N glycosylation

Further modifications are different in plants and mammals. In plants, an alpha 

3-linked fucosylation to the first core GlcNAc, and a beta 1,2-linked xylosylation to the first core mannose residue, are mediated by specific glycosyltransferases (for a review, see Ref [58]). These structures are common for plants, including mosses [59, 60]. 

The major drawback for plant-derived complex biopharmaceuticals is that the plant-specific xylosyl and fucosyl residues are attached to the core structure of N-gly-cans. Although Ghargelegue et al. [62] observed no immunogenic effects of a plant-derived murine monoclonal antibody in an animal study based on a mouse model, both residues are described in the Htera-ture as structures with high immunogenic potential [8, 9]. 

Gonsequently, major efforts were made to overcome this limitation. One approach is based on an observation by von Schae-wen et al. [63]. These authors isolated a mutant strain of Arabidopsis thaliana (cgl) which showed a loss of GNTI activity. AU N-glycan structures isolated from this mutant strain were related to high mannose-type, and no complex-type structures were detected. The loss of complex-type N-gly-cans was accompanied by the loss of the 

Taken together, humanized glycosylation at least sufficient for antibody production was achieved in genetically engineered moss strains, without any negative influence on growth rates in bioreactor cultures or on their secretion capacity for recombinant proteins. 

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Purines, N-alkyl-N-phenyl-synthesis, 5, 576 Purines, alkylthio-hydrolysis, 5, 560 Mannich reaction, 5, 536 Michael addition reactions, 5, 536 Purines, S-alkylthio-hydrolysis, 5, 560 Purines, amino-alkylation, 5, 530, 551 IR spectra, 5, 518 reactions, 5, 551-553 with diazonium ions, 5, 538 reduction, 5, 541 UV spectra, 5, 517 Purines, N-amino-synthesis, 5, 595 Purines, aminohydroxy-hydrogenation, 5, 555 reactions, 5, 555 Purines, aminooxo-reactions, 5, 557 thiation, 5, 557 Purines, bromo-synthesis, 5, 557 Purines, chloro-synthesis, 5, 573 Purines, cyano-reactions, 5, 550 Purines, dialkoxy-rearrangement, 5, 558 Purines, diazoreactions, 5, 96 Purines, dioxo-alkylation, 5, 532 Purines, N-glycosyl-, 5, 536 Purines, halo-N-alkylation, 5, 529 hydrogenolysis, 5, 562 reactions, 5, 561-562, 564 with alkoxides, 5, 563 synthesis, 5, 556 Purines, hydrazino-reactions, 5, 553 Purines, hydroxyamino-reactions, 5, 556 Purines, 8-lithiotrimethylsilyl-nucleosides alkylation, 5, 537 Purines, N-methyl-magnetic circular dichroism, 5, 523 Purines, methylthio-bromination, 5, 559 Purines, nitro-reactions, 5, 550, 551 Purines, oxo-alkylation, 5, 532 amination, 5, 557 dipole moments, 5, 522 H NMR, 5, 512 pJfa, 5, 524 reactions, 5, 556-557 with diazonium ions, 5, 538 reduction, 5, 541 thiation, 5, 557 Purines, oxohydro-IR spectra, 5, 518 Purines, selenoxo-synthesis, 5, 597 Purines, thio-acylation, 5, 559 alkylation, 5, 559 Purines, thioxo-acetylation, 5, 559... 

Glycosydation AChE and BChE carry 3 and 9, respectively, N-glycosylation consensus sequences attaching carbohydrate residues to the core protein via asparagines. Different molecular forms of the enzymes in various tissues, show different number and composition of carbohydrate residues. N-glycosylation at all sites was shown to be important for effective biosynthesis, secretion and clearance of ChEs from the circulation. Altered patterns of AChE glycosylation have been observed in the brain and cerebrospinal fluid of Alzheimer s disease (AD) patients, with potential diagnostic value. 

N-Glycosyl derivatives are conveniently named as glycosylamines. In the case of complex heterocyclic amines, such as nucleosides, the same approach is used. 

Fischer projection of acyclic form, 56-57 glycosides, 132-135 C-glycosyl compounds, 139-140 N-glycosyl derivatives, 137-139 glycosyl halides, 136-137 glycosyl residues, 125 isotopic substitution and isotopic labelling, 91 me so forms, 59 optical rotation, 59 parent structure choice, 53... 

Keywords aza-Diels-Alder reactions, N-glycosyl imines as dienophiles, piperidine... 

The genes encoding the polypeptide backbones of a number of mucins derived from various tissues (eg, pancreas, small intestine, trachea and bronchi, stomach, and salivary glands) have been cloned and sequenced. These studies have revealed new information about the polypeptide backbones of mucins (size of tandem repeats, potential sites of N-glycosylation, etc) and ultimately should reveal aspects of their genetic control. Some important properties of mucins are summarized in Table 47-8. 

The process of N-glycosylation can be broken down into two stages (1) assembly of Dol-P-P-oligosaccha-ride and transfer of the oligosaccharide and (2) processing of the oligosaccharide chain. 

Some Glycan Intermediates Formed During N-Glycosylation Have Specific Functions... 

Fig. 1.—Amino Acid Sequence for Glycophorins and A. [Diamonds represent points of 0-glycosylation. The single N-glycosylation point occurs at Asn-26. The data were obtained from Refs. 8 and 19.]...

The diamonds in Fig. 1 represent the point of O-glycosylation by tetrasac-charide 1. Asn-26 indicates the point of N-glycosylation by the complex oligosaccharide 2. 

One potential N-glycosylation site is present in all endopolygalacturonases. This N-glycosylation sequon is conserved throughout whereas PGI has two sequons, the second one at a more C-terminal location. 

The affinity chromatography on ConA - cellulose indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results). This method was usefull for purification of these enzymes from other protein inpurities but was completely uneffective by separation of individual forms (Fig. 4). 

Figure 3. Schematic representation of the PGII, PGI, PGC [13] and PGE proteins from A. niger, indicating the putative processing sites for the signal peptide ( ) and the mono- and dibasic processing site for the propeptide ( ). The position of introns (lA, IB and IC) are indicated ( [) and variation of amino acids number is shown in different parts of protein. The putative N-glycosylation sites are marked ( ).

PGE was isolated as desribed in Material and Methods. SDS-PAGE electrophoresis of purified protein showed a single band migrating at approximately 60 kDa. This observation is not in the agreement with the calculated molecular weight of 35 584. However a similar effect has been observed previously in case of PGI and PGC. Apart of the N-glycosylation which plays role in all PGs (Fig. 3), O-glycosylation may also be present as indicated by the band size shift after a treatment of PGE with O.IM NaOH (data not shown). 

D-Gal — hydroxy-L-histidine,4950 d-G1cA — hydroxy-L-tryptophan,51 d-GlcA — hydroxy-L-phenylalanine,51 d-G1cA — L-Ser,51 and carbohydrates N-glycosylated to the a-amino group of the N-terminal portion of proteins.51-53 Most of these compounds will be discussed in more depth later in this article, in terms of model compounds for oligosaccharide linkages to proteins. 

III. Structural Analysis of Glycopeptides and Glycoproteins Containing N-Glycosyl Linkages... 

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