N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines

Summary The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5bl-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of α 1–3-linked mannose.     

Three-dimensional structure of a cell wall glycoprotein

Electron microscopy and computer image analysis have been used to determine the three-dimensional structure of the crystalline glycoprotein cell wall layer of the alga Lobomonas piriformis. Images of negatively stained specimens, tilted through a range of angles up to 70 °, were combined to give a map of the molecular envelope to a resolution of 2.0 nm. The cell wall layer consists of crystalline plates the centres and edges of which display distinctly different but isomorphous structures. A comparison of three-dimensional reconstructions of the two areas shows the difference probably to be due to a conformational change of one of the glycoprotein subunits. The structure consists of two sets of dimers composed of rod-shaped subunits which lie with their long axes approximately in the plane of the crystal. The centre-edge transition may have significance in the pathway of accretion of new subunits during cell wall growth.     

Extensin—a major cell wall glycoprotein

Abstract The structure of extensin is described in detail. It has a hydroxyproline-rich backbone, which contains repeating peptides glycosylated by short side chains and it adopts a polyproline II helical conformation. The glycoprotein is synthesized intracellularly and soluble precursors are secreted to the wall, where they are bound, perhaps, by the formation of isodityrosine cross-links. The various hypotheses, including the most recent ‘warp and weft’ model, which have been suggested to explain the attachment of extensin to the other wall polymers are discussed. The possible functions of extensin in defence and in the control of extension growth are described in addition to its probable structural role. Other glycoproteins which resemble extensin are also mentioned.     

Effect of different cell culture conditions on the polypeptide integrity and N-glycosylation of a recombinant model glycoprotein

The effect of different short-term controlled cell culture conditions on the product quality of a genetically engineered human interleukin-2 N-glycosylation variant protein expressed from a baby hamster kidney cell line (BHK-21) has been investigated. A perfused 2-L stirred tank reactor was used. Products purified from the culture supernatant of cells grown under experimentally initiated nutrient limitations (glucose, amino acids, pO(2)) were characterized by their HPLC-elution profile, SDS-PAGE and western blotting, amino acid sequencing as well as for their N-linked carbohydrates, using "HPAEC-PAD fingerprinting" and methylation analysis. The glycoprotein products secreted from cells under the different culture conditions (kept for 24 h, after an adaption time period of 48 h) showed an almost identical oligosaccharide pattern. By contrast, short-term changes of the culture condition led to considerable differences in the ratio of glycosylated to unglycosylated protein forms. Significant amounts of NH(2)-terminally truncated polypeptide forms were observed. They lacked proponderantly the first two amino acids; however, under certain culture conditions forms lacking up to eight NH(2)-terminal amino acids were detected. (c) 1995 John Wiley & Sons, Inc.     

N-glycosylation controls the function of junctional adhesion molecule-A

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells. JAM-A serves many roles and contributes to barrier function and cell migration and motility, and it also acts as a ligand for the leukocyte receptor LFA-1. JAM-A is reported to contain N-glycans, but the extent of this modification and its contribution to the protein’s functions are unknown. We show that human JAM-A contains a single N-glycan at N185 and that this residue is conserved across multiple mammalian species. A glycomutant lacking all N-glycans, N185Q, is able to reach the cell surface but exhibits decreased protein half-life compared with the wild- type protein. N-glycosylation of JAM-A is required for the protein’s ability to reinforce barrier function and contributes to Rap1 activity. We further show that glycosylation of N185 is required for JAM-A–mediated reduction of cell migration. Finally, we show that N-glycosylation of JAM-A regulates leukocyte adhesion and LFA-1 binding. These findings identify N-glycosylation as critical for JAM-A’s many functions.     

