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Glycosylation Phosphorylation

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Glycosylation Phosphorylation Protein structure and function The Function of Proteins Enzymes biological catalysts. Immuno- antibodies of immune system. globulins Transport move materials around hemoglobin for O2. Regulatory hormones, control metabolism. Structural coverings and support skin, tendons, hair, nails, bone. Movement muscles, cilia, flagella. 1 DNA Protein The life cycle of a protein 2 Functional protein Families Science (2001), 15 % intermediary and nucleic acids metabolism 15-20 % structure, protein metabolism (cytoskeleton, chaperones, mediator of degradation) 20-25 % signal transduction or DAN binding protein 40 % gene encodes protein product are unknown function General flow scheme for proteomic analysis Experiment Sample Proteome Clinical Proteins mixture separation digestion protein Peptide mixture digestion peptide Mass Spectrometer identification Data analysis 3 Amino acid 2 amino acids peptide polypeptide 1o 2 o 3o 4o 一級primary 二級secondary 三級tertiary 四級quaternary Nelson & Cox (2000) Lehninger Principles of Biochemistry H pK α-carboxylic acid α-amino group pK2 1 H2N C COOH R The general structural formula of amino acids - pK1 values of the α-carboxylic acid groups lie in a small range around 2.2, above 3.5 COOH COO 2+ pK2 values of the α-amino group lie in a small range around 9.4, below 8.0 NH2 NH H pK pK2 1 +- + H N C COO Amino group = -NH3 3 Carboxyl group = -COO- R • Under normal cellular conditions amino acids are zwitterions (dipolar ions): How well it happens is based on pH and the type of amino acid. Called a zwitterion 4 Acidic environment Neutral environment Alkaline environment pK2 ~ 9 + + NH2 H NH2 H NH2 R-C-H R-C-H R-C-H COOH COO- COO- pK ~ 2 1 5.5 +1 0 -1 Isoelectric point Juang RH (2004) BCbasics 5 Amino Acids Have Buffering Effect pH 12 pK 9 ★ 2 + NH2 H 6 H-C-R Isoelectric point = pI COO- pK1 + pK2 3 ★ 2 pK1 0 [OH] → Juang RH (2004) BCbasics Four aliphatic (脂肪族) amino acid structures (non-polar) CH CH3 Isoleucine (I) CH2 (Ile) CH3 6 Aromatic (芳香族) amino acid structures 7 Methionine and cysteine First a.a. disulfide bond Non-poar aliphatic Formation of cystine 8 Side Chains with Alcohol Groups • Serine (Ser, S) and Threonine (Thr, T) have uncharged polar side chains Glycosylation phosphorylation Phosphorylation Phosphorylation is reversible and is used in many pathways to control activity. Enzymes that add a phosphate to a hydroxyl side chain are commonly called kinases. Enzymes that remove a phosphate from a phosphorylated side chain are called phosphatases 9 Glycosylation There are two basic types of glycosylation which occur on: (a)N-linked: asparagine (b)O-linked: serine and threonine The biological function of protein glycosylation 1. Covalently attached to the polypeptide as oligosaccharide chains containing 4 to 15 sugars 2. Sugars frequently comprise 50% or more of the total molecular weight of a glycoprotein 3. Most glycosylated proteins are either secreted or remain membrane- bound 4. Glycosylation is the most abundant form of post-translational modification 5. Glycosylation confers resistance to protease digestion by steric protection 6. Important in cell-cell recognition 10 N-linked glycosylation on asparagine (Asn) side chains: • an alkali-stable bond between the amide nitrogen of asparagine and the C-1 of an amino sugar residue • occurs co-translationally in the endoplasmic reticulum (ER) during synthesis • lipid-linked oligosaccharide complex is transferred to polypeptide by oligosaccharyl transferase • target sequence or consensus site on protein is Asn-X-Ser/Thr • further processing in Golgi apparatus Examples: Heavy chain of immunoglobulin G (IgG) Hen ovalbumin Ribonuclease B N-LINKED OLIGOSACHHARIDES There are two types which both share a common pentasachharide core (shaded yellow in the next two slides): High-mannose type Complex type 11 A COMPLEX TYPE N-LINKED OLIGOSACCHARIDE O-linked glycosylation on serine (Ser) or threonine (Thr) side chains • an alkali-labile bond between the hydroxyl group of serine or threonine and an amino sugar • carried out by a class of membrane-bound enzymes called glycosyl transferases which reside in the endoplasmic reticulum (ER) or the Golgi apparatus • nucleotide-linked monosaccharides added to protein side chain one at a time • Linked one at a time to OH of Ser, Thr or OH in modified amino acids (e.g. Hydroxylysine of collagen) • Example: Blood group antigens on erythrocyte surface: •The A antigen and B antigen are pentasaccharides which differ in composition of the 5th sugar residue •The O substance is a tetrasaccharide which is missing the 5th residue and does not elecit an antibody response (non-antigenic). 