Post-Translational Modifications: Phosphorylation & Glycosylation

🧬 Post-Translational Modifications (PTMs)

• Proteins achieve chemical variety after translation via:

  • Binding cofactors

  • Post-translational modifications (PTMs)

  • Incorporating non-standard amino acids

• PTMs often regulate protein function or localisation.

✳ Examples of PTMs:

• Phosphorylation (activation/inactivation)

• Glycosylation

• Ubiquitination

• S-Nitrosylation

• Methylation

• Acetylation

• Lipidation

• Proteolytic cleavage (e.g. zymogens like trypsinogen → trypsin)

🔁 Phosphorylation

🧪 Definition:

Addition of a phosphate group (PO₄³⁻) to specific amino acids — reversible covalent modification

🧠 Why is it important?

• Regulates protein activity

• Alters charge, conformation, binding properties

• Used in signalling cascades (e.g. fight or flight)

• Amplifies signals

🔄 Reversible process:

• Kinases add phosphate (from ATP)

• Phosphatases remove phosphate

🧬 Amino acids phosphorylated (in eukaryotes):

• Serine (pS) – ~95%

• Threonine (pT)

• Tyrosine (pY) – ~1%, key in Receptor Tyrosine Kinases (RTKs)

⚙ Mechanism (for serine/threonine):

1. Enzyme removes H⁺ from hydroxyl group

2. Nucleophilic attack on ATP’s γ-phosphate

3. Phosphate transferred → forms phospho-serine/threonine

4. ADP is released

ΔG°’ = –50 kJ/mol → energetically favourable

📦 Kinase Specificity:

• Some are highly specific

• Others are multifunctional

• Recognise consensus sequences, e.g.

  • PKA: Arg–Arg–X–Ser–Z
    (X = small, Z = large hydrophobic)

📌 Domain recognition:

• pY: SH2 and PTB domains

• pS: WW and MH2 domains

• pT: FHA domains

WW domain:

• ~40 residues, recognises pS/pT motifs

• Present in 98+ human proteins

🔄 Effects of phosphorylation:

• Conformational change → alters activity

• Creates binding sites → recruits signalling proteins
  → Critical in signal transduction

🧪 Example: Protein Kinase A (PKA) Pathway

🧬 Trigger: Epinephrine → β-adrenergic GPCR
➡ ↑ cAMP
➡ Binds to PKA regulatory subunits
➡ Releases catalytic subunits → active PKA

🧬 Effect: Fight or flight (metabolic activation)

🧬 Clinical links:

Cushing’s Syndrome:

• Mutation → PKA always active (even without cAMP)

• Results in excess cortisol secretion

Cystic Fibrosis:

• Phosphorylation of CFTR → allows ATP binding

• Opens Cl⁻ channel

• Mutation affects regulation → CF symptoms

🍬 Glycosylation

🧪 Definition:

Covalent addition of carbohydrate chains (sugars) to proteins — non-reversible

Occurs on:

• Asparagine (Asn) – N-linked

• Serine/Threonine – O-linked

🧬 Types of glycoproteins:

1. Glycoproteins: mostly protein (e.g. membrane proteins)

2. Proteoglycans: mostly carbohydrate (e.g. extracellular matrix)

3. Mucins: high sugar content; lubricants

🏭 Where it happens:

N-linked glycosylation

📍 Occurs in ER + Golgi
🧬 Core: 5 sugars (3 mannoses + 2 GlcNAc)
🧬 Built on dolichol, transferred by oligosaccharyltransferase

🧬 Motif: Asn-X-Ser/Thr

O-linked glycosylation

📍 Occurs in Golgi
🧬 Simpler sugars than N-linked
🧬 On serine or threonine

🧠 Why is glycosylation important?

✔ Stability – increases protein lifespan
✔ Solubility – sugars are hydrophilic
✔ Folding quality control – only properly folded proteins proceed
✔ Cell signalling – sugars act as ligands
✔ Protein-protein interaction

🧬 Proteins that bind carbohydrates = lectins

⚙ Enzymes involved:

• Glycosyltransferases – add sugars

• Glycosidases – remove sugars

🧬 Clinical relevance:

• Congenital glycosylation disorders → often lethal

• HIV envelope glycoprotein:

  • Heavily glycosylated

  • Binds to host cells & facilitates membrane fusion

🧠 Quick Comparison Table