Carbohydrates: Glycoconjugates

Question 1

Define the terms glycoconjugate and proteoglycan.

Answer 1

Glycoconjugates: Polysaccharides covalently attached to other molecule types
- Peptide structures: Peptidoglycans
- Proteins: Proteoglycans, Glycoproteins
- Lipids: Glycolipids

Proteoglycans:
- Glycosaminoglycan/Protein Glycoconjugates
- Heteropolysaccharide (Glycosaminoglycan (GAG)) portion is major component
- “Core Proteins” covalently linked to the glycosaminoglycans via glycosidic bonds to Ser side chains
- Often secreted into extracellular matrix or are integral membrane structures

Question 2

Proteoglycans: List the elements that compose a typical proteoglycan.

Answer 2

Proteoglycans: Glycosaminoglycan/Protein Glycoconjugates
- The heteropolysaccharide (glycosaminoglycan (GAG)) portion is the major component of these molecules.
- “Core proteins” are covalently linked to the glycosaminoglycans via glycosidic bonds to Ser side chains

Syndecan:
- Proteoglycans involved in cell binding, receptor, cytoskeletal interactions
- Core protein is anchored in the cell membrane via a single alpha helix

Multiple glycosaminoglycans (GAGs) attached on the extracellular (N-terminal) side
- Chondroitin Sulfate
- Heparan sulfate (3-5)
- Connections via glycosidic linkages to Serine side chains

Question 3

Syndecan:
- Proteoglycans involved in ?
- Core protein is anchored in the cell membrane via a ?

Multiple glycosaminoglycans (GAGs) attached on the ? side
- Chondroitin Sulfate
- Heparan sulfate (3-5)
- Connections via ? bonds to ? side chains

Glycosaminoglycans are the site of ? interactions
- Overall structure (of carbohydrate) is ? owing to ? charges

Answer 3

Syndecan:
- Proteoglycans involved in cell binding, receptor, cytoskeletal interactions
- Core protein is anchored in the cell membrane via a single alpha helix

Multiple glycosaminoglycans (GAGs) attached on the extracellular (N-terminal) side
- Chondroitin Sulfate
- Heparan sulfate (3-5)
- Connections via glycosidic linkages to Serine side chains

Glycosaminoglycans are the site of intermolecular (binding) interactions
- Overall structure (of carbohydrate) is extended owing to negative charges

Heparan sulfate (HS) and Chondroitin Sulfate are attached via the trisaccharide link to a serine side chain

Xylose (Xyl) of the trisaccharide attached via anomeric carbon to serine of core protein

Attachment site sequence: SGxG

Question 4

Describe the most common type of linkage between the core protein and the glycosaminoglycan in a proteoglycan.

Answer 4

O-linked glycosylation is a
- type of post-translational modification in which a carbohydrate chain is covalently attached to the hydroxyl group of a serine or threonine amino acid residue in a protein
- In the case of proteoglycans, the GAG chains are attached to the core protein through O-linked glycosylation bonds, specifically to the hydroxyl group of serine or threonine residues in the core protein.

Attachment Site has general consensus sequence: SGxG

Question 5

Describe general features of the modification of heparan sulfate in proteoglycans.

Answer 5

Heparan Sulfate:
- Initially derived from N-Acetylglucosamine (GlcNAc) and Glucuronic Acid (GlcA).
- Some portions remain unmodified disaccharide repeats (NA domains).

Some portions become heavily derivatized, in particular sulfated (NS domains).
- Enzymes modify the monosaccharides after synthesis.
- Overall charge become more negative through these regions.
- Variations exist between different cell types.

Heparin columns similar to Cation Exchangers (Neg Stationary Phase)
- Can be used as Affinity Chromatography colums
- Cation Exchange chromatography

Question 6

Using syndecan as a model, describe different ways that proteoglycans may function in cells.

Answer 6

NS Domain:
- NS-domains bind to dif proteins & affect their activity
- Binding largely mediated through electrostatic/polar interactions
- Interactions occur in dif ways: Allostery/Enhanced binding effects // acting as co-receptors for extracellular ligands // Cell-surface localization

Ternary complex binding with Heparan Sulfate (HS):
Thrombin/Antithrombin interaction enhanced by interaction with HS
- Antithrombin undergoes conformational change upon binding to HS/heparin that allows it to act as an inhibitor (for multiple blood clotting proteins)

Binding of lipoprotein Lipase:
- to Heparan-Sulfate proteoglycans will increase its concentration/localization near the membrane when the proteoglycan is membrane-bound

HS/ligand/receptor complexes may also be associated with endocytosis of receptor complexes
- Shedding of HS-containing structures will dissociate bound ligands (and HS) from the cell surface

Syndecan:
- Proteoglycans involved in cell binding, receptor, cytoskeletal interactions
- Core protein is anchored in the cell membrane via a single alpha helix

Multiple glycosaminoglycans (GAGs) attached on the extracellular (N-terminal) side
- Chondroitin Sulfate
- Heparan sulfate (3-5)
- Connections via glycosidic linkages to Serine side chains

Glycosaminoglycans are the site of intermolecular (binding) interactions
- Overall structure (of carbohydrate) is extended owing to negative charges

Question 7

Define the terms glycoprotein and glycolipid.

