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Post-Translational Modification

Proteolytic Cleavage
Most proteins undergo proteolytic cleavage following translation. The simplest form of this is the removal of the initiation methionine. Many proteins are synthesized as inactive precursors that are activated under proper physiological conditions by limited proteolysis. Pancreatic enzymes and enzymes involved in clotting are examples of the latter. Inactive precursor proteins that are activated by removal of polypeptides are termed, proproteins.
Proteins that are membrane bound or are destined for excretion are synthesized by ribosomes associated with the membranes of the endoplasmic reticulum (ER). The ER associated with ribosomes is termed rough ER (RER). This class of proteins all contain an N-terminus termed a signal sequence or signal peptide. The signal peptide is usually 13-36 predominantly hydrophobic residues. The signal peptide is recognized by a multi-protein complex termed the signal recognition particle (SRP). This signal peptide is removed following passage through the endoplasmic reticulum membrane. The removal of the signal peptide is catalyzed by signal peptidase. Proteins that contain a signal peptide are called preproteins to distinguish them from proproteins. However, some proteins that are destined for secretion are also further proteolyzed following secretion and, therefore contain pro sequences. This class of proteins is termed preproproteins.
A complex example of post-translational processing of a preproprotein is the cleavage of prepro-opiomelanocortin (POMC) synthesized in the pituitary. This preproprotein undergoes complex cleavages, the pathway of which differs depending upon the cellular location of POMC synthesis.
Another is example of a preproprotein is insulin. Since insulin is secreted from the pancreas it has a prepeptide. Following cleavage of the 24 amino acid signal peptide the protein folds into proinsulin. Proinsulin is further cleaved yielding active insulin which is composed of two peptide chains linked togehter through disulfide bonds..
Still other proteins (of the enzyme class) are synthesized as inactive precursors called zymogens. Zymogens are activated by proteolytic cleavage such as is the situation for several proteins of the blood clotting cascade.
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Membrane associated carbohydrate is exclusively in the form of oliogsaccharides covalently attached to proteins forming glycoproteins, and to a lesser extent covalently attached to lipid forming the glycolipids. Glycoproteins consist of proteins covalently linked to carbohydrate. The predominant sugars found in glycoproteins are glucose, galactose, mannose, fucose, GalNAc, GlcNAc and NANA. The distinction between proteoglycans and glycoproteins resides in the level and types of carbohydrate modification. The carbohydrate modifications found in glycoproteins are rarely complex: carbohydrates are linked to the protein component through either O-glycosidic or N-glycosidic bonds. The N-glycosidic linkage is through the amide group of asparagine. The O-glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine. The linkage of carbohydrate to hydroxylysine is generally found only in the collagens. The linkage of carbohydrate to 5-hydroxylysine is either the single sugar galactose or the disaccharide glucosylgalactose. In ser- and thr-type O-linked glycoproteins, the carbohydrate directly attached to the protein is GalNAc. In N-linked glycoproteins, it is GlcNAc.

O-linkage to GalNAc
N-linkage to GlcNAc
The predominant carbohydrate attachment in glycoproteins of mammalian cells is via N-glycosidic linkage. The site of carbohydrate attachment to N-linked glycoproteins is found within a consensus sequence of amino acids, N-X-S(T), where X is any amino acid except proline. N-linked glycoproteins all contain a common core of carbohydrate attached to the polypeptide. This core consists of three mannose residues and two GlcNAc. A variety of other sugars are attached to this core and comprise three major N-linked families:
  • 1. High-mannose type contains all mannose outside the core in varying amounts.
  • 2. Hybrid type contains various sugars and amino sugars.
  • 3. Complex type is similar to the hybrid type, but in addition, contains sialic acids to varying degrees.
Structures of carbohydrates on the 3 major classes of glycoprotein
Most proteins that are secreted or bound to the plasma membrane are modified by carbohydrate attachment. The part that modified, in plasma membrane-bound protiens, is the extracellular portion of plasma membrane bound proteins that is modified. Intracellular proteins are less frequently modified by carbohydrate attachment.
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Mechanism of Carbohydrate Linkage to Protein
The protein component of all glycoproteins is synthesized from polyribosomes that are bound to the endoplasmic reticulum. The processing of the sugar groups occurs cotranslationally in the lumen of the ER and continues in the Golgi apparatus for N-linked glycoproteins. Attachment of sugars in O-linked glycoproteins occurs post-translationally in the Golgi apparatus.

