Maria Tuhoy Lecture 1 Notes PDF

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Understand N- and O-glycan biosynthesis and protein glycosylation in the Secretory pathway in Eukaryotic cells...

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MARIA TUHOY LECTURE 1 NOTES:

1. Understand N- and O-glycan biosynthesis and protein glycosylation in the Secretory pathway in Eukaryotic cells The lumen of the ER is continuous with that of the outer nuclear membrane. mRNA within the cytoplasm is targeted by ribosomes which translate the protein in the cytoplasm snd then transport it into the ER or it can be partially translated and then can continue to occur as the protein is translocated into the ER lumen (co-translationally). N-glycosylation: N-glycosylation occurs within both the lumen of the ER and the Golgi. It is mainly responsible for protein folding. It involves glycosidic bonding of N-glycans to the amide nitrogen of the arginine residue of nascent polypeptides.

Formation of the dolichol sugar molecule Glc3Man9GlcNAc2: N-glycosylation biosynthesis involves the formation of a precursor 14-sugar oligosaccharide molecule. A dolichol lipid carrier is embedded in the ER membrane. A nucleotide sugar donor UDPGlcNAc drives the attachement of 2 GlcNAc residues onto the dolichol moleculae. Furthermors, a nucleotide sugar donor GDP-Man drives the further addition of five mannose sugars to the structure.Therefore,a dolichol sugar molecule Glc3Man9GlcNAc2 is produced. This occurs within the cytoplasmic region of the ER and is catalysed by the enzymes GlcNAc-1-phosphtransferase and GlcNAc transferase respectively.

Formation of N-glycan precursor molecule: The entire Glc3Man9GlcNAc2 structure is flipped into the lumen of the ER, catalysed by the enzyme flippase. The dilcohol0linked sugars Dolichol-P-Man and Dolichol-P-Glc drive the attactchment of

four mannose residues and three glucose residues respectively. These reactions are catalysed by the enzymes mannosyl transferase and glucosyl transferase respectively.

Attatchment to nascent proteins: This occurs within the lumen of the ER and the nergy provided for this is derived from the c=cleavage of the pyrophosphate group that links the glycan precursor to the dolichol molecule under the action of the enzyme phosphatase. The precursor N-glycan is trasnfered to asparagine residues along the surface of proteins with the consensus sequence Àsn-X-Ser/Thr/Cys, where X represent any amino acid except Proline. The enzyme oligosaccharyltransferase (OST)catalyzes this reaction.

N-glycosylaiton is a co-translational event because transfer of the precursor N-glycan to proteins occurs whilst the nascent unfolded protein is being translocated into the lumen of the ER.

Protein folding:

Trimming is a hydrolysis reaction that involves the splitting of precursor oligosaccharide residues. It can occur in both the ER or Golgi but they serve different roles in these different compartments. In the ER, trimming facilitates protein folding. Glucosidase I removes a terminal glucose molecule. Glucosidase II removes two inner glucose molecules from the precursor oligosaccharide which signals that the protein is ready for transport put of the ER.

The chaperones calnexin and calreticulum bind to the glucose molecule of the precursor oligossacharide retaining them within the ER, maintaining the unfolded state of the protein and presenting them to protein folding proteins such as ERp57 which facilitate protein folding. This facilitate cleavage of the glucose molecule catalysed by the glucosidase enzyme preventing its interaction with calnexin thus facilitating protein folding. They can cause release of the the gylcoptoein from the chaperones or the chaperones may already have dissociated from the glycoprotein. Properfly folded proteins are then transported to the Golgi apparatus. Misfolded proteins are recognized and reglycosylsted by the enzyme UDP-glucose: glycoprotein glucoslytrasnferase (UGGT) introducing a calnexin binding site to facilitate protein folding and transport to the Golgi. This process is continued until the protein is properly folded or if this does not work they are transported to the ER-associated degradation (ERAD).

ERAD: The mannose residues of the protein are cleaved by mannosidases. Further deglycosylation occurs before trasnprt to ERAD.

Further N-glycosylation within the secretory pathway: Properly folded proteins are transported to the Golgi for further modification. Further modification begins in the cis-Golgi under the action of mannosidases which cleave mannose residues from the precursor N-glycan catalysed by the enzyme mannosiadases IA, IB/IC. Precurose glycans that remain in this glycosylated form are known as high-mannose oligossacharides. Glycans can be processed further within the medial Golgi to produce glycans with only 3 mannose residues and an extra GlcNAc molecules. These are known as complex oligosaccharides. Further modification can occur with the trans-Golgi under the action of transferases that attach sialic acid, galactose and GlcNAc residues to the presuro glycan to produce highly branched glycans known as hybrid oligosaccharides.

O-glycosylation in the secretory pathway: Biosynthesis involved in mucin-type O-glycosylation This involves attachment of GlcNAc to the serine/threonine residues of proteins around prolie/serine/threonine residue-rich regions within the Golgi(therefore after N-glycosylation). This is driven by the nucleotide sugar donor UDP-GlcNAc. This is catalysed by the enzyme GalNAc transferase. No consistent consenceus sequence has been identified for this attathcment. Them Gal residues are attached, driven by UDP-Glc and catalysed by galatosyl transferase. The enzyme sialytransferase catalyzes the addition of sialic of two sialic acid residues to form a core 1 Oglycan.The enzyme fucosyl transferase and glucosyltransferase and galactosyl transferase catalyse the the addition of a fucose residue and multiple GlcNAc and Gal residues onto the protein to form a core 2 O-glycan. Furthermore, core 3-7 protiens require a combination of specific enzymes to catalyze specific O-glycan formation. Therefore, unlike N-glycosylation which occurs at once under the action of oligosaccharyltrasnferase, O-glycosylation involves the addition of sugar molecules one at a time to initiate particular trasnoprt pathways.