Heterogeneity and cell type-specific localization of a cell wall glycoprotein from carrot suspension cells

EP1, an extracellular protein from carrot (Daucus carota) cell suspensions, has been partially characterized by means of an antiserum and a cDNA clone. In both embryo and suspension cultures different molecular mass EP1 proteins were detected, some of which (31, 32, 52, and 54 kilodaltons) were bound to the cell wall and released into the medium, whereas others (49, 60, and 62 kilodaltons) were more firmly bound to the cell wall and could be extracted with a salt solution. Immunoprecipitation of in vitro translation products revealed a single primary translation product of 45 kilodaltons, suggesting that EP1 heterogeneity is due to differential posttranslational modification. In seedlings organ-specific modification of EP1 proteins was observed, a phenomenon which did not persist in suspension cultures initiated from different seedling organs. In culture EP1 proteins were only found to be associated with vacuolated, nonembryogenic cells, and on these cells they were localized in loosely attached, pectin-containing cell wall material. Purified 52/54 kilodaltons EP1 proteins did not alleviate the inhibitory effect of the glycosylation inhibitor tunicamycin on somatic embryogenesis.     

The structure of a glycopeptide isolated from the yeast cell wall

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.     

The chaotrope-soluble glycoprotein GP2 is a precursor of the insoluble glycoprotein framework of the Chlamydomonas cell wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.     

The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall

Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.     

Identification of a glycoprotein involved in cell cycle progression in yeast

The molecular events of start, the regulatory step that commits yeast cells to DNA replication, have recently begun to be investigated. One of the gene products required for completion of start has been found to have a significant structural homology with oncogenes endowed with protein kinase activity. Our experiments provide data on the biosynthetic pathway of a previously identified labile protein (p100, molecular weight 100,000, isoelectric point of approximately 4.8-5) involved in cell cycle progression at start, which appears to be specifically made during the release from cell cycle arrest of a temperature-sensitive mutant (cdc25) of Saccharomyces cerevisiae. On two-dimensional gel, p100 migrates very close to another 100-kDa labile protein (p100*) which behaves as a cell cycle modulated protein with reduced synthesis in G1. Pulse and chase labeling of protein with [35S]methionine suggests that both p100 and p100* are processed to a protein (p115) of slightly higher molecular weight (Mr = 115,000). Peptide mapping analysis indicates that p100 and p100 yield identical maps and that both p100 and p100* are very much similar to p115. p115 is a glycosylated protein as shown by a labeling experiment with [3H]glucosamine and by the fact that the synthesis of both p100 and p115 is inhibited if cells are cultured in the presence of tunicamycin. A protein having the same heterogeneous aspect of migration on sodium dodecyl sulfate-polyacrylamide gel and the same apparent molecular weight and isoelectric point of p115 is abundantly present in a preparation of membranes from S. cerevisiae and the isolated radioactive p115 comigrates with it. Taken together these results favor the idea that terminal glycosylation of both p100 and p100* gives rise to the fully glycosylated p115 protein which appears to be a membrane-associated protein.     

Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata

Gametophytic self-incompatibility, a mechanism that preventsinbreeding in some families of flowering plants, is mediatedby the products of a single genetic locus, the S-locus. Theproducts of the S-gene in the female sexual tissues of Nicotianaalata are an allelic series of glycoproteins with RNase activity.In this study, we report on the microheterogeneity of N-linkedglycosylation at the four potential N-glycosylation sites ofthe S²-glycoprotein. The S-glycoproteins from N.alata containfrom one to five potential N-glycosylation sites based on theconsensus sequence Asn-Xaa-Ser/Thr. The S²-glycoprotein containsfour potential N-glycosylation sites at Asn²⁷, Asn³⁷, Asn¹³⁸and Asn¹⁵⁰, designated sites I, n, IV and V, respectively. SiteIII is absent from the S₂-glycoprotein. Analysis of glycopeptidesgenerated from the S₂-glycoprotein by trypsin and chymotrypsindigestions revealed the types of glycans and the degree of microheterogeneitypresent at each site. Sites I (Asn²⁷) and IV (Asn¹³⁸) displaymicroheterogeneity, site II (Asn³⁷) contains only a single typeof N-glycan, and site V (Asn¹⁵⁰) is not glycosylated. The microheterogeneityobserved at site I on the S₂-glycoprotein is the same as thatobserved at the only site, site I, on the Srglycoprotein (Woodwardet al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylationconsensus sequence at site I is conserved in all S-glycoproteinsfrom other species of self-incompatible solanaceous plants,glycosylation at this site may be important to their function.No other post-translational modifications (e.g. O-glycosylation,phosphorylation) were detected on the S₂-glycoprotein. fertilization microheterogeneity N-glycans plants RNase     

Proteolytic activity of a yeast cell wall lytic Arthrobacter species

AIMS: To investigate properties of the proteolytic activity of a yeast cell wall lytic soil bacterium identified as an Arthrobacter species. METHODS AND RESULTS: The organism was grown at pH 7.5 and 30 degrees C in shake flasks on media with different complex subtrates. Highest proteolytic activity assayed with azocaseine was detected in media with wheat gluten. In addition, l-leucine, l-alanine exopeptidase activity and esterase activity were found. The proteolytic activity showed stability up to pH 12, with a maximum at pH 11. The temperature optimum was at 55 degrees C, but there was a loss in enzyme activity of 50% within 2 h. The proteolytic activity was inhibited by 3,4-dichloroisocumarin, whereas there was little or no effect with EDTA, pepstatin A or E64. CONCLUSIONS: The proteolytic activity is highly alkaline stable. The formation of the enzyme can be induced by media with high protein content.     

The humanization of N-glycosylation pathways in yeast

Yeast and other fungal protein-expression hosts have been extensively used to produce industrial enzymes, and are often the expression system of choice when manufacturing costs are of primary concern. However, for the production of therapeutic glycoproteins intended for use in humans, yeast have been less useful owing to their inability to modify proteins with human glycosylation structures. Yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and thereby compromises the efficacy of most therapeutic glycoproteins. Several approaches to humanizing yeast N-glycosylation pathways have been attempted over the past decade with limited success. Recently however, advances in the glycoengineering of yeast and the expression of therapeutic glycoproteins with humanized N-glycosylation structures have shown significant promise - this review summarizes the most important developments in the field.     

Acid-treated yeast cell wall as a binder displaying function of disintegrant

This investigation examined the application of acid-treated yeast cell wall (AYC) as a binder functioning as a disintegrant. Acetylsalicylic acid (ASA) was granulated with AYC, hydroxypropylcellulose (HPC), polyvinylpyrrolidone (PVP), or pullulan (PUL) and compressed into a tablet in the absence of disintegrant. Particle size and angle of repose of the granules, tensile strength, disintegration time, and water absorption behavior of the tablets and ASA release profiles from the tablets were measured. The surface of AYC-granules was observed with a scanning electron microscope. As was the case with the granules of HPC, PVP, or PUL, D50 of the granules of AYC increased with increasing AYC addition percentage, indicating that it is possible to granulate ASA with AYC. Tablets incorporating HPC, PVP, and PUL failed to disintegrate within 30 minutes at all percentages of binder addition because in the case of the HPC, PVP, or PUL tablets in the dissolution medium, water scarcely penetrated into the inner region of the tablet, causing no disintegration. In the case of the AYC tablets, disintegration was not detected at 3% or less of AYC. When AYC was equal to or greater than 5%, AYC tablets disintegrated in approximately 4 minutes and rapid ASA release from the tablets was observed. These results may have been caused by the following. In the case of the AYC 3% granules, ungranulated aspirin powder remained, but in the case of the AYC 5% granules, ASA powder was granulated and covered with AYC. Water absorption was observed initially; however, a plateau was reached in the case of the AYC 3%-tablet. In contrast, in the cases of the AYC 5% and more tablets, water absorption was greater and increased with time. The angle of repose of the AYC 5% granules was 25.7°, which represented high fluidity. The tablets produced by compressing the granules demonstrated sufficient tensile strength greater than 0.8 MPa. The tablets rapidly disintegrated and rapid ASA release was obtained. AYC functioned as a binder at granulation; additionally, AYC served as a disintegrant in the dissolution of drug from the tablets. These results indicate that AYC affords high utility as a unique pharmaceutical additive possessing contrary functions such as binding and disintegration.