functional-proteins-and-genes

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Carbohydrates and glycobiology

Tymoczko et al. Biochemistry – A Short Course Chapter 10

Glycobiology: the study of the structure, function and biology of carbohydrates

• Carbohydrate structure and function • Lectins • Roles of glycobiology in human health and disease

Carbohydrates: any carbohydrates (monomers, oligomers, polymers or glycoconjugates)

Glycans: the carbohydrates attached to a wide variety of biological molecules through glycosylation

Glycosylation: the process of attaching carbohydrates to other molecules such as proteins and lipids

Protein glycosylation is the enzyme-catalysed covalent attachment of carbohydrates to a protein

Approximately half of all proteins typically expressed in a cell undergo this modification

From genome to proteins, and protein glycosylation

Carbohydrates in proteins impart an additional level of 'information content' to underlying protein structures

It is estimated that for most genomes about 1% of the coding regions are glycosyltransferases and glycosidases

Glycosyltransferases: to add sugars on Glycosidases: to cleave sugars off

Why glycosylation for proteins?

 Protein folding  Conformation stabilisation  Protein function  Protein secretion and targeting  Protein-protein interaction  Disruption of glycosylation cause disease

such as congenital muscular dystrophy (disruption of O-glycans)

 Cell to cell interaction  Signal transduction  Microbial infection

When to think about the involvement of sugars in cell-cell interaction, we are talking about sugars on the cell membrane.

The cells are decorated with sugars

When to think about the involvement of sugars in protein transport, we are mainly talking about Intracellular events

GlcNAc Man Gal

Glc

GalNAc

Sialic acid

Glycosylation reactions

Essentials of Glycobiology

Second Edition

asparagine (N-linkage)

serine or threonine (O-linkage)

11

Draw asparagine Draw serine and threonine Draw asparagine residue in a protein

Draw serine and threonine residues in a protein

Draw asparagine linked with a glucose Draw serine and threonine linked with a glucose

Draw N-acetylglucosamine linked to asparagine in a protein Draw N-acetylgalactosamine linked to serine in a protein

ABO blood type is determined by two glycosyltransferases

O blood type

oligosaccharide chain attached to either lipid or protein on RBC

A blood type

B blood type

Glycoprotein = sugars + protein (predominated by the protein)

Proteoglycan = polysaccharide + protein (predominated by carbohydrates) Mucin (proteins in mucus) = sugars + protein (predominated by carbohydrates)

N-linked protein glycosylation in the endoplasmic reticulum (ER) is a conserved three-phase process in eukaryotic cells. It involves the assembly of an oligosaccharide on a lipid carrier, dolichylpyrophosphate and the transfer of the oligosaccharide to selected asparagine residues of polypeptides

in the lumen of the ER

glycosyltransferases oligosaccharyltransferase

O-linked glycosylation

N-linked glycosylation

Nucleotide-activated sugars

N-Linked Protein glycosylation

1. Assembly of the precursor oligosaccharide...

IMPORTANT POINTS:

–  Assembly takes place on the carrier lipid dolichol, anchored in the ER membane.

–  A pyrophosphate bridge joins the 1st sugar (N-acetylglucosamine) to the dolichol.

–  Sugars are added singly and sequentially.

–  The glycan assembly flips from the cytosolic side to the ER lumen.

–  More sugar molecules are added to the assembly.

Now in the second step, attachment

2. En-bloc transfer of the oligosaccharide to the protein

One step transfer, catalyzed by oligosaccharyl transferase, which is bound to the membrane at the translocator.

Covalently attached to certain asparagines in the polypeptide chain (said to be “N-linked” glycosylation).

Attaches to NH2 side chain of Asn but only in the context:

Asn-x-Ser or Asn-x-Thr

Finally modification of oligosaccharide

3. Modification of the oligosaccharide by removal of sugars...

Transport from the ER to Golgi

• The N-linked glycoproteins leave the ER and travel to the Golgi Apparatus.

• They travel in membrane vesicles that arise from special regions of membranes that are coated by proteins.

The Golgi Apparatus has two major functions: 1. Modifies the N-linked oligosaccharides and adds O-linked oligosaccharides. 2. Sorts proteins so that when they exit the trans Golgi network, they are delivered to the correct destination.

Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various Golgi compartments.

Ribosomes bind the ER

Proteins sorted by secretory pathway start synthesis on a free ribosome in cytoplasm.

Ribosome directed to rough ER docks with ER membrane & protein synthesis continues.

Newly synthesised protein transported through membrane to ER.

How does the ribosome interact with ER??

Getting proteins into the ER

Signal sequence

• Machinery required to direct ribosome to ER & to translocate nascent protein across ER membrane consists of 4 components:

1. Signal sequence – sequence of 9 – 12 hydrophobic residues near amino terminus.

Can be cleaved by signal peptidases 2. Signalrecognitionparticle–recognizessignal

sequence on nascent polypeptide chain as emerges from ribosome. It binds this sequence & ribosome & shepherds ribosome with nascent polypeptide chain to ER

Signal sequence

3. SRP receptor – Integral membrane protein. SRP binds this receptor 4. Translocon – this is a channel across the membrane.

Channel opens when translocon & ribosome bind to each other Once ribosome bound to ER, protein synthesis reactivated – nascent protein now directed through ER membrane

Where does Dolichol come from?

• Dolichol is an isoprenoid compound synthesized by the same metabolic route as cholesterol. In vertebrate tissues, dolichol contains 18-20 isoprenoid units (90-100 carbons total). Dolichol is phosphorylated by a kinase that uses CTP to form dolichol Phosphate. Dolichol phosphate is the structure upon which the carbohydrate moieties of N- linked glycoproteins are built. After assembly on dolichol phosphate, the carbohydrate structure is transferred to an asparagine residue of a target protein having the sequence Asn-x-Ser/Thr, where X is any amino acid.

Dolichols function as a membrane anchor for the formation of the oligosaccharide

O-linked glycosylation

The O-glycosidic mechanism is not as complex as that of N-glycosylation. Proteins trafficked into the Golgi are most often O-glycosylated by N-acetylgalactosamine

(GalNAc) transferase, which transfers GalNAc to the -OH group of serine or threonine,

and then may add more sugars in a step-wise manner.

O-linked glycosylation

N-linked protein glycosylation

O-linked protein glycosylation

Cotranslationally or posttranslationally

Posttranslationally

Complex oligosaccharides (assembly of oligosaccharides first, then en bloc transfer to Asn in proteins)

Simple oligosaccharides (step-wise addition of monosaccharides to Ser ot Thr in proteins)

ER and Golgi

Mainly Golgi

Attached to asparagine residue

Attached to serine or threonine residue

Consensus sequence: NXS/T

No consensus sequence

Such as antibodies

Such as mucins

Lectin

Carbohydrates-binding proteins. Therefore it binds to glycoproteins

They bind carbohydrates with specificity and promote molecular recognition

Lectin in action