O-linked sugars: The synthesis of O-linked glycoproteins occurs via the stepwise addition of nucleotide-activated sugars directly onto the polypeptide. The nucleotide-activated sugars are coupled to either UDP, GDP (as with mannose) or CMP (for instance, NANA). The attachment of sugars is catalyzed by specific glycoprotein glycosyltransferases. Evidence indicates that each specific type of carbohydrate linkage in O-linked glycoproteins is the result of a different glycosyltransferase.

N-linked sugars: As indicated earlier, the three major classes of N-linked carbohydrate modifications are high-mannose, hybrid and complex. The major distinguishing feature of the complex class is the presence of sialic acid, whereas the hybrid class contains no sialic acid.

In contrast to the step-wise addition of sugar groups to the O-linked class of glycoproteins, N-linked glycoprotein synthesis requires a lipid intermediate: dolichol phosphate. Dolichols are polyprenols (C80-C100) containing 17 to 21 isoprene units, in which the terminal unit is saturated.

The black bracket denotes the isoprene unit. The phosphate in dolichol phosphate is attached to the hydroxyl.

The oligosaccharide unit is attached to dolichol phosphate through a pyrophosphate bond. The sugars used for N-linked glycoprotein synthesis are activated by coupling to nucleotides, as in the synthesis of O-linked glycoproteins. GlcNAc is coupled to UDP, and Man to GDP. The first reaction involves the formation of GlcNAc-P-P-dolichol with the release of UMP from the nucleotide-activated sugar, UDP-GlcNAc. The second GlcNAc and Man transferase reactions proceed via sugar transfer from the nucleotide-activated suger directly to GlcNAc-P-P-dolichol. From this point, additional mannoses are added to the Man-GlcNAc-GlcNAc-P-P-dolichol by transfer from Man-P-dolichol; formed from dolichol phosphate and GDP-Man. Once the oligosaccharide core unit is complete, it is transferred to an N residue in the protein. As indicated above this N residue is found within N-X-S(T) consensus sequence.
After the oligosaccharide core is transferred to the protein, additional modifications take place through the action of glycosyltransferases as well as through the removal of certain glycosyl residues. These modifications occur as the protein migrates through the Golgi apparatus to the cell surface.
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Lysosomal Targeting of Enzymes
Enzymes that are destined for the lysosomes (lysosomal enzymes) are directed there by a specific carbohydrate modification. During transit through the Golgi apparatus a residue of a-N-acetylglucosamine-1-phosphate (GlcNAc-1-P) is added to carbon 6 of one or more specific mannose residues that have been incorporated into these enzymes. The GlcNAc is activated by coupling to UDP and is transferred by UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase (GlcNAc phosphotransferase), yielding a phosphodiester intermediate: GlcNAc-1-P-6-Man-protein. A second reaction (catalyzed by GlcNAc 1-phosphodiester-N-acetylglucosaminidase) removes the GlcNAc, leaving mannose residues phosphorylated in the 6 position: Man-6-P-protein. A specific Man-6-P receptor is present in the membranes of the Golgi apparatus. Binding of Man-6-P to this receptor targets proteins to the lysosomes.
Two distinct Man-6-P receptors have been identified. Both are integral membrane proteins. One receptor is large with a molecular weight of approximately 275,000 Daltons. The other receptor is smaller with a molecular weight of approximately 46,000 Daltons. Structural similarities between these two receptors indicates they are derived from a single ancestral gene with the larger receptor arising through multiple gene duplications. The large receptor binds 2 mol of Man-6-P and the smaller 1 mol of Man-6-P per subunit. The larger receptor does not require divalent cations for ligand binding and is termed the cation-independent Man-6-P receptor, CI-MPR. In some species (however, not humans) the smaller receptor does require cation for ligand binding and is termed the CD-MPR. The human CI-MPR also binds the nonglycosylated polypeptide hormone insulin-like growth factor II (IGF-II) and has thus been termed the Man-6-P/IGF-II receptor. The IGF-II and Man-6-P binding sites on the receptor are distinct from each other. Evidence indicates that both receptors function to target newly synthesized lysosomal enzymes to the lysosomes.
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Clinical Significances of Glycoproteins
Glycoproteins on cell surfaces are important for communication between cells, for maintaining cell structure and for self-recognition by the immune system. The alteration of cell-surface glycoproteins can, therefore, produce profound physiological effects, of which several are listed below.
Structure of the ABO Blood Group Carbohydrates
R represents the linkage to protein in the secreted forms, sphingolipid in the cell-surface bound form
  • 2. The truncation of erythrocyte surface glycoproteins leads to cell clumping, as in congenital dyserythropoietic anemia type II. Also referred to as HEMPAS (hereditary erythroblastic multinuclearity with positive acidified-serum test).

