Protein Glycosylation 1

Course Information

Module: BIOL2012 Topic: Glycosylation and its Influence on Glycoprotein Structure and Function Lecture Title: Protein Glycosylation 1 Date: 16th March 2026 Lecturer: Dr Joel Allen, University of Southampton

Introduction to Glycobiology

Glycobiology is defined as the specialized study of the structure, biosynthesis, and overarching biology of saccharides. These saccharides, often referred to as sugar chains or glycans, are found extensively distributed throughout the natural world. This field examines how these complex carbohydrate structures interact with mechanical and biological systems to influence health and disease.

Complexity Beyond Genetic Encoding

Protein structure and function are not solely determined by the genetic code. While the central dogma (Gene → RNA → Peptide → Protein) provides the primary amino acid sequence, glycosylation represents a significant post-translational modification that adds layers of functional complexity.

Types of Glycan Attachment

The attachment of glycans to proteins is often dictated by specific protein sequence motifs or domains:

• N-linked Glycosylation: Attachment occurs at the nitrogen atom of an Asparagine ($\text{Asn}$) residue. This process usually requires a specific consensus sequence (sequon) denoted as $\dots ext{NxS/Tx} …$, where $\text{N}$ is Asparagine, $\text{x}$ is any amino acid except Proline, and $\text{S/T}$ is either Serine or Threonine.

• O-linked GalNAc Glycosylation: Attachment occurs at the oxygen atom of Serine ($\text{Ser}$) or Threonine ($\text{Thr}$) residues. These are frequently associated with $\text{STP}$ (Serine, Threonine, Proline) domains.

Clinical and Economic Importance of Glycoproteins

Glycoproteins, particularly monoclonal antibodies (mAbs), represent a massive sector of the pharmaceutical industry. The market value for these treatments is projected to reach approximately $\$300 imes 10^9 \, \text{USD}$ in 2025.

Historical Timeline and Successful Therapeutics

• 1975: Successful test of anti-lymphoma antibodies.

• 1981: Development of the first mAb.

• 1986: Muromonab-CD3 (Orthoclone OKT3) - anti-CD3 for kidney transplantation rejection treatment.

• 1994: Abciximab (Reopro) - anti-GP IIb/IIIa for the prevention of blood clots in angioplasty.

• 1997: Rituximab (Rituxan) - anti-CD20 for Non-Hodgkin lymphoma.

• 1998: Trastuzumab (Herceptin) - anti-HER2 for breast cancer; Infliximab (Remicade) - anti-TNF$\alpha$ for Crohn's disease.

• 2002: Adalimumab (Humira) - anti-TNF$\alpha$ for Rheumatoid Arthritis (RA).

• 2003: Omalizumab (Xolair) - anti-IgE for asthma.

• 2004: Bevacizumab (Avastin) - anti-VEGF for colorectal cancer (CRC); Cetuximab (Erbitux) - anti-EGFR for CRC.

• 2006: Ranibizumab (Lucentis) - anti-VEGF-A for Macular degeneration; Panitumumab (Vectibix) - anti-EGFR for CRC.

• 2007: Eculizumab (Soliris) - anti-C5 for Paroxysmal nocturnal hemoglobinuria.

• 2009: Denosumab (Prolia) - anti-RANKL for bone loss.

• 2010: Ipilimumab (Yervoy) - anti-CTLA-4 for metastatic melanoma.

• 2011: Belimumab.

• 2012: Pertuzumab (Perjeta) - anti-HER2 for BC.

• 2013: Brentuximab vedotin (Adcetris) - anti-CD30 for Hodgkin lymphoma; Gazyva.

• 2014: Nivolumab (Opdivo) and Pembrolizumab (Keytruda) - anti-PD-1 for melanoma and NSCLC; Ramucirumab (Cyramza) - anti-VEGFR2 for GC.

• 2015: Reslizumab (Cinqair) - anti-IL-5 for asthma; Dinutuximab (Unituxin) - anti-GD2 for neuroblastoma; Necitumumab (Portrazza) - anti-EGFR for NSCLC.

• 2016: Atezolizumab (Tecentriq) - anti-PD-L1 for bladder cancer.

• 2017: Avelumab (Bavencio) - anti-PD-L1 for MCC; Durvalumab (Imfinzi) - anti-PD-L1 for bladder cancer; Brodalumab (Siliq) - anti-IL-17R for plaque psoriasis.

• 2018: Erenumab (Aimovig) - anti-CGRPR for migraine; Ibalizumab (Trogarzo) - anti-CD4 for HIV; Burosumab (Crysvita) - anti-FGF23 for XLH.

• 2019: Caplacizumab (Cablivi) - anti-vWF for acquired aTTP; Romosozumab (Evenity) - anti-Sclerostin for osteoporosis.

