Post -translational modifications

The lantibiotics differ extensively from the class II bacteriocins in that they contain post-translationally modified amino acids, as for example dehydrated amino acids and lanthionine residues, forming intramolecular thioether bridges [39, 184]. The chemical modification reactions leading to the typical lanthionines were first proposed by Ingram [185] and are assumed to be catalyzed by specific enzymes encoded in the lantibiotic gene cluster. In the lantibiotic lactocin S, p-alanine residues were discovered, probably by conversion of dehydrated serine residues via a dehydrogenation reaction [82]. In some 

The presence of two genes, nisB and nisC, encoding 993- and 414-residue proteins without significant homology to other known proteins, but conserved in several lantibiotic operons, has made them strong candidates for post-trans-lational modifications in the maturation pathway of lantibiotics [40]. Limited similarity between NisB and E. coli IlvA, a threonine dehydratase, was reported and hence a dehydratase function for NisB was suggested [40]. Mutation studies of NisB, NisC, EpiB, EpiC, and SpaB indicated that these proteins were essential for nisin, epidermin and subtilin biosynthesis, respectively [40,86,87,190]. As no precursors have been identified and characterized in these mutants, conclusions about the reaction that is catalyzed by these proteins remain speculative [40]. Secondary-structure predictions and experimental evidence confirmed that NisB and SpaB are both membrane-bound [100]. 

The complex N-linked oligosaccharide contains the j8-Man-di-A-acetylchitobiose (Man-/3-l,4-Glc N Ac-/3-l,4-Glc N Ac-Asn) core structure and also consists of a variable number of outer chains containing sialic acid (sial), galactose (Gal) and fu-cose (fuc) residues linked to the core. Usually, 2a-mannose (Man) residues are at- 

Many biologically active secreted peptides are also amidated at their carboxyl terminal, and acetylated at their amino-terminal. The consequences of these modifications are (a) to reduce the susceptibility of these peptides to degradative actions of extracellular aminopeptidases and carboxypeptidases after their secretion and (b) to influence the biological activity of the peptides. Corticotropin-releasing factor, gastrin, cholecystokinin and vasopressin require the C-terminal amide for full activity [54—56]. Acetylation of the N-terminus of a-MSH is necessary for activity, whereas acetylation of /3-endorphin inhibits its opioid activity [57], The enzymes responsible for acetylation have been identified from bovine and rat intermediate lobes [57] and enzymes with a-amidation activity have been reported in preparations of pituitary secretory granules [54,55]. 

FIGURE 10.14 MALDI-TOF mass spectrum of a 5% portion of the peptide products isolated from a single resin bead. Reprinted with permission from reference 22. 

FQPHHH. The mass differences between the capped species is straightforward. However, the difference in mass between the intact species at m/z 1267.5 and the first capped species at m/z 1243.4 must account for the mass of the CAP (113.0 Da). Thus, 1267.5 - 1243.4 + 113.0 = 137.1, which corresponds to a histidine residue. 

Expressed proteins are often modified by post-translational processing, which may include cleavages at specific residues chemical modifications to the amino or carboxy terminus or to specific residues formation of disulfide bonds or the addition of phosphate, sulfete, fatty acyl groups, or carbohydrate. Chemical modifications may occur as well during sample isolation and preparation. When the sequence of the protein is known or can be inferred from the gene sequence, the type and location of specific modifications can often be deduced from simple mass differences between the expected and observed peaks in an enzymatic map. Thus, Table 10.4 shows a list of common modifications, their sites, and the mass differences that would be observed. 

C-terminal amidation Terminal -COOH Replaced by -CONHj -1 

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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. 

Post-translational modifications to proteins are biochemical in origin and alter the measured molecular mass relative to that calculated for an untranslated sequence. 

Yeast. The advantages of expression in yeast include potentially high level production of proteins, the abiUty to have expressed proteins secreted into the media for ease of purification, and relatively low cost, easy scale-up. A disadvantage is that plasmid instabiUty may be a problem which can lead to low product yield. Whereas post-translational modification occurs in yeast, proteins are quite often hyperglycosylated. This is generally a problem with expression in Saccharomyces cerevisiae but not for the more recently used yeast host Pichiapastoris (25) (see Yeasts). 

Insect Cells. In this system the cDNA is inserted into the genome of an insect vims, baculovims. Insect cells, or Hve insect larvae, are then infected with the vims. In this way advantage is taken of the vims s natural machinery for repHcation utilizing the insect cell. This is one of the best systems available for high level production of native protein having post-translational modifications similar to those seen in mammalian cells. Disadvantages of this system include lytic—batch variations, comparatively slow growth, and cosdy scale-up. 

MammaBan. For mammalian proteins, mammalian cells offer the most natural host for expression. Problems of incorrect processing and post-translational modification are avoided using these cells. Mammalian cells are usually grown in continuous cell culture, reducing the variabiUty in results (see Cell CULTURE technology). Moderate-level production of native protein is possible. The procedure, however, is slow and very cosdy, and the level of protein expression is low. Thus large-scale production of proteins in mammalian cells is not practical. When low quantities of protein are sufficient, this system offers the several advantages described. 

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

After their synthesis (translation), most proteins go through a maturation process, called post-translational modification that affects their activity. One common post-translational modification of proteins is phosphorylation. Two functional classes of enzymes mediate this reversible process protein kinases add phosphate groups to hydroxyl groups of serine, threonine and tyrosine in their substrate, while protein phosphatases remove phosphate groups. The phosphate-linking... 

Proteosomal degration is the process by which improperly folded proteins or proteins with altered post-translational modifications are removed from a cell before they have a detrimental effect on cellular function. This is performed in small organelles known as proteosomes. Proteins are targeted for destruction in the proteosome by having a number of small ubiquitin molecules added. 

Poor Metabolizer Phenotype Population Pharmacokinetics Positron Emission Tomography Post-translational Modification Potassium Channels Potassium Competitive Acid Blockers PP... 

The Location of Post-Translational Modifications Using LC-MS Data from an Enzyme Digest 170... 

Post-translational modification may affect the biological activity of a protein and the location of such modifications is an extension of sequencing. 

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

Myllyharjn J (2005) Intracellular Post-Translational Modifications of CoUagens. 247 115-148... 

Very few post-translational modifications have been found on tropoelastin. However, hydroxylation of 25% of the proline residues is observed [10]. The enzymatic modification of proline to hydroxyproline (Hyp) is performed by prolyl hydroxylase [11]. The purpose of this hydroxylation remains unclear and it is even proposed that Hyps in tropoelastin are a by-product of collagen hydroxylation as this occurs in the same cellular compartment [8]. 

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