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Glycobiology I

BIOL 30603 Lecture 21

Revised Central Dogma

  • Glycobiology: A field of biology that studies the structure and function of glycoconjugates and the proteins that specifically interact with them.

Review: Protein Orientation

  • Protein Orientation:
    • Conserved once inserted into the membrane.
  • Key Components:
    • Cargo molecules
    • CYTOSOL, DONOR COMPARTMENT, LUMEN, TARGET COMPARTMENT
    • Process of Fusion and Budding.

Review: From the ER to the Golgi

  • Vesicles leaving the ER:
    • Formed at the ER exit sites that concentrate secretory cargoes.

Main Functions of the Golgi Apparatus

  • Function:
    • Modify endomembrane proteins.
    • Sort and package these proteins for secretion.

Glycoconjugates

  • Definition:
    • Covalent attachment of one or more sugars to a protein or lipid.
    • Also referred to as a glycan.
  • Glycosylation:
    • Most proteins synthesized in the rough ER are glycosylated by the addition of a common N-linked oligosaccharide.
  • N-linked refers to sugars added to the amino acid Asparagine (N).
  • Importance of Glycosylation:
    • Major biosynthetic function of the ER.

Glycosylation Mechanism

  • Pre-formed Precursor Oligosaccharide:
    • Transferred to proteins in the ER.
    • Asn-X-Ser or Asn-X-Thr where X is any amino acid except Proline.
  • Key Structures:
    • Dolichol-linked oligosaccharides involved in the glycosylation process.

N-Linked Glycosylation in the ER

  • Protein Folding:
    • Only properly folded proteins with the structure (Man)<em>8(GlcNAc)</em>2(Man)<em>8(GlcNAc)</em>2 can exit the ER to the cis Golgi.
    • Glycosidases: Remove 3 Glu and 1 Man.
    • Calnexin: An ER chaperone and lectin that binds to oligosaccharides on incompletely folded proteins.
    • Role of Enzymes:
    • Glycosidases remove sugars
    • Transferases add sugars.

Degradation Pathway in the ER

  • Components Involved:
    • AAA-ATPase, N-glycanase, E3 ubiquitin ligase, chaperones, and disulfide isomerase play roles in targeting and recognition of misfolded proteins.
  • Degradation Process:
    • Ubiquitin is tagged for degradation via a polyubiquitin chain.
    • Proteasome is involved in the degradation of misfolded proteins.

Addition and Initial Processing of N-linked Oligosaccharides

  • Oligosaccharide Structure:
    • Original N-linked oligosaccharide processed as follows:
    • Resulting in structures like $(Glc)3(Man)$(-n)(GlcNAc)_2.
  • Final Form:
    • Modified structure delivered to the cis-Golgi.

Oligosaccharide Processing in Golgi Compartments

  • Steps Involved:
    • Phosphorylation of oligosaccharides on lysosomal proteins.
    • Various processing events including removal of Man and addition of GlcNAc, Gal, and NANA (N-acetylneuraminic acid).
  • Sorting:
    • Occurs in different compartments: Cis Golgi network, cis, medial, trans cisternae, and trans Golgi network.

Two Classes of N-Linked Oligosaccharides

  • Complex Oligosaccharides:
    • Generated when the original N-linked oligosaccharide in the ER is trimmed and further sugars added.
  • High-Mannose Oligosaccharides:
    • Generated when the original N-linked oligosaccharide is trimmed but no new sugars are added.

Assembly of Complex Oligosaccharides

  • Enzymatic Breakdown Process:
    1. First step involves glucosidase I and II that modify oligosaccharides.
    2. Mannosidase enzymes in Golgi facilitate further processing.
    3. Complex oligosaccharides are formed using UDP and CMP sugar donors.
  • Endo H Sensitivity:
    • Differentiates between high-mannose and processed complex forms.

Glycosylation and the Lysosome

  • Mannose 6-Phosphate (M6P) Tag:
    • Added to lysosomal proteins in the Golgi.
    • M6P directs proteins to lysosomes.

Mannose 6-Phosphate Addition to Lysosomal Hydrolases

  • Key Processes:
    • A signal patch is recognized by specific phosphotransferase, transferring a phosphate group to mannose in the oligosaccharide.
  • Final Outcome:
    • Lysosomal hydrolases are tagged with a GlcNAc-P attached to mannose.

Transport of Lysosomal Hydrolases

  • Transport Mechanism:
    • Involves M6P receptors interacting with lysosomal hydrolases.
    • Receptors ensure precise delivery of precursor proteins to lysosomes, facilitated by clathrin-coated vesicle transport.
  • Dissociation:
    • Occurs at acidic pH, where the phosphate group is removed.

Lysosomal Storage Diseases

  • Overview:
    • Result in accumulation of undigested substrates in lysosomes.
    • Example: I-cell disease, caused by a defective GlcNAc phosphotransferase gene, leading to the impairment of lysosomal hydrolase targeting.
  • Implications:
    • Most lysosomal hydrolases are secreted and found in blood, and treatments for I-Cell disease are currently unavailable.
    • Fatal prognosis, typically by age 6.