Answer 7

Glycoproteins: Protein/Carbohydrate conjugates
Carbohydrate Portion often differs from those in proteoglycans
- Smaller (oligosaccharides)
- More branched
- Greater variety of saccharides

Carbohydrate covalently attached via serine, threonine or asparagine side chains
- Ser/Thr: O-Linked
- Asn: N-Linked

Glycolipids: Lipid/Carbohydrate conjugates
- Can be used as recognition structures

Question 8

Describe the two types of linkages between protein and the oligosaccharide in a glycoprotein.

Answer 8

O-Linked Glycosylation
- linked via O-glycosyl linkage to the oxygen of a Ser or Thr side chain

N-Linked Glycosylation
- via N-glycosyl linkage to the nitrogen of the Asn side chain
- Proceeds during synthesis in Rough ER (Signal sequences at N-terminus direct synthesis to ER)
- Carbohydrate attachment occurs on lumen side of ER membrane
- Initial glycosylation in ER is processed further in ER and golgi (maturation) to determine final location and glycosylation structure
- Glycosylated = trafficked to be extracellular or membrane protein

Question 9

Outline some of the possible roles of the oligosaccharide in a glycoprotein structure.

Answer 9

Oligosaccharides in glycoproteins and glycolipids can be used as recognition structures for intermolecular interactions
- Protein targeting/sorting
- Cell/cell interactions
- signaling
- tissue development

Rice source of structural variation
- Multiple monosaccharides and derivatives
- Branched structures
- Glycosidic Configuration variations

Glycosylation of Proteins:
- Leads to change in the chemical nature of proteins
- Polarity and Solubility
- Folding (changing tertiary structure)

Interactions with other proteins:
- Recognition (sorting, Cell-cell signaling)
- Protection from proteolysis
- Antigenicity (recognition by antibodies)

Question 10

Function and role of lectins and be able to provide an example.

Answer 10

Lectins:
-Proteins that bind to carbohydrates (oligosaccharides) with high specificity.
-Found in all organisms
-Roles in cell-cell recognition, signalling, adhesion processes and intracellular targeting of newly synthesized proteins.
-Protein/Ligand interaction
- Equilibrium binding process.
- Oligosaccharides (or portions of them) interact with binding sites

Example Specificity:
Galectin-2
* Binding of lactose (Gal(β
1→4)Glc) to Galectin-2.
* Hydrogen bond interactions are shown as dotted lines.
* Van der Waals interactions will also form between non-polar groups (in this image, with Trp65).
* Glycoproteins with this sugar structure as part of a larger oligosaccharide will also bind.
* Lectin:carbohydrate binding interactions (Kd) are sensitive to ionic strength and pH

Question 11

Describe how complementarity and specificity can be achieved using sugars and lectins.

Answer 11

Example Specificity:
Galectin-2
* Binding of lactose (Gal(β1→4)Glc) to Galectin-2.
* Hydrogen bond interactions are shown as dotted lines.
* Van der Waals interactions will also form between non-polar groups (in this image, with Trp65).
* Glycoproteins with this sugar structure as part of a larger oligosaccharide will also bind.
* Lectin:carbohydrate binding interactions (Kd) are sensitive to ionic strength and pH

Complementarity and specificity can be achieved using sugars and lectins through a process called lectin-carbohydrate recognition. Lectins are a diverse group of proteins that have the ability to bind to specific carbohydrate structures, such as glycoproteins and glycolipids, through their carbohydrate recognition domains (CRDs)

Question 12

If the disaccharide Glc(β1→4)Glc was used instead of lactose (Gal(β1→4)Glc) with Galectin-2, which interactions would NOT be affected?
a) with His 45
b) w/ Asn 47
c) w/ Trp 65
d) All are affected

Answer 12

d) All are affected
Changing to glucose -> change is at C4
- Lactose is C4 antomer of glucose
- OH @ C4 moved to equatorial position = breaks H-bonds and puts OH near Trp impacting interactions

Example Specificity:
Galectin-2
* Binding of lactose (Gal(β1→4)Glc) to Galectin-2.
* Hydrogen bond interactions are shown as dotted lines.
* Van der Waals interactions will also form between non-polar groups (in this image, with Trp65).
* Glycoproteins with this sugar structure as part of a larger oligosaccharide will also bind.
* Lectin:carbohydrate binding interactions (Kd) are sensitive to ionic strength and pH