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Glycoproteins
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.
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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. When an analysis of proteins in the public databases is carried out, it can be shown that approximately 65% of all the proteins contain at least one occurrence of the Asn-X-Ser/Thr consensus. 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.
Open squares: GlcNAc; open circles: mannose; open diamonds: galactose; filled squares: fucose; filled triangles: sialic acid the greek symbols α and β followed by numbers refers to the type of linkage.
Structures of oligosaccharides of 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 is modified, in plasma membrane-bound proteins, is the extracellular portion of the protein that is modified. Intracellular proteins are less frequently modified by carbohydrate attachment. However, the attachment of carbohydrate to intracellular proteins confers unique functional activities on these proteins. Linkage of carbohydrate to cytosolic and/or nuclear proteins occurs via O-linkage and involves attachment of GlcNAc to serine or threonine residues. The linkage is catalyzed by the enzyme O-GlcNAc transferase, OGT. Several transcription factors and RNA polymerase II have been shown to be modified by O-GlcNAc linkage.
back to the topMechanism of Carbohydrate Linkage to Protein
The protein component of all glycoproteins is synthesized from polyribosomes that are bound to the endoplasmic reticulum (ER). 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. Sugars used for glycoprotein synthesis (both N-linked and O-linked) are activated by coupling to nucleotides. Glucose and GlcNAc are coupled to UDP and mannose is coupled to GDP.
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. In the image below the black bracket denotes the isoprene unit which can be repeated numerous times. The phosphate moiety of dolichol phosphate is attached to the –OH group.
As indicated, the formation of the GlcNAc-β-Asn linkage in proteins occurs in the endoplasmic reticulum (ER) through cotranslational addition of a preassembled carbohydrate core structure that is delivered via the carbohydrate-dolichol lipid intermediate. The preassembled carbohydrate core structure comprises three terminal residues of glucose attached to a branched cluster of nine mannose residues that are in turn attached to two GlcNAc residues attached to dolicholpyrophosphate. The structure is abbreviated Glc3Man9GlcNAc2–PP–dolichol. This structure is commonly referred to as the lipid-linked oligosaccharide (LLO) whereas the oligosaccharide structure itself is termed the en bloc oligosaccharide. In mammalian cells the importance of the terminal glucose residues is evident from the fact that transfer of Man9GlcNAc2–PP–dolichol is some 25-times less efficient than the complete structure. In addition, structures that contain three terminal glucose residues, but not the complete Man9GlcNAc2 structure, are efficiently transferred to protein by oligosaccharyltransferase. Synthesis of the en bloc dolichol–PP–oligosaccharide unit begins on the cytoplasmic face of the ER membrane and prior to transfer to the protein, the structure “flips” to the luminal side.
Pathway of Synthesis and Transfer of the LLO unit in the ER.
Immediately following transfer of the en bloc oligosaccharide unit to the protein, processing and alteration of the composition of the oligosaccharide ensues and continues as the protein passes through the ER then into and through the Golgi apparatus. Initially, the terminal glucose is removed through the action of glucosidase I (GI), a membrane bound enzyme recognizing α-1,2-linked glucose. The remaining two glucose residues are then removed by glucosidase II (GII), a soluble enzyme recognizing α-1,3-inked glucose. After removal of the glucose residues, the action of α-mannosidases removes several mannose residues as the protein progresses to the Golgi. The action of the various glucosidases and mannosidases leaves N-linked glycoproteins containing a common core of carbohydrate consisting of three mannose residues and two GlcNAc. Through the action of a wide range of glycosyltransferases and glycosidases a variety of other sugars are attached to this core as the protein progresses through the Golgi. These latter reactions generate the three major families of N-linked glycoproteins described above.
back to the topLysosomal 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 GlcNAc-1-phosphate (GlcNAc-1-P) is added to the carbon-6 hydroxyl group of one or more specific mannose residues that have been added to 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 (MPR) is present in the membranes of the Golgi apparatus. Binding of Man-6-P to this receptor targets proteins to the lysosomes.
Two distinct MPRs have been identified and both are members of the P-type lectin family. Both are type I integral membrane glycoproteins that contain an N-terminal extracellular domain, a single transmembrane domain and a C-terminal cytoplasmic domain. One receptor is large with a molecular weight of approximately 300kDa, the other receptor is smaller with a molecular weight of approximately 46kDa. 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 extracellular portion of the larger receptor contains 15 repeating elements, each of which is highly similar to the extracellular domain of the smaller receptor. Both receptors exist as dimers embedded in the membrane.
