Protein modifications
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- Are proteins just proteins normally?
- No; the majority of proteins contain covalently attached molecules.
- What are 5 molecules commonly added to proteins?
-
-Ubiquitin
-Phosphate
-Carbohydrates
-ADP-ribose
-Fatty acids/lipids/choleterol - Why is ubiquitin added to proteins?
- It targets proteins for degradation in proteosomes.
- What does phosphate addition do to proteins?
- Activates/inactivates many enzymes
- What is the term for addition of carbohydrates to protein?
- Glycosylation.
- WHY modify proteins?
- To increase the functional diversity of the limited gene pool.
- How many genes are in the human genome?
- ~35,000
- What are 2 types of hydrophilic protein modifications?
-
1. Oligosaccharide addition (glycosylation)
2. ADP-ribosylation - What are 2 types of hydrophobic protein modifications?
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-Fatty acid addition
-Prenylation (Cholesterol addition) - Give 2 examples of physiologic conditinos associated w/ hydrophilic modification:
-
-ABO blood groups
-Influenza virus - What are the effects of Hydrophobic protein modification (fatty acid/prenylation)?
- Membrane association - proteins can act as membrane surface molecules.
- What is the definition of an oligosaccharide?
- Polymer containing 2 or more monosaccharides joined by O-glycosidic linkages.
- Why is glycosylation important?
- It is one of the principal co- and post-translational modifications of membrane proteins, secreted proteins, and the majority of proteins made in rough ER.
- What are the 2 types of glycosylation and what is the gist of each?
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N-linked: glycosylation of amide N of asparagine residues
O-linked: glycosylation of hydroxy oxygen of Ser and Thr residues. - Why glycosylate proteins?
-
-Assist in folding
-To give stability
-Assist in cell-cell adhesion - What is the main purpose of N-glycosylation of proteins?
- To assist in proper protein folding.
- What is the 1st step in N-linked glycosylation?
- Addition of a precursor molecule to the target protein.
- What does this precursor molecule consist of?
-
3 Glucose
9 mannose
2 N-acetylglucosamine - What happens after addition of the precursor molecule to the protein??
- The complex is taken up by the carrier molecule DOLICHOL
- What is the purpose of dolichol?
- To transfer the precursor-protein complex to the correct point on the protein's polypeptide chain.
- What happens as the complex is transferred by Dolichol?
- It is translocated into the ER lumen.
- What happens in the ER lumen?
- An oligosaccharide chain is attached to Asparagine.
- What catalyzes the oligosacch. transfer?
- Oligosaccharyl transferase
- Can an oligosacch. be transferred to just any Asn?
- No; must be in the tripeptide sequences Asn-X-Ser or Asn-X-Thr
- What can X be?
- Any amino acid except Pro
- What is the sequence containing Asn called?
- Glycosylation Sequon
- What happens after the glycosylation reaction?
- Removal of the 3 glucose molecules in the precursor component - if the protein folded correctly.
- Once the protein has correctly folded what happens to the glycoprotein?
- Transport to Golgi for further processing like mannose removal.
- Ok that's the end of wikipedia notes
- ok
- Recap: what type of modification is addition of oligosaccharides to protein?
- Hydrophilic
- What is an oligosaccharide?
- A polymer of sugar molecules containing 2 or more monosaccharides joined by O-glycosidic linkages
- Of the carbs generally found in mammals, which is not usually in glycolipids/GAGs?
- Mannose
- Which 2 are epimers?
- GalNac and Glcnac
- What are the 3 predominant sugars in glycosaminoglycans?
-
-Xylose
-Glucaronic acid
-Iduronic acid - What is the significant feature that makes these 3 sugars good for GAGs?
- They are negatively charged.
- What must happen to monosaccharides before they can be added to protein?
- Acitvation - to make NT sugars
- What are nucleotide sugars?
-
Activated, hi-energy compounds
-The immediate precursors of glycoprotein/lipid synth. - Where does sugar activation occur?
- In the cytoplasm - EXCEPT FOR SIALIC ACID
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Where does activation of sialic acid occur?
What does it produce? -
Nucleus
-Product is CMP-sialic acid - What is the actual reaction for NT sugar synthesis
- NTP + Sugar 1-phosphate -> Sugar NDP + PPi
- What is the actual reaction for CMP-sialic acid?
- CTP + sialic acid -> CMP-sialic acid + PPi
- What are 2 methods in which NT sugars can be synthesized?
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-De novo
-Epimerization - What is the major in vivo source of monosaccharide components in glycoproteins and glycolipids?
- GLUCOSE
- What is a major requirement of NT sugar synthesis?
- ENERGY - Lots of ATP
- What enzymes catalyze glycosylation?
- Glycosyltransferases
- Where are Glycosyltransferases found?