12 Protein glycosylation takes place in the ER and Golgi The endoplasmic reticulum- ER – A continuous cytoplasmic network studded with ribosomes and functions as a transport system for newly synthesized proteins. The Golgi complex – An organelle consisting of stacks of flat membranous vesicles that modify, store, and route products of the ER. N-linked glycosylation begins in the ER and continues in the Golgi apparatus (via dolichol phosphate). O-linked glycosylation takes place only in the Golgi apparatus. In the Golgi: 1. O-linked sugar units are linked to proteins. 2. N-linked glycoproteins continue to be modified. 3. Proteins are sorted and are sent to- lysosomes secretory granules plasma membrane according to signals encoded by amino acid sequences. GLYCOSYLATION Glycosylation starts in the endoplasmic reticulum and continues in the Golgi. Glycosylated proteins, probably half of all the proteins in animals, include: Most proteins in the extracellular matrix Most proteins on the plasma membrane and in the lysosome Most proteins in blood (albumin is a notable exception) Coat proteins of viruses made in animal cells Function of glycosylation: Makes proteins more hydrophilic Increases stability by decreasing protease accessiblity of the backbone Provides specific recognition handles for targeting Glycosylation is not limited to proteins. Lipids are glycosylated (glycolipids) altering their presentation in the membrane and some glycolipids (e.g. gangliosides) can have messenger character. The liver makes extensive use of glycosylation during detoxification of foreign substances. 13 Precursor synthesis The oligosaccharide is assembled sugar by sugar onto the carrier lipid dolichol High energy pyrophosphate bond (We shall discuss only the synthesis of a complex N-linked oligosaccharide.) 1. Synthesis of Nucleotide Sugars Every sugar is first linked to a nucleotide. This is accomplished by a series of reactions whose details need not concern us here. 2. Synthesis of Lipid-linked Oligosaccharides In this stage, which takes place in the endoplasmic reticulum, the oligosaccharide is assembled onto a very hydrophobic lipid: dolichol phosphate. 3. Transfer of the Oligosaccharide to the Protein The enzyme protein-oligosaccharyltransferase transfers the oligosaccharide en bloc onto an asparagine residue of the protein. This reaction also takes place in the ER. 4. Processing of the Protein-Bound Oligosaccharide 14 Biosynthesis of dolichol pyrophosphoryl oligosaccharide precursor Strongly hydrophobic lipid (79-95 carbon) Oligosaccharide side chain may promote folding and stability of glycoproteins Consensus: Asn-X-Ser/Thr Addition & processing of N-linked oligosaccharides in r-ER of vertebrate cells 15 N-glycosylation: Oligosaccharide precursor is attached to the protein co-translationally Consensus: Asn-X-Ser/Thr Red: GlcNAc Blue: mannose Green: Glucose Mannose 6-Phosphate directs a protein to the lysosome • M6P is generated in the cis-Golgi in a 2-step process • This sorting signal recruits adaptor proteins and clathrin 16 Newly synthesized proteins destined for the lysosome are first transported to the late endosome. The acid hydrolases in the lysosome are sorted in the TGN based on the chemical marker mannose 6-phosphate. Hydrolases are transported to the late Adaptins bridge endosome which later the M6P receptor matures into a lysosome. to clathrin. Acidic pH causes hydrolase to dissociate from the receptor. M6P receptor is recycled back to the TGN. 17 Formation of Mannose 6-phosphate tag at golgi complex. The phosphate is added in the Golgi This was first attached in the ER. The creation of the M6P marker in the Golgi relies on recognition of a signal patch in the tertiary structure of the hydrolase. Patients with a disease called inclusion-cell disease have cells lacking hydrolases in their lysosomes. Instead, the hydrolases are found in the blood. These patients lack GlcNAc phosphotransferase. Without the M6P-tag, the acid hydrolases are transported to the plasma membrane instead of the late endosome. 18 Structures of histidine, lysine and arginine Structures of aspartate, glutamate, asparagine and glutamine Glycosylation 19 Families of Amino Acids • The common amino acids are grouped according to whether their side chains are: – Acidic: aspartate, glutamate (tyrosine) – Basic: lysine, arginine, (histidine) polar – (natural) uncharged polar: serine, threonine, glutamine, asparagine, (glycine) – Nonpolar: (glycine), alanine, valine, leucine, Methioine, cysteine, proline, phenylalanine, tryptophan • Hydrophilic amino acids (uncharged polar) are usually on the outside of a protein whereas nonpolar residues cluster on the inside of protein • Basic or acidic amino acids are very polar and are generally found on the outside of protein molecules Ampholyte contains both positive and negative groups on its molecule Uncommon a.a. Prothrombin Ca2+ binding protein Plant
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