  • 3. Several viruses, bacteria and parasites have exploited the presence of cell-surface carbohydrates, principally associated with protein (glycoproteins), using them as portals of entry into the cell.

    • A. Human immunodeficiency virus (HIV), the causative agent of AIDS, gains entry into cells of the immune system by attaching to a class of cellular receptors known as the chemokine receptors, most notably CXCR4 and CCR5. For more information on chemokines and their receptors visit the C.O.P.E site.
    • B. Members of the poxvirus family of viruses gain entry into cells, most frequently migratory leukocytes, by attaching to chemokine receptors including CCR1, CCR5 and CXCR4 (Science [1999] vol. 286 pp. 1968-1971).
    • C. Dystroglycan (DG) is a component of the dystrophin-glycoprotein complex. It is a laminin receptor encoded by a single gene and cleaved by postranslational processing into two proteins, peripheral membrane a-DG and transmembrane b-DG. a-DG interacts with laminin-2 in the basal lamina and b-DG binds to dystrophin containing cytoskeletal proteins in muscle and peripheral nerves. DG is involved in agrin- and laminin-induced acetylcholine receptor clustering at neuromuscular junctions, morphogenesis, early development, and the pathogenesis of muscular dystrophies. Recent evidence (Science (1998) vol. 282 pp. 2076-2079 and 2079-2081) demonstrates that a-DG present on Schwann cell membranes is the receptor for Mycobacterium leprae and also serves as the receptor for the arenavirus class of pathogens. Arenaviruses cause hemorrhagic fever in humans. Lymphocytic choriomeningitis virus (LCMV), Lassa fever virus (LFV), Oliveros and Mobala (all members of the arenavirus family) all bind to a-DG. The specificity of this interaction was demonstrated by the resistance to LCMV infection of cells harboring a null mutation in DG.
    • D. Rhinoviruses utilize attachment to ICAM-1 (intercellular adhesion molecule-1) to gain entry into cells.
    • E. The pathogenic human parvovirus, B19, attaches to the erythrocyte-specific cell-surface globoside identified as erythrocyte P antigen to infect erythrocytes.
    • F. The malarial parasite Plasmodium vivax, binds to the erythrocyte chemokine receptor known as the Duffy blood group antigen (also known as the erythrocyte receptor for interleukin-8) to infect erythrocytes.
    • G. The MN blood group system is a well-characterized set of erythrocyte surface antigens that represent the variable carbohydrate modifications of the trans-membrane glycoprotein, glycophorin. Glycophorin is the cellular receptor for influenza virus as well as the receptor for erythrocyte invasion by the malarial parasite Plasmodium falciparum.
    • H. Helicobacter pylori is the bacterium responsible for chronic active gastritis and gastric and duodenal ulcers; it is also the causative agent for one of the most common forms of cancer in humans, adenocarcinoma. This bacterium attaches to the Lewis blood group antigen on the surfaces of gastric mucous cells.
    • I. Rabies virus binds to cells through interactions with neural cell adhesion molecule (N-CAM).
    • J. The receptor for fibroblast growth factor (FGF) has been reported to be the portal of entry for human herpes virus Type I. Recent new evidence indicates that the portal of entry for human herpes simplex Type I viruses is 3-O-sulfated heparan sulfate (Cell 99:13-22, 1999).
    • K. Human herpesvirus 6 (HHV-6) infection occurs in virtually all persons within the first 2 years of life and persists the entire lifetime. In immunocompromised patients HHV-6 causes opportunistic infections and is the causative agent of exanthema subitum. HHV-6 has been linked to multiple sclerosis and to the progression of AIDS. The cellular receptor for HHV-6 is the cell-surface type-I glycoprotein, CD46 (Cell 99:817-827, 1999).
  • 4. Some glycoproteins are tethered to the membrane by a lipid linkage: the protein is attached to the carbohydrate through phosphatidylethanolamine (PE) linkage, and the carbohydrate is in turn attached to the membrane via linkage to phosphatidylinositol (PI), which anchors the structure within the membrane. The linkage is called a glycosylphosphotidylinositol (GPI) anchor, and proteins that are anchored in this way are termed glypiated proteins. The disease, paroxysmal nocturnal hemoglobinuria, results from the loss of the erythrocyte surface glycoprotein, decay-accelerating factor, (DAF). DAF prevents erythrocyte lysis by complement. When this factor is lost from the erythrocyte surface, abnormal hemolysis occurs, with the end result of hemoglobin accumulation in the urine.
Structure of the GPI linkage
Other important GPI linked proteins are the enzymes acetylcholinesterase, intestinal and placental alkaline phosphatase and 5'-nucleotidase, the cell adhesion molecule N-CAM (neural cell adhesion molecule) and the T-cell markers Thy-1 and LFA-3 (lymphocyte function associated antigen-3).

