1. Dr. Mythreye Karthikeyan 803-576-5806 karthike@mailbox.sc.edu
MEDICAL BIOCHEMISTRY Lecture Chp Synthesis of Glycosides, Lactose, Glycoproteins, and Glycolipids
Dr. Mythreye Karthikeyan

2. Patient Diagnosis
To help support herself through medical school, Erna Nemdy works evenings in a hospital blood bank. She is responsible for assuring that a compatible donor blood is available to patients who need blood transfusions. As part of her training, Erna has learned that the external surfaces of all blood cells contain large numbers of antigenic determinants. These determinants are often glycoproteins or glycolipids that differ from one individual to another. As a result, all blood transfusions expose the recipient to many foreign immunogens. Most of these, fortunately, do not induce antibodies, or they induce antibodies that elicit little or no immunologic response. For routine blood transfusions, therefore, tests are performed only for the presence of antigens that determine whether the patient's blood type is A, B, AB, or O, and Rh(D)-positive or -negative.

3. Metabolism of UDP-Glucose
An activated sugar nucleotide
Precursor for:
Glycogen
Lactose
UDP-glucuronate and glucuronides
Carbs chains in proteoglycans, glycoproteins, and glycolipids

4. Reactions of UDP-Glucose
Catalyzed by sugar transferase (a.k.a. glycosyltransferase)
Sugar transferred from nucleotide sugar to alcohol (or other nucleophile) to form glycosidic bond
UDP as a leaving group provides energy for formation of new bond
Glycogen synthase is an example of a glycosyltransferase
This is an example of transfer of a sugar to a nucleophilic amino acid residue on a protein (Ser). Other transferases transfer a sugar from a nucleotide sugar to a hydroxyl group of other sugars.

5. Metabolism of UDP-Glucuronate
Formed from UDP-glucose
Glucuronate can be obtained in the diet
Precursor of:
Glycosaminoglycans (GAG)
Iduronate (epimer of glucuronate)
UDP-xylose - also in GAGs
Incorporated into:
Bilirubin to make bilirubin diglucuronide (soluble)
Steroids, drugs, xenobiotics to make glucuronides

6. Formation of Glucuronate and Glucuronides
Glucuronate formed by oxidation of alcohol at C6 of glucose to an acid by an NAD+-dependent dehydrogenase
Glucuronide formed by creation of glycosidic bond between anomeric -OH of glucuronate and -OH group of non-polar compound

7. Glucuronides and Excretion
Addition of glucuronate to non-polar compounds like drugs, xenobiotics, and bilirubin adds negative charge and increases solubility
Aids in excretion of compounds in bile or urine
Some compounds degraded and excreted as urinary glucuronides
Estrogen (female sex hormone)
Progesterone (steroid hormone)
Triiodothyronine (thyroid hormone)
Acetylaminofluorene (xenobiotic carcinogen)
Meprobamate (drug for sleep)
Morphine (painkiller)
Barbiturates
Tylenol & Aspirin

8. Formation of Bilirubin Diglucuronide
Bilirubin is degradation product of heme - only slightly soluble in plasma
“Unconjugated” or “free” bilirubin transported to liver bound to serum albumin
In liver, 2 glucuronate residues transferred from UDP-glucuronate to 2 carboxyl groups on bilirubin
Forms “conjugated” bilirubin, a.k.a bilirubin diglucuronide - more soluble form transported into bile for excretion

9. Normal Bilirubin Metabolism
75% of bilirubin derived from destruction of hemoglobin from RBCs
Unconjugated bilirubin carried to liver by albumin
Conjugated to more soluble form
Excreted in bile
Converted to excretion products in feces and urine

10. Bilirubin Measurements
Blood tests can separately measure:
Indirect bilirubin (nonconjugated form that is bound to serum albumin)
Direct bilirubin (conjugated, water-soluble form, i.e. bilirubin diglucuronide)
Total bilirubin (sum of indirect and direct levels)
If total levels high, need to determine levels of direct and indirect to find cause for elevation of total

11. Bilirubin Excretion and Jaundice
Bilirubin may increase in blood for several reasons:
Premature infants (Low levels of conjugating enzyme)
Liver disease (poor conjugation or biliary excretion, or both)
Excessive hemolysis (G6PD deficiency)
A failure of the liver to transport, store, or conjugate bilirubin results in the accumulation of unconjugated bilirubin in the blood. Jaundice, the yellowish tinge to the skin and the whites of the eyes (sclera) experienced by Erin Galway, occurs when plasma becomes supersaturated with bilirubin (>2 to 2.5 mg/dL), and the excess diffuses into tissues.
Bruise