2. Understand the importance of glycosyltransferases & glycosidases in this process – Evolutionary significance of glycosylation

Non-Secretory Protein glycosylation: O-GlcNAcylation:

O-GlcNAcylation occurs primarily in the cytoplasm , mitochondria and the nucleus. Addition of a GlcNAc molecule -covently attached from the sugar donor UDP-GlcNAc to ser/thr residues of proteins catalysed by GlcNAc-transferase. It is a reversible process catalysed by the enzyme N-acetylglucosaaminidase which cleaves GlcNAc which can be replaced by the GlcNAc transferase. There is no consistent concensus sequence for this modification but a general preference for proline/alanine/serine/threonine.

GlcNAc may modulate the degree of phosphorylation of proteins by binding to these proteins preventing their phosphorylation at specific residues.

O-GlcNAc is a nutrient sensor:

Glucose in the blodd stream modules the concentration of the monosaccharide sugar O-GlcNAc. Therefore, increased glucose concentration increases O-GlcNAc modification of proteins.

Synthesis of UDP-GlcNAc:

With insulin resistance, the pancreas produces more and more insulin until the pancreas can no longer produce sufficient insulin for the body's demands, and then blood sugar rises.

Increased glucose concentration within the hexosamine biosynthesis pathway has bee shown to be linked to insulin resistance that leads to type II diabetes. Overexpression of O-GlcNAc in muscke and adipose tissue is associated with insulin resistance. Subsequenctly overexpression of GlcNAc transferase and N-acetylglucosamidinase can cause insulin resistance. Many proteins involved in insulin signalling are O-GlcNAc modified but the role of this modification is currently unknown.

For example, O-GlcNAc modification of the enzyme that catalyses synthesis of glycogen from glucose, glycogen synthase, occurs in the presence of high glucose concentration. This makes the enzyme less sensitive to a protein phosphatase 1 which responds to insulin ,therby potentially contributing to insulin resistance.

Cells lacking the enzyme GlcNAc transferase are not viable. Therefore, it is essential to cell phisology.

O-GlcNAc has also been shown to play roles in cell cycle regulation and control of many cellular processes through currently unknow molecular mechanisms- includes transcription, translation, neural development, response to replication stress.

In transcription, the unphosphorylated form of RNA polymerase binds to transcription factors and its phosphorylation stimulates its release to form the elongation complex that drives synthesis of mRNA. RNA polymerase II and RNa polymerase II transcription factors are highly )-GlcNAc modified and the rmoval of the GlcNAc modidifcations enable their phosphorylation and thus facilitates elongation and RNA synthesis. Thus, GlcNAc acts as a modulator of cellular processe through modulation of phosphorylation status of proteins.

O-GlcNAc modification also plays important roles in maitniaing neural function. For example, in Alzheimers disease the protein tau is hyperphosphorylated causing microtubule disfunction and neural disruption. Eveident shows that O-GlcNAc may regulate the phosphorylation of tau abd decreased addition of O-GlcNAc to tau may stimulate hyperphsoprylation related to the disease. Inhbition of N-acetylglucosaminidase (removes GlcNAc) has been shown to decrease phosphorylation of tau at ser/thr residues indicating the important role of O-GlcNAc negative regulation of tau phosphorylation.

Understand the role of Lectins as ‘translators’ of the sugar (glycan) code Lectins are carbohydrate-binding proteins.

Lectins were first identified in plants whrer they play an important role in defending plants against predators and pests. They are commonly multivalent oligomers. Therefore, they are capable of binding to multiple sites of carbodhydrates. The first lectin described was concanavalin (Con A) which binds with high affinity to mannosecontaining oligossachharides due to the presence of several branched trimannose structures which it has a high affinity for. It is involved in stimulating cell divison from G0 stage (quinescence) of the cell cycle. They are involved in the lateral crosslinking growth factor receptors thorugh glycan binding on each receptor.

Their high specificity for glycans makes them ideal for specific glycan detection on western blots or tissues.

Lectins can crosslink glycans present on the surface of cells It can also induce activation of receptors through lateral crosslinking of glycans between two receptors!

Galectins: Are ~130 aa soluble proteins that bind to β-galactosidases. 15 mammalian galectins gave currently been identififed.

Galectins bind to β-galactosidases.

There are a variety of differernt galectin present in human cells. They can be classified into three major groups: 1. Prototypical (Galectins 1,2,7,10,13,14) 2. Chimeric( Galectin-3) 3. Tandem repeat (Galectin 4,8,9,12) Galectins contain a highly conserved carbohydrate-recognition domain (CRD) of ~ 130aa, however only a part of the domain comes in direct contact with the carbohydrate molecule. The galectins are made up of different amino acid sequences but contain invariant residues involved in glycan binding.

15 human galectins have been classified to date and can be classified into 3 groups: 1. Group 1: Galectins containing one CRD. They can exist as homodimers. Contain protypical galectins. 2. Group 2: Galectins containing two distinct CDR’s within a single polypeptide, connected by a ~70aa peptide domain. Include tandem repeat gelectins. 3. Group 3: the Galectin-3 is the only currently identified chimeric galectin in vertebrates made up of proline-glycine repeats and forms oligomers.

Galectins are important in activation of signalling cascades. They can initiate this through formation of crosslinks between galactose-containing glycans of glycoconjugates such as glycoproteins and glycolipids present on the cell surface. Therefore, they can cause clustering of a multiple glyconjugates to form a lattice-like arrangement. They can also bridge two cells together or bridge cells to extracellular matrix proteins. Within this structure, galectins have specific affinity for specific oligossacharodes....