Glycosylation modifications

In Section II (on the biosynthesis of oligosaccharides), emphasis is placed on the biosynthesis of the dolichol-linked sugars, because the inhibitors mainly interfere with steps in the dolichol cycle of protein glycosylation. Modification of the oligosaccharide side-chain, once it has been transferred from the carrier lipid to the protein, is not discussed in any great detail. 

Glycosyl Modification Combined with Metabolic Control by Unnatural Sugars. 1861... 

Y.B. Chao, W.M. Scovell and S.B. Yan, Protein ScL, 1994, 3, 2452-2454, High mobility group protein, HMG-1, contains insignificant glycosyl modification. 

B. Chackerian, L. M. Rudensey, J. Overbaugh, Specific N-linked and O-linked glycosylation modifications in the envelope VI domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies. J. Virol. 1997, 71, 7719-7727. 

While electrospray is used for molecules of all molecular masses, it has had an especially marked impact on the measurement of accurate molecular mass for proteins. Traditionally, direct measurement of molecular mass on proteins has been difficult, with the obtained values accurate to only tens or even hundreds of Daltons. The advent of electrospray means that molecular masses of 20,000 Da and more can be measured with unprecedented accuracy (Figure 40.6). This level of accuracy means that it is also possible to identify post-translational modifications of proteins (e.g., glycosylation, acetylation, methylation, hydroxylation, etc.) and to detect mass changes associated with substitution or deletion of a single amino acid. 

FIGURE 9.20 The glycosyl phosphatidylinositol (GPI) moiety is an elaborate lipidanchoring group. Note the core of three mannose residues and a glucosamine. Additional modifications may include fatty acids at the inositol and glycerol —OH groups. 

Both ChEs undergo several post-translational modifications, including glycosylation and glycosylphosphatidy-linositolation (GPI), phosphorylation and carbamylation. 

Evolution has provided the cell with a repertoire of 20 amino acids to build proteins. The diversity of amino acid side chain properties is enormous, yet many additional functional groups have been selectively chosen to be covalently attached to side chains and this further increases the unique properties of proteins. Diese additional groups play a regulatory role allowing the cell to respond to changing cellular conditions and events. Known covalent modifications of proteins now include phosphorylation, methylation, acetylation, ubi-quitylation, hydroxylation, uridylylation and glycosyl-ation, among many others. Intense study in this field has shown the addition of a phosphate moiety to a protein... 

In the Koenigs-Knorr method and in the Helferich or Zemplen modifications thereof, a glycosyl halide (bromide or chloride iodides can be produced in situ by the addition of tetraalkylammonium iodide) is allowed to react with a hydrox-ylic compound in the presence of a heavy-metal promoter such as silver oxide, carbonate, perchlorate, or mercuric bromide and/or oxide,19-21 or by silver triflu-oromethanesulfonate22 (AgOTf). Related to this is the use of glycosyl fluoride donors,23 which normally are prepared from thioglycosides.24... 

From a mass spectrometry perspective, these modifications, such as phosphorylation or glycosylation, manifest themselves as an increase in the molecular weight of both the parent protein and also of the polypeptides (produced by enzymatic digestion) containing the modification. 

Post-translation modification Changes that occur to proteins after peptide-bond formation has occurred, e.g. glycosylation and acylation. 

Posttranslational modifications of S-layer proteins include cleavage of N- or C-terminal fragments, glycosylation, and phosphorylation of amino acid residues. 

DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes. However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of prohne and lysine, and disulfide bond formation that accompany mamra-tion. While Edman sequencing can detect the presence of most posttranslational events, technical hmitations often prevent identification of a specific modification. 

Mammalian proteins are the targets of a wide range of covalent modification processes. Modifications such as glycosylation, hydroxylation, and fatty acid acylation introduce new structural features into newly synthesized proteins that tend to persist for the lifetime of the protein. Among the covalent modifications that regulate protein function (eg, methylation, adenylylation), the most common by far is phosphorylation-dephos-phorylation. Protein kinases phosphorylate proteins by... 

There are extensive additional tissue-specific modifications of these peptides that affect activity. These modifications include phosphorylation, acetylation, glycosylation, and amidation. 

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