12. Newborns and Bili Light Therapy
Many (60%) full-term newborns develop jaundice, termed neonatal jaundice. This is usually caused by an increased destruction of red blood cells after birth (the fetus has an unusually large number of red blood cells) and an immature bilirubin conjugating system in the liver. This leads to elevated levels of nonconjugated bilirubin, which is deposited in hydrophobic (fat) environments. If bilirubin levels reach a certain threshold at the age of 48 hours, the newborn is a candidate for phototherapy, in which the child is placed under lamps that emit light between the wavelengths of 425 and 475 nm. Bilirubin absorbs this light, undergoes chemical changes, and becomes more water-soluble. Usually, within a week of birth, the newborn's liver can handle the load generated from red blood cell turnover.

13. Jaundice and Galactosemia-
Galactose-1-phosphate X
UDP-glucose
UDP-galactose
Galactose 1-P accumulation inhibits phosphoglucomutase causing:
Hypoglycemia (glycogenolysis is blocked)
Jaundice - UDP-glucuronate isn’t synthesized from glucose-6P - nonconjugated (aka indirect) bilirubin builds up

14. Synthesis of UDP-Galactose from Glucose
Formation of UDP-galactose from UDP-glucose is an epimerization at C4
Epimerase uses NAD+ to oxidize -OH to ketone (=O), then reduces it back to -OH to reform on other side of carbon
Reversible rxn
UDP-galactose needed for lactose synthesis

15. Synthesis of Lactose Lactose = glucose + galactose
Only synthesized in mammary for short periods during lactation
Lactose synthase catalyzes transfer of galactose from UDP-galactose to glucose (NOT UDP-glucose) to form glycosidic bond
Lactose synthase has 2 subunits
Galactosyltransferase (enzyme)
-Lactalbumin (regulatory subunit)
Synthesized after childbirth in response to prolactin
Lowers Km of galactosyltransferase for glucose (1200 mM  1 mM) to increase rate of lactose synthesis
Without -lactalbumin, Galactosyltransferase normally transfers galactosyl units to glycoproteins
-Lactalbumin acts as “specifier” protein by altering substrate specificity

16. Formation of Sugars for Glycolipid and Glycoprotein Synthesis
Transferases produce oligosaccharide and polysaccharide side chains for glycolipids and attach sugars residues to glycoproteins
Transferases are specific for sugar moiety and for donating nucleotide (e.g. UDP, CMP, GDP)
Sugar-nucleotides used for glycoprotein, proteoglycan, and glycolipid formation shown in Table on right
Large variety - allows for relatively specific and different functions
Examples of Sugar Nucleotides that are Precursors for Transferase Reactions
UDP-glucose
UDP-galactose
UDP-glucuronic acid
UDP-xylose
UDP-N-acetylglucosamine
UDP-N-acetylgalactosamine
CMP-N-acetylneuraminic acid
GDP-fucose
GDP-mannose

17. Pathways for the Interconversion of Sugars
ALL of different sugars found in glycosaminoglycans, gangliosides, glycoproteins, glycolipids, can be synthesized from GLUCOSE
Dietary glucose, fructose, galactose, mannose, etc enters common pool from which other sugars derived (reversible rxns)
Activated sugar transferred from nucleotide sugar (e.g. UDP-glucose) to form glycosidic bond with another sugar or amino acid
See next slide

19. Synthesis of Amino Sugars
Amino sugars used in synthesis of glycosaminoglycans are all derived from glucosamine 6-phosphate
Amino sugar synthesis:
Amino group transferred from amide of glutamine to fructose 6-P to make glucosamine-6-P
Amino sugar can then be N-acetylated at amino group by transfer of an acetyl group from acetyl CoA

20. Mannose Minor component of the diet
Interconverted with glucose by epimerization (like galactose)
Interconversion can take place from fructose-6-P to make mannose-6-P
Interconversion can take place from derivatized sugars

21. Location of Glycoconjugates
Glycoconjugates are located primarily:
on the cell surface
in membrane-enclosed vesicles inside the cell
in the extracellular matrix
in extracellular, plasma proteins