- In the membranes of the ER and Golgi body
- What exactly do glycosyltransferases do?
- Transfer sugar from an activated donor (NT sugar) to an acceptor target (protein or other).
- What is the important characteristic of glycosyltransferases?
-
Their specificity for:
1. Sugar transferred
2. Substrate transferred TO
3. Type of linkage created - How many glycosyltransferases are there?
- Over 300
- What do the glycosyltransferases do in N-glycosylation?
- Build up the oligosaccharide chain precursor molecule.
- How many glycosyltransferases operate in this process?
- 7 - on the cytosolic face of RER.
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Where does ea. take place:
-N glycosylation
-O glycosylation -
N - in the RER lumen, processing in Golgi
O - in the Golgi - Where do NT sugars tend to be?
- Cytosol - so they need transporters for uptake into the RER and golgi
- How do glycoproteins get to their destination after synthesis?
- By vesicular transport from the lumen to the cytosol.
- What are the 3 major types of carbohydrate-protein linkages?
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1. N-linkage btwn GlcNAc and Asn
2. O-linkage btwn GalNAc and Ser(Thr)
3. O-linkage betwn Xyl and Ser - Where are the first 2 types of linkages found?
- In membrane and secretory proteins.
- Where is O linkage of Xyl-Ser found?
- In proteoglycans
- What is the requirement of N-linked GlcNAc and Asn?
- -Must be linked to the sequence Asn-X-Ser/Thr
- What is the 1st step in Biosynthesis of N-linked sugars?
- Preassembly of an oligosacch. chain to Dolichol phosphate.
- What is dol-P?
- Dolichol phosphate; a membrane isoprenoid derivative.
- What is a major regulator of Dolichol phosphate synthesis?
- Cholesterol metabolism; Mevalonic acid is the precursor of both dol P and cholesterol.
- What is step 2 of N-glycosylation?
- Translation of proteins in RER
- What is step 3 of N-glycosylation?
- Oligosaccharyltransferase catalyzes the co-translational transfer of precursor oligosacch. chain from dol-P to the Asn residue.
- Where does the Asn residue hve to be located again?
- In the sequence Asn-X-Ser/Thr
- Where does step 3 occur?
- in the lumen of the ER.
- Where does the building up of the oligosaccharide precursor occur?
-
-First 7 steps (2 glu + 5 Mannoses) on RER face
-Last 7 steps (4 more mannoses + 3 Glc) IN RER lumen - So what 2 different enzyme types are needed for N-linkage?
-
-14 Glycosyltransferases - for making the precursor
-1 Oligosaccharyltransferase - to transfer precursor to Asn. - What happens after the actual N-linkage is made?
- Vesicular transport from RER lumen to golgi for processing and further modifications.
- What kind of modifications occur on the glycoproteins in golgi regions?
- Chaperones recognize specific sugars and assist in specific protein folding.
- Ok. Recap; where are O-glycosidic linkages between GalNAc and Ser/Thr found?
- In secretory and membrane glycoproteins.
- How is O-linked glycosylation different from N-linked?
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-No specific sequon recogn'n.
-No preformed precursor
-No common core structure
-Not cotranslational
-Stepwise addition of sugars
-No glycosidases -
Where does each function:
-Glycosidases
-Glycosyltransferases -
Glycosidases - ER and golgi
Glycosyltransferses - Golgi - How is o-linked glycosylation SIMILAR to n-linked?
- Both use the action of glycosyltransferases to make the oligosaccharide chain.
- What 2 types of O-linkages exist again?
-
1. GalNAc - Ser/Thr
2. Xyl - Ser - Where are Xyl-Ser linkages found?
- In Proteoglycans
- What are proteoglycans?
- Glycoproteins that contain a spcf type of oligosaccharide called GAGs
- What are GAGs?
- Glycosaminoglycans - neg charged polysaccharides made of repeating disacch. units which are sulfated.
- How many GAGs are commonly found in Proteoglycans?
- MANY - like 100 or more
- What 4 GAGs are used in proteoglycans?
-
-Hyaluronic acid
-Chondroitin sulfate
-Heparin/heparin sulfate
-Dermatan sulfate - How are GAGs attached to proteoglycans?
- Via covalent links - EXCEPT HYLAURONIC ACID
- What is the common linkage region for the GAGs except hylauronic acid?
- GlcUA-Gal-Gal-Xyl-Ser
- What is the purpose of Hyaluronic acid if not covalently linked to the proteoglycan?
- Serves as the backbone scaffold for proteoglycans.
- What is a proteoglycan macromolecule made of?
-
Core - hylauronic acid
-Link proteins
-GAGs bound to core protein - What are the functions of proteoglycans?
- -Bind H2O via highly neg charged sugars to support joints.