Structural Fundamentals of Glycans

Glycans are not linear like DNA or simple peptides; they consist of branched, extended chains attached to protein structures (such as the Fc protein structure, PDB: 1H3Y). These chains are comprised of monosaccharide subunits with varying chemical structures.

Key Monosaccharide Building Blocks

• N-Acetyl-D-glucosamine (GlcNAc): A derivative of glucose with an acetylated amino group.

• D-Mannose (Man): An epimer of glucose commonly found in N-linked glycan cores.

• D-Galactose (Gal): An isomer of glucose often found in the antennae of complex N-glycans.

• L-Fucose (Fuc): A deoxyhexose frequently added to the core GlcNAc or as a terminal modification.

• D-Glucose (Glc): The most basic hexose.

• N-Acetyl-D-galactosamine (GalNAc): Common in O-linked glycans.

• D-Xylose (Xyl): A pentose sugar common in proteoglycan cores.

• D-Glucuronic acid (GlcA): An oxidized form of glucose (hexuronate).

• N-Acetylneuraminic acid (NeuAc): A common form of Sialic acid in humans, often found at the terminal positions of glycan chains.

Symbol Nomenclature for Glycans (SNFG)

To simplify complex chemical structures, glycobiologists use standard color-coded shapes:

The Glycosidic Bond

Monosaccharides link together via glycosidic bonds formed by a dehydration reaction ($-H_2O$).

• Example: Lactose (Gal$\beta 1-4$Glc) is formed by a bond between the $\text{C-1}$ of Galactose and the $\text{C-4}$ of Glucose.

• Reducing vs. Non-reducing End: The reducing end of a glycan is the end with a free anomeric carbon that can potentially reduce metal ions; the non-reducing ends are the terminal tips of the glycan branches.

Biological Case Study: Viral Infection and Sialic Acid Linkages

The specific arrangement and linkage of monosaccharides (stereochemistry and connectivity) profoundly influence biological function. This is critical in the mechanism of the Influenza virus.

Haemagglutinin and Receptor Specificity

Influenza viral haemagglutinin binds to sialic acid on host cells. The type of linkage between sialic acid and the preceding galactose determines the virus's host range:

• $\alpha 2,6$ Linkage (Human Receptor): Found predominantly in the human upper respiratory tract (nose, trachea). Seasonal human flu viruses prefer this linkage, allowing for efficient human-to-human transmission.

• $\alpha 2,3$ Linkage (Avian Receptor): Primarily found in the intestinal tract of birds. In humans, these receptors are located deep within the lungs.

Pandemic Potential and Intermediate Hosts

• Transmission Barriers: Avian flu (e.g., H5N1) can be lethal to humans because it infects deep lung tissue, but it lacks easy transmission because it does not bind the $\alpha 2,6$ receptors in the upper respiratory tract.

• Pandemic Mutants: For an avian flu to trigger a pandemic, it must mutate to recognize $\alpha 2,6$ receptors.

• Pigs as "Mixing Vessels": Pigs are significant intermediate hosts because their tracheas possess both $\alpha 2,3$ and $\alpha 2,6$ receptors, allowing for the co-infection and reassortment of avian and human viral strains.

Structural Detail of a Complex N-linked Glycan

A representative "bisialylated biantennary glycan" consists of a core structure attached to Asparagine ($\text{Asn}$). The full chemical name reflects the precise linkages:

NeuAc̑̑̑̑2−3Gal̑̑̑̑1−4GlcNAc̑̑̑̑1−2Man̑̑̑̑1−3(NeuAc̑̑̑̑2−3Gal̑̑̑̑1−4GlcNAc̑̑̑̑1−2Man̑̑̑̑1−6)Man̑̑̑̑1−4GlcNAc̑̑̑̑1−4GlcNAc̑̑̑̑1−Asn

Cellular Localization and Classes of Glycosylation

Glycans are found in diverse cellular and physiological locations:

• Luminal/Extracellular: Attached to proteins in the ER/Golgi lumen or secreted into the extracellular matrix and serum.

• Plasma Membrane: Linked to proteins (glycoproteins) or lipids (glycosphingolipids).

• Cytoplasmic/Nuclear: Specific modifications like O-GlcNAc occur on proteins within the cytoplasm or nucleus.

Major Classes of Glycosylation

1. N-glycans: Attached to Asparagine (N).

2. O-glycans: Attached to Serine (S) or Threonine (T).

3. Proteoglycans: Large structures involving glycosaminoglycans (GAGs) like Heparan sulfate, Chondroitin sulfate, Dermatan sulfate, and Hyaluronan.

4. GPI-anchored Glycoproteins: Proteins anchored to the membrane via a Glycosylphosphatidylinositol (GPI) tail.

5. Glycosphingolipids: Sugars attached to a lipid base.

6. O-GlcNAc Glycoproteins: Intracellular glycosylation signaling.

7. Free GAGs: Such as Hyaluronan, which exists independently of a protein core.