22. Glycoconjugates Glycoconjugates serve as information carriers
Act as destination labels for some proteins
Acts as mediators of specific cell-cell interactions
Act as mediators of interactions between cell and extracellular matrix
Three general types:
Proteoglycans - macromolecules with one or more glycosaminoglycan (GAG) chains joined covalently to a membrane protein or secreted protein
Major component of connective tissue (e.g. collagen)
Glycoproteins - one or several oligosaccharides of varying complexity joined covalently to a protein
Form highly specific sites for recognition and high-affinity binding
Glycolipids - membrane lipids in which hydrophilic head groups are oligosaccharides
Act as specific sites for recognition of carbohydrate-binding proteins

23. Glycoproteins vs. Proteoglycans
Carb portion less monotonous - does not have repeating disaccharides
Carbs form short highly-branched chains
Proteoglycan
Carb portion greater in mass than protein portion
Carb dominates structure
Carb forms long, linear, unbranched chains with repeating disaccharides (GAGs)

24. Glycoprotein Structure
N-linked glycoprotein
Sugar attached at its anomeric carbon through a glycosidic link to protein residue
O-linked = attached to -OH of Ser/Thr or -OH group of hydroxylysine (collagen)
N-linked = attached to amide N of Asn
NANA = N-acetylneuramine
Gal = galactose
GlcNAc = N-acetylglucosamine
Man = mannose
Fuc = fucose

25. Glycoprotein Function
Most proteins in blood are glycoproteins
Hormones, enzymes, antibodies, blood clotting
Milk proteins - lactalbumin
Structural components of extracellular matrix - e.g. collagen
Secretions of mucus-producing cells - e.g. salivary mucin
Sugar H-bonds with water
All are O-linked
On cell membrane - act as hormone receptors, as transport proteins, as cell attachment and cell-cell recognition sites
Lysosomal enzymes that degrade various types of cellular and extracellular material
(NANA)
Charge-charge repulsions makes mucins highly extended, taking up space and forming a viscous solution

26. Glycoprotein Secretion and Segregation
Glycoproteins are transported from the Golgi complex to their final destination, either 1) the cell membrane, 2) secretion outside the cell, or 3) inside lysosomes (mannose 6-P recognition)

27. O-linked Glycoprotein Synthesis
Protein portion synthesized in ER
Sugar chains attached to protein in lumen of ER or in Golgi complex
GalNAc attached to Ser/Thr
Stepwise addition of sugars from sugar nucleotide donor (e.g. UDP-Gal, UDP-GalNAc, CMP-sialic acid) to non-reducing end

28. N-linked Oligosaccharide Structure
2 classes of N-linked oligosaccharides:
High mannose - simple - only 2 GlcNAc and up to 9 Mannose
Complex - more complex composition - terminal trisaccharides added to core
Same core structure - 2 GlcNAc and 3 Man
Glycoproteins with terminal Man-6-P are directed to lysosome

29. N-linked Glycoprotein Synthesis
Requires a lipid carrier (dolichol phosphate) to form activated oligosaccharide
Oligosaccharide then transferred as branched sugar chain to ASN on protein
Dolichol phosphate is an integral lipid of the ER membrane
Dolichol phosphate
n = 17 in humans

30. N-linked Glycoprotein Synthesis
Individual sugars added to dolichol phosphate one at a time by specific glycosyl transferases, then oligosaccharide donated to protein.

31. Processing of N-linked Oligosaccharides from High Mannose to Complex Forms
Oligosaccharide transferred from dolichol phosphate to protein while protein translated in ER
Sugars removed and added as glycoprotein moves from ER through Golgi complex
Oligosaccharide pruned in ER and Golgi to core structure of 2 GlcNAc and 3 Man
Core structure elongated by addition of one or more sugars in trans-Golgi to make complex oligosaccharide
new protein
ribosome

32. I-Cell (Inclusion Cell) Disease
Deficiency in machinery that targets lysosomal enzymes to lysosome
Deficient phosphotransferase required for tagging terminal mannose with phosphate group
Lysosomal enzymes require Man-6-P as terminal sugar for proper intracellular trafficking
Enzymes secreted to extracellular space instead of transported to lysosome
Lysosomes engorged with undigested substrate, which accumulate and form inclusion bodies
Death in infancy