The large receptor binds two moles of Man-6-P and the smaller binds one mole of Man-6-P per subunit, thus 4 and 2 moles of Man-6-P per dimer, respectively. The bovine and murine versions of the smaller receptors require divalent cations for ligand binding and thus the receptor has been termed the cation-dependent Man-6-P receptor (CD-MPR). However, the human counterpart may not require cations for its activity. The larger receptor does not require divalent cations for ligand binding and is therefore, commonly referred to as the cation-independent Man-6-P receptor (CI-MPR). However, the CI-MPR has been shown to bind the nonglycosylated polypeptide hormone, insulin-like growth factor 2 (IGF-2) and as such the larger MPR is more frequently identified as IGF-2/MPR. The IGF-2/MPR is available at the cell surface and its role in binding IGF-2 is to target this hormone for degradation in the lysosomes. In addition to IGF-2, the IGF-2/MPR has been shown to bind a diverse array of Man-6-P-containing proteins as well as several nonglucosylated proteins. Although IGF-2/MPR and CD-MPR exhibit distinct activities, both receptors function to target newly synthesized lysosomal enzymes to the lysosomes.
back to the topClinical 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.
1. The ABO blood group antigens are the carbohydrate moieties of glycolipids on the surface of cells as well as the carbohydrate portion of serum glycoproteins. When present on the surface of cells the ABO carbohydrates are linked to sphingolipid and are therefore of the glycosphingolipid class. When the ABO carbohydrates are associated with protein in the form of glycoproteins they are found in the serum and are referred to as the secreted forms. Some individuals produce the glycoprotein forms of the ABO antigens while others do not. This property distinguishes secretors from non-secretors, a property that has forensic importance such as in cases of rape. For more information of blood group antigens, including ABO visit the blood group antigen gene mutation database at NCBI.
R represents the linkage to protein in the secreted forms, sphingolipid (ceramide) in the cell-surface bound form, open square = GlcNAc, open diamond = galactose, filled square = fucose, filled diamond = GalNAc. The linkage in the glycolipid form may include a glucose in a β-1,3 or β-1,4 to the initial galactose residue.
Structure of the ABO blood group carbohydrates
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.
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 α-DG and transmembrane β-DG. α-DG interacts with laminin-2 in the basal lamina and β-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. Evidence has shown that α-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 α-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 MNS 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. 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.
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; also known as CD55 = cluster of differentiation protein 55). 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. 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).
Shown in the Figure below is the structure of the GPI linkage in the T-cell protein Thy-1. However, it is important to note that there are a variety of different constituents in the GPI anchors of different proteins and the process of producing the GPI anchor is a post-translational event that is controlled at the cellular level.
Line represents the outer surface of the membrane. Squiggles represent the lipid portion of the GPI linkage embedded in the membrane. Open circles = mannose, filled diamonds = GalNAc, filled squares = fucose. Filled pentagons = ethanolamine, solid circles with P = phosphates.
The GPI linkage of the T-cell marker Thy-1
5. 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.
back to the topClinical Significance of Defective Glycoprotein Degradation
The proper degradation of glycoproteins has important 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. Numerous proteins and sphinogolipids harbor similar carbohydrate modifications. The enzymes that remove these sugar residues are the same for both glycoproteins and glycolipids and as such there is often overlapping phenotypes in diseases that were originally identified as being caused by defects in glycoprotein degradation or glycolipid degradation.
The following Figure shows the locations of the actions of several glycosidases involved in glycoprotein metabolism. The structures of the carbohydrates in a typical complex oligosaccharide cluster are included (see Figure above of the 3 major classes of glycoprotein). However, when linked as they would be by the indicated bonds (e.g. α-2,3, or 6 indicated for sialic acid linkage to galactose) there would be loss of H2O. The bonds are indicated for each linkage by the solid line between structures. Enzyme names are in green and diseases associated with defects in the indicated enzymes are in blue. Each of the disease names in the image can be clicked to go to a descriptive page of that disease. The Table below the Figure lists some of these diseases as well as the affected enzyme and classic symptoms of the disease. Note that as indicated in the above paragraph, many lysosomal storage diseases (e.g. Tay-Sachs) resulting from defective enzymes that metabolize both glycolipids and glycoproteins are defined by one or the other defective pathway (see the Sphingolipids page for more information).
| Disease | Enzyme Deficiency | Symptoms/Comments |
| Aspartylglucosaminuria |
aspartylglucosaminidase (N-aspartyl-β-glucosaminidase) |
progressive mental retardation, delayed speech and motor development, coarse facial features |
| β-Mannosidosis | β-mannosidase | primarily neurological defects, speech impairment |
| α-Mannosidosis | α-mannosidase | mental retardation, dystosis multiplex, hepatosplenomegaly, hearing loss, delayed speech |
| GM1 Gangliosidosis | β-galactosidase | also identified as a glycosphingolipid storage disease or lysosomal storage disease |
| Sandhoff disease | β-hexosaminidases A and B | also identified as a glycosphingolipid storage disease or lysosomal storage disease |
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Sialidosis (also identified as Mucolipidosis I) |
neuraminidase (sialidase) | myoclonus, congenital ascites, hepatosplenomegaly, coarse facial features, delayed mental and motor development |
| Fucosidosis | α-fucosidase | progressive motor and mental deterioration, growth retardation, coarse facial features, recurrent sinus and pulmonary infections |
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Congenital Disorders of Glycosylation (CDG)
Congenital disorders of glycosylation (CDG) represent a constellation of diseases that result from defects in the N-linked glycosylation pathway. These diseases are clustered into two broad categories. Group I CDG diseases are defined by alterations/deficiencies in the synthesis and/or transfer of the dolichol-pyrophosphate oligosaccharide precursor to Asn residues in substrate proteins. Group II CDG diseases are defined as those that result from defects in subsequent N-linked glycan processing. It is important to note that these disorders are only reflective of deficiencies of N-glycosylation and that diseases/disorders are known to result from deficiencies in O-glycosylation, GPI-linkage and the biosynthesis of proteoglycans, all of which involve carbohydrate addition and remodeling in the context of a protein backbone. For more information on both N-linked and O-linked glycosylation defects see the Congenital Disorders of Glycosylation page.
