-linked or O-linked carbohydrate chains are involved in the sperm receptor activity of pig zona pellucida. The elimination of N-linked carbohydrate chains from endo-beta-galactosidase-digested PZP3 alpha by digestion with N-glycanase markedly reduced its inhibitory effect on sperm-egg binding in an in vitro competition assay, whereas the elimination of O-linked carbohydrate chains by alkali treatment hardly reduced the inhibitory effect. These results indicate that N-linked carbohydrate chains of PZP3 alpha play a major role in mediating the sperm binding of zona pellucida in pig.

Numerous studies have shown that glycosylation of the alpha-subunit of human chorionic gonadotropin (alpha hCG) is essential for the biological activity of this hormone. To obtain detailed insight into the function of N-glycosylation, the availability of site-specifically and fully deglycosylated alpha-subunits obtained under non-denaturing conditions is a prerequisite. NMR spectroscopy in combination with FAB-mapping demonstrates that only Asn52 of the alpha-subunit is accessible to digestion by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F under native conditions. Treatment of native alpha hCG with endo-beta-N-acetylglucosaminidase B results in full deglycosylation yielding alpha hCG with one GlcNAc residue at both Asn52 and Asn78.

PC-1 is an ecto-enzyme possessing alkaline phosphodiesterase I and nucleotide pyrophosphatase activities. In this paper, we demonstrate the expression, biochemical characterization and biosynthesis of human PC-1. Previously, there has been uncertainty concerning which of two methionine residues is the initiator. It is now shown that expression of PC-1 is much greater if the first methionine residue is present, and that the sequence between the two methionine residues is translated in both human and mouse, in both transfected cells and cells naturally expressing PC-1. The first methionine residue is therefore the initiator. Human PC-1 is capable of autophosphorylation, and conditions are described in which PC-1 is the only labelled phosphoprotein on the plasma membranes of intact cells, allowing the demonstration that the mature membrane form of human PC-1 is approximately 10 kDa larger than that of the mouse form. Pulse-chase biosynthetic studies and treatment with two different endoglycosidases show that most of this difference is due to N-linked oligosaccharides. The polypeptide backbone of human PC-1 is 20 amino acids longer than that of the mouse PC-1, with most of the difference in polypeptide chain length being in the cytoplasmic domain. The revised cytoplasmic domain of human PC-1 has 76 amino acids, while the mouse cytoplasmic domain has 58 amino acids. Optimal alignment of mouse and human cytoplasmic domains reveals areas of sequence conservation in which the third bases vary. It is suggested that these regions of conservation may point to functionally important sequences in the cytoplasmic domain.

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum, and the presence in the infected erythrocyte of the respective precursors. In in vitro cultures, P. falciparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or O-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into O-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. falciparum, using exogenous glycosyl donors and acceptors. Our studies show that O-glycosylation is the major form of protein glycosylation in intraerythrocytic P. falciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A., Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372, 645].

The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit. The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry. The following carbohydrate structures could be established: [formula: see text] The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115-122], having 3, 5 and 6 as common elements. The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B. cellulosolvens.

The ability of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked beta 1----2 to beta-mannose and fucose linked alpha 1----3 to the innermost N-acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of alpha 1----3-linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that alpha 1----3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.

alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.

A detailed study of the oligosaccharide specificity of the almond enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, was undertaken by comparing the rate of release of intact oligosaccharide chains from defined glycopeptides of all significant classes. The oligosaccharide of a trisialo-triantennary pentaglycopeptide from fetuin was released at the highest rate. A procedure was developed for the isolation of this glycopeptide in high yield from 5 g fetuin. Sequence analysis established the structure as Leu-Ala-Asn(CHO)-Cys-Ser. The Cys(Cm) and the Cys(Ae) derivatives of the glycopeptide were reacted with 4-(dimethylamino)-azobenzene-4'-sulfonyl (dabsyl) chloride to yield a monosubstituted and a disubstituted glycopeptide respectively. This chromophore confers high sensitivity at 436 nm on a pentapeptide backbone having minimal bonds for protease cleavage. A procedure was developed wherein these dabsyl derivatives were used in a high-performance liquid chromatography assay. The dabsyl-pentapeptide was retarded significantly from the dabsyl-glycopeptide and provided a sensitive method (1-2 nmol) of detection of peptide-N4-(N-acetylglucosaminyl)asparagine amidase activity. Enzyme was detected in crude extracts of all eight seed sources surveyed. The enzyme from Pisum sativum was partially purified and its properties were compared with the corresponding enzyme from almonds.

The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)