33. Glycolipid Structure and Function
Sugar derivatives of sphingolipids
Involved in intracellular communication
Mainly on outer surface of plasma membrane
Cerebrosides
Ceramide w/ monosaccharide (Glu or Gal)
Gangliosides
Ceramide w/ complex oligosaccharide
High concentrations in neural cells
NANA
NANA

34. Lysosomal Pathway for Ganglioside GM1 degradation
Various enzymes may be missing in specific lipid storage diseases

35. Patient Diagnosis
Jay Sakz's psychomotor development has become progressively more abnormal (see Chapter 15). At 2 years of age, he is obviously mentally retarded and nearly blind. His muscle weakness has progressed to the point that he cannot sit up or even crawl. As the result of a weak cough reflex, he is unable to clear his normal respiratory secretions and has had recurrent respiratory infections.

36. Lysosomal Storage Diseases
The sphingolipidoses affect mainly the brain, the skin, and the reticuloendothelial system (e.g., liver and spleen). In these diseases, complex lipids accumulate. Each of these lipids contains a ceramide as part of its structure. The rate at which the lipid is synthesized is normal. However, the lysosomal enzyme required to degrade it is not very active, either because it is made in deficient quantities as a result of a mutation in a gene that specifically codes for the enzyme or because a critical protein required to activate the enzyme is deficient. Because the lipid cannot be degraded, it accumulates and causes degeneration of the affected tissues, with progressive malfunction, such as the psychomotor deficits that occur as a result of the central nervous system involvement seen in most of these storage diseases.
Glycolipids are degraded by exoglycosidases in lysosomes
Defects in glycolipid degradation lead to lysosomal storage diseases
also called sphingolipidoses or gangliosidoses
Glycolipids accumulate in lysosomes
Causes lysosome to lyse and release enzymes or prevents normal functioning
Affects central nervous system

37. Jay Sakz has Tay-Sachs Disease
Tay–Sachs disease, the problem afflicting Jay Sakz, is an autosomal recessive disorder that is rare in the general population (1 in 300,000 births), but its prevalence in Jews of Eastern European extraction (who make up 90% of the Jewish population in the United States) is much higher (1 in 3,600 births). One in 28 Ashkenazi Jews carries this defective gene. Its presence can be discovered by measuring the tissue level of the protein produced by the gene (hexosaminidase A) or by recombinant DNA techniques. Skin fibroblasts of concerned couples planning a family are frequently used for these tests.
Carriers of the affected gene have a reduced but functional level of this enzyme that normally hydrolyzes a specific bond between an N-acetyl-D-galactosamine and a D-galactose residue in the polar head of the ganglioside.
No effective therapy is available. Enzyme replacement has met with little success because of the difficulties in getting the enzyme across the blood–brain barrier.

38. Lysosomal Storage Diseases
Examples of Defective Enzymes in Some Gangliosidoses (also called sphingolipidoses)
Disease
Enzyme Deficiency
Accumulated Lipid
Clinical Symptoms
Tay–Sachs disease
Hexosaminidase A
Cer–Glc–Gal(NeuAc) : GalNAc GM2 ganglioside
Mental retardation, blindness, cherry-red spot on macula, muscular weakness, death at 2-3 yrs
Fabry’s disease
α-Galactosidase
Cer–Glc–Gal :Gal globotriaosylceramide
Skin rash, kidney failure
Metachromatic leukodystrophy
Arylsulfatase A
Cer–Gal :OSO33- sulfogalactosylceramide
Mental retardation and psychological disturbances in adults, demyelination
Krabbe’s disease
β-Galactosidase
Cer :Gal galactosylceramide
Mental retardation, myelin almost absent
Gaucher’s disease
β-Glucosidase
Cer :Glc glucosylceramide
Enlarged liver and spleen, erosion of long bones, mental retardation in infants
Niemann-Pick disease
Sphingomyelinase
Cer :P–choline sphingomyelin
Enlarged liver and spleen, mental retardation, fatal in early life
Farber’s disease
Ceramidase
Acyl :sphingosine ceramide
Hoarseness, dermatitis, skeletal deformation, mental retardation, fatal in early life
NeuAc, N-acetylneuraminic acid; Cer, ceramide: Glc, glucose; Gal, galactose; Fuc, fucose.
:, site of deficient enzyme hydrolysis reaction