  • 5. The proper degradation of glycoproteins has medical relevance. Degradation occurs within lysosomes and requires specific lysosomal hydrolases, termed glycosidases. Exoglycosidases remove sugars sequentially from the non-reducing end and exhibit restricted substrate specificities. In contrast, endoglycosidases cleave carbohydrate linkages from within and exhibit broader substrate specificities. Several inherited disorders involving the abnormal storage of glycoprotein degradation products have been identified in humans. These disorders result from defects in the genes encoding specific glycosidases, leading to incomplete degradation and subsequent over-accumulation of partially degraded glycoproteins. As a general class, such disorders are known as lysosomal storage diseases and include the diseases known as mucolipidoses that result from incomplete degradation of the carbohydrate portions of glycolipids.

  • 6. Defects in the proper targeting of glycoproteins to the lysosomes can also lead to clinical complications. Deficiencies in the enzyme responsible for the transfer of GlcNAc-1-P to Man residues (GlcNAc phosphotransferase) in lysosomal enzymes leads to the formation of dense inclusion bodies formation in the fibroblasts. Two disorders related to deficiencies in the targeting of lysosomal enzymes are termed I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III, also called mucolipidosis-HI). I-cell disease is characterized by severe psychomotor retardation, skeletal abnormalities, coarse facial features, painful restricted joint movement, and early mortality. Pseudo-Hurler polydystrophy is less severe; it progresses more slowly, and afflicted individuals live to adulthood.

Enzyme Defects in Degradation of
Asn-GlcNAc Type Glycoproteins

DiseaseEnzyme DeficiencySymptoms/Comments
aspartylglycosaminuriaaspartylglycosaminidaseprogressive mental retardation, delayed speech and motor development, coarse facial features
b-Mannosidosisb-Mannosidaseprimarily neurological defects, speech impairment
a-Mannosidosisa-Mannosidasemental retardation, dystosis multiplex, hepatosplenomegaly, hearing loss, delayed speech
GM1 Gangliosidosisb-Galactosidasealso identified as a glycosphingolipid storage disease
GM2 Gangliosidosis
(Sandhoff-Jatzkewitz disease)
b-N-acetylhexosaminidases A and Balso identified as a glycosphingolipid storage disease
(also identified as Mucolipidosis I)
myoclonus, congenital ascites, hepatosplenomegaly, coarse facial features, delayed mental and motor development
Fucosidosisa-Fucosidaseprogressive motor and mental deterioration, growth retardation, coarse facial features, recurrent sinus and pulmonary infections
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OMIM links for additional Defects in Glycoprotein Degradation
Many proteins are modified at their N-termini following synthesis. In most cases the initiator methionine is hydrolyzed and an acetyl group is added to the new N-terminal amino acid. Acetyl-CoA is the acetyl donor for these reactions. Some proteins have the 14 carbon myristoyl group added to their N-termini. The donor for this modification is myristoyl-CoA. This latter modification allows association of the modified protein with membranes. The catalytic subunit of cyclicAMP-dependent protein kinase (PKA) is myristoylated.
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Post-translational methylation occurs at lysine residues in some proteins such as calmodulin and cytochrome c. The activated methyl donor is S-adenosylmethionine.
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Post-translational phosphorylation is one of the most common protein modifications that occurs in animal cells. The vast majority of phosphorylations occur as a mechanism to regulate the biological activity of a protein and as such are transient. In other words a phosphate (or more than one in many cases) is added and later removed.
Physiologically relevant examples are the phosphorylations that occur in glycogen synthase and glycogen phosphorylase in hepatocytes in response to glucagon release from the pancreas. Phosphorylation of synthase inhibits its activity, whereas, the activity of phosphorylase is increased. These two events lead to increased hepatic glucose delivery to the blood.
The enzymes that phosphorylate proteins are termed kinases and those that remove phosphates are termed phosphatases. Protein kinases catalyze reactions of the following type:

ATP + protein <----> phosphoprotein + ADP

In animal cells serine, threonine and tyrosine are the amino acids subject to phosphorylation. The largest group of kinases are those that phsophorylate either serines or threonines and as such are termed serine/threonine kinases. The ratio of phosphorylation of the three different amino acids is approximately 1000/100/1 for serine/threonine/tyrosine.
Although the level of tyrosine phosphorylation is minor, the importance of phosphorylation of this amino acid is profound. As an example, the activity of numerous growth factor receptors is controlled by tyrosine phosphorylation.
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Sulfate modification of proteins occurs at tyrosine residues such as in fibrinogen and in some secreted proteins (eg gastrin). The universal sulfate donor is 3'-phosphoadenosyl-5'-phosphosulphate (PAPS).
Since sulfate is added permanently it is necessary for the biological activity and not used as a regulatory modification like that of tyrosine phosphorylation.
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Prenylation refers to the addition of the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group to acceptor proteins, both of which are isoprenoid compounds derived from the cholesterol biosynthetic pathway. The isoprenoid groups are attached to cysteine residues at the carboxy terminus of proteins in a thioether linkage (C-S-C). A common consensus sequence at the C-terminus of prenylated proteins has been identified and is composed of CAAX, where C is cysteine, A is any aliphatic amino acid (except alanine) and X is the C-terminal amino acid. In order for the prenylation reaction to occur the three C-terminal amino acids (AAX) are first removed and the cysteine activated by methylation in a reaction utilizing S-adenosylmethionine as the methyl donor.
Important examples of prenylated proteins include the oncogenic GTP-binding and hydrolyzing protein Ras and the g-subunit of the visual protein transducin, both of which are farnesylated. Numerous GTP-binding and hydrolyzing proteins (termed G-proteins) of signal transduction cascades have g-subunits modified by geranylgeranylation.
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Vitamin C-Dependent Modifications
Modifications of proteins that depend upon vitamin C as a cofactor include proline and lysine hydroxylations and carboxy terminal amidation. The hydroxylating enzymes are identified as prolyl hydroxylase and lysyl hydroxylase. The donor of the amide for C-terminal amidation is glycine.
The most important hydroxylated proteins are the collagens. Several peptide hormones such as oxytocin and vasopressin have C-terminal amidation.
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Vitamin K-Dependent Modifications
Vitamin K is a cofactor in the carboxylation of glutamine residues. The result of this type of reaction is a g-carboxyglutamate (called a gla residue).

.Structure of a gla residue

The formation of gla residues within several proteins of the blood clotting cascade is critical for their normal function. The presence of gla residues allows the protein to chelate calcium ions and thereby render an altered conformation and biological activity to the protein. The coumarin-based anticoagulants, warfarin and dicumarol function by inhibiting the carboxylation reaction.
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Selenium is a trace element and is found as a component of several prokaryotic and eukaryotic enzymes that are involved in redox reactions. The selenium in these selenoproteins is incorporated as a unique amino acid, selenocysteine, during translation. A particularly important eukaryotic selenoenzyme is glutathione peroxidase. This enzyme is required during the oxidation of glutathione by hydrogen peroxide (H2O2) and organic hydroperoxides.
Structure of the selenocysteine residue
Incorporation of selenocysteine by the translational machinery occurs via an interesting and unique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is brought into the ribosome by a dedicated specific elongation factor. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.
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This article has been modified by Dr. M. Javed Abbas.
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20:43 21/12/2002