CDG-Ia is the most commonly occurring CDG, with appearance in individuals of European ancestry being highest. Although there is considerable variability in the clinical phenotypes observed in CDG-Ia patients, there is always some level of psychomotor retardation. In addition, children are ataxic and have skeletal abnormalities consisting of long limbs and short torsos. Due to defective synthesis of coagulation factors by the liver (primarily factor XI, antithrombin III, protein C and protein S), patients have severe coagulation defects. Adding to the situation is hepatomegaly with consequent liver dysfunction. CDG-Ia results from mutations in phosphomannomutase 2 (PMM2) the enzyme that is required to convert Man-6-P to Man-1-P used in the generation of GDP-Man. Over 60 mutations in PMM2 have been identified that either decrease enzyme activity or stability.
CDG-IIc is more commonly referred to as leukocyte adhesion deficiency syndrome II (LAD II). LAD II belongs to the class of disorders referred to as primary immunodeficiency syndromes as the symptoms of the disease manifest due to defects in leukocyte function. Symptoms of LAD II are characterized by unique facial features, recurrent infections, persistent leukocytosis, defective neutrophil chemotaxis and severe growth and mental retardation. The genetic defect resulting in LAD II is in the pathway of fucose utilization leading to loss of fucosylated glycans on the cell surface. An additional feature of LAD II is that individuals harbor the rare Bombay (hh) blood type at the ABO locus as well as lack the Lewis blood group antigens. The Bombay blood type is characterized by a deficiency in the H (referred to as the O-type), A and B antigens due to loss of the fucose residue. Each of these blood group antigens contains a Fuc-α-1,2-Gal modification that is the final carbohydrate addition to these antigens. These fucosylation reactions are catalyzed by α-1,2-fucosyltransferase which is encoded by the H and Se loci. The defective neutrophil chemotaxis is due to the loss of a selectin ligand on these cells. This ligand is the sialylated Lewisx antigen, another blood group antigen.
The recurrent infections seen in LAD II patients are the result of the defective neutrophil function. Neutrophils are involved in innate immunity responses to bacterial infection. To carry out their role in host defense mechanisms, neutrophils must adhere to the surface of the endothelium at the site of inflammation which is an event mediated by cell surface adhesion molecules. The selectin family (E-, L-, and P-selectins) of animal lectins are necessary to mediate the initial process of neutrophil adherence to the endothelium. The selectins recognize sialylated fucosylated lactosamines typified by the Lewisx antigen. Once neutrophils adhere and roll along the surface of the endothelium (due to vascular flow), the integrin family of adhesion molecules allow for firm adherence followed by tissue penetration. A related disorder, termed LAD I, is caused by the absence of CD18 which is the β2 subunit of the leukocyte integrin found on the surface of neutrophils and monocytes.
Because there is widespread loss of fucosylated antigens in LAD II patients, each of which can be formed through the actions of several fucosyltransferases, the role of these enzymes in the disease could be ruled out. In addition, normal levels of the α-1,2-, α-1,3- and α-1,4-fucosyltransferases were observed in the serum of LAD II individuals. These observations indicated that the pathways to GDP-fucose synthesis or utilization by the fucosyltransferases in the Golgi must be deficient in LAD II. Fucose can be converted to GDP-fucose by salvage of free fucose (exogenous or derived through glycoconjugate degradation) or by epimerization of GDP-mannose. In order for GDP-fucose to be utilized by Golgi fucosyltransferases it must first be transported into the Golgi from the cytosol where it is synthesized. Examinations of the enzymes involved in the synthesis and transport of GDP-fucose have been undertaken. While the activity of one of the enzymes of GDP-mannose epimerization (GDP-D-mannose 4,6-dehydratase, GMD) has been shown to be reduced in LAD II patients, it has been determined that the major defect causing LAD II is an impairment in the transport of GDP-fucose into the Golgi. This latter reaction is catalyzed by the GDP-fucose transporter encoded by the FUCT1 gene (also identified as solute carrier family 35, member C1: SCL35C1).
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Michael W. King, Ph.D / IU School of Medicine / miking at iupui.edu