39. Blood Group Substances
The blood group substances are oligosaccharide components of glycolipids and glycoproteins found in most cell membranes. Those located on red blood cells have been studied extensively. A single genetic locus with two alleles determines an individual's blood type. These genes encode glycosyltransferases involved in the synthesis of the oligosaccharides of the blood group substances.
Most individuals can synthesize the H substance, an oligosaccharide that contains a fucose linked to a galactose at the nonreducing end of the blood group substance (see Fig ). Type A individuals produce an N-acetylgalactosamine transferase (encoded by the A gene) that attaches N-acetylgalactosamine to the galactose residue of the H substance. Type B individuals produce a galactosyltransferase (encoded by the B gene) that links galactose to the galactose residue of the H substance. Type AB individuals have both alleles and produce both transferases. Thus, some of the oligosaccharides of their blood group substances contain N-acetylgalactosamine and some contain galactose. Type O individuals produce a defective transferase, and, therefore, they do not attach either N-acetylgalactosamine or galactose to the H substance. Thus, individuals of blood type O have only the H substance.

40. Glycolipids as Determinants of Blood Groups
ABO blood group antigens are complex carbs present on BOTH glycolipids and glycoproteins of RBC membrane
Oligo core called “H substance”
Type A person has transferase that attaches GalNAc to H substance
Type B person has transferase that attaches Gal to H substance
Type AB person has both transferases
Type O person has neither transferase

41. Glycolipids as Determinants of Blood Groups
Type A person
Antibodies against B antigen
Type B person
Antibodies against A antigen
Type AB person
Neither A nor B antibodies
Type O person
Both A and B antibodies
Which one is universal donor?
Which one is universal recipient?

42. Blood Serum/Plasma vs. Cells
Serum = liquid portion of coagulated blood after centrifugation - contains anti-A and/or anti-B antibodies
Plasma = liquid portion of uncoagulated blood after centrifugation – similar to serum but also contains fibrinogens
Red blood cells have A and/or B antigens on surface
Whole blood
Blood separated into plasma and cells
Transfusions usually done by transfer of packed RBCs- i.e. RBCs, but not plasma (with antibodies), are transfused
For incompatibility testing (type and cross-match) of blood, the serum of the recipient (which would contain antibodies) is mixed with RBCs from the donor. Agglutination of the RBCs indicates incompatibility.

43. Erna Nemdy and Blood Groups
During her stint in the hospital blood bank, Erna Nemdy learned that the importance of the ABO blood group system in transfusion therapy is based on two principles (Table 30.4). (a) Antibodies to A and to B antigens occur naturally in the blood serum of persons whose red blood cell surfaces lack the corresponding antigen (i.e., individuals with A antigens on their red blood cells have B antibodies in their serum, and vice versa). These antibodies may arise as a result of previous exposure to cross-reacting antigens in bacteria and foods or to blood transfusions. (b) Antibodies to A and B are usually present in high titers and are capable of activating the entire complement system. As a result, these antibodies may cause intravascular destruction of a large number of incompatible red blood cells given inadvertently during a blood transfusion. Individuals with type AB blood have both A and B antigens and do not produce antibodies to either. Hence, they are “universal” recipients. They can safely receive red blood cells from individuals of A, B, AB, or O blood type. (However, they cannot safely receive serum from these individuals, because it contains antibodies to A or B antigens.) Those with type O blood do not have either antigen. They are “universal” donors; that is, their red cells can safely be infused into type A, B, O, or AB individuals. (However, their serum contains antibodies to both A and B antigens and cannot be given safely to recipients with those antigens.

44. ABO Blood Group Summary
Characteristics of the ABO Blood Groups
Red cell type O A B AB
Possible genotypes OO
AA or AO
BB or BO
Antibodies in serum
Anti-A & B
Anti-B
Anti-A
None
Frequency (in Caucasians)
45%
40%
10% 5%
Can accept blood types
A, O
B, O
A, B, AB, O
The second important red blood cell group is the Rh group. It is important because one of its antigenic determinants, the D antigen, is a very potent immunogen, stimulating the production of a large number of antibodies. The unique carbohydrate composition of the glycoproteins that constitute the antigenic determinants on red blood cells in part contributes to the relative immunogenicity of the A, B, and Rh(D) red blood cell groups in human blood.
For more info about Rh: