Post-translational modification

• Threonine and serine are the most likely amino acids to be post-translationally modified due to their hydroxyl groups
• Post-translational modification is beneficial as it reduces the need for additional genes and provides a rapid mechanism for altering protein function
• Histones can have the following post-translational modifications:

  • Methylation
  • Acetylation
  • Ubiquitination
  • Phosphorylation
• All cells have a sugar coat called the glycocalyx. The glycocalyx allows the cell to recognise other cells to determine if they should interact
• Glycans presented on the surface of one cell are recognised and bound by lectins presented on the surface of another cell
• Glycans are biomolecules made up of monosaccharides
• The differences between monosaccharides are usually differences in hydroxyl orientation - as such, lectins are highly specific to the slight differences in hydroxyl orientation
• Symbol for alpha-D-mannose:

  Green circle

  
• Symbol for alpha-D-N-acetylneuraminic acid:

  Purple diamond

  
• Symbol for alpha-D-N-glycolylneuraminic acid:

  Light blue diamond

  
• Symbol for beta-D-glucose:

  Blue circle

  
• Symbol for alpha-D-N-acetylglucosamine:

  Blue square

  
• Symbol for alpha-L-fucose

  Red triangle

  
• Symbol for alpha-D-N-acetylgalactosamine:

  Yellow square

  
• Symbol for beta-D-xylose:

  Orange star

  
• Symbol for beta-D-galactose:

  Yellow circle

  
• Humans cannot oxidise NeuAc to NeuGc due to a partial gene deletion resulting in an inactive hydroxylase
• Glycosidic bond formation happens via dehydration reaction, resulting in a glycosidic linkage that can be in the alpha or beta formation
• Glycosidic bond formation requires enzymatic action as it is not energetically favourable and a number of different bond orientations are possible - leads to formation of branched glycans
• Glycoproteins are the most common type of glycosylation in the human body; the majority of proteins are glycosylated
• A high proportion of secreted and membrane bound proteins are glycosylated as their hydrophilicity is favourable
• N-glycosylation involves linking a sugar to an amide nitrogen in the side chain of an asparagine residue
• O-glycosylation involves linking a sugar to an oxygen in the side chain of serine or threonine
• The consensus sequence for N-glycosylation is Asn-X-Ser/Thr where:

  • The sugar is linked to the Asn
  • X is any amino acid except proline
• N-glycans are added to Asn residues as the nascent protein is emerging from the ribosome on the surface of the rough endoplasmic reticulum - protein synthesis takes place at these ribosomes to allow for N-glycosylation
• Due to the hydrophilic nature of glycans, the nascent protein preparing to be glycosylated remains unfolded until N-glycosylation has taken place
• The conserved structure of N-glycans consists of two N-acetylglucosamine molecules joined to three D-mannose in a V-shape:

  
• The end N-acetylglucosamine is linked to a dolichol lipid in the ER membrane prior to linking to the protein
• The D-mannose glycans link to variable antennae regions made up of variable other glycans
• Process of N-glycan biosynthesis (organelles):

  1. Synthesis of lipid-linked precursor oligosaccharide in the ER
  2. At the same time as the protein is ejected into the ER from the surface-bound ribosome, the N-glycan is added to Asn in the Asn-X-Ser/Thr consensus sequence
  3. Initial trimming of glycans from precursor prior to exocytosis
  4. Addition of terminal sugar residues in Golgi
  5. Secretion of mature glycan/delivery to plasma membrane
• N-glycan biosynthesis (biochemical):

  1. Biosynthetic precursor (linked to Asn) is cleaved by glucosidase
  2. Mannosidases cleave to form high mannose N-glycans - can be terminal
  3. Can cleave with glycosyltransferases to form hybrid N-glycans (terminal) OR N-acetylglucosaminyltransferase I (next step)
  4. Mannosidases cleave again - product goes on to undergo branching and elongation to form complex N-glycans
• High mannose N-glycans always contain between five and nine mannose residues
• Important when biosynthesising N-glycans to form beta glycosidic bonds - these form straight, linear glycans
• Most antennae are made up of N-acetylglucosamine and galactose residues linked by beta-glycosidic linkages
• On top of the GlcNAc/Gal antennae "flagpoles", alpha-linked sugars are added to dictate functionality
• Addition of an alpha-linked sugar to the top of the antennae causes a kink to form, meaning no more sugars can sequentially be addede
• Terminal alpha-linked sugars such as fucose and GalNAc are recognised by lectins and antibodies
• As the glycosidic bond is more flexible than the peptide bond, they take up a larger hydrodynamic volume and take up more space in free solution
• Three types of N-glycans:

  • High mannose
  • Hybrid
  • Complex
• O-glycosylation occurs on Ser and Thr residues - no consensus sequence but preferably near Pro and tandem Ser/Thr repeats