Download MEDICAL BIOCHEMISTRY Lectures 35-36 Chp. 26

MEDICAL BIOCHEMISTRY
Lecture 39-40
Chp. 30 - Synthesis of Glycosides, Lactose,
Glycoproteins, and Glycolipids
Dr. Mythreye Karthikeyan
803-576-5806
[email protected]
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.
Metabolism of UDP-Glucose
• An activated sugar
nucleotide
• Precursor for:
– Glycogen
– Lactose
– UDP-glucuronate
and glucuronides
– Carbs chains in
proteoglycans,
glycoproteins, and
glycolipids
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.
Metabolism of UDP-Glucuronate
• Formed from UDPglucose
• 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
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
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
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
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
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
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
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.
Jaundice and Galactosemia
Galactose
Galactose-1-phosphate
X
UDPglucose
UDPgalactose
• 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
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
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
1. Galactosyltransferase (enzyme)
2. -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
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
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
Synthesis of Amino Sugars
• Amino sugars used in
synthesis of
glycosaminoglycans are all
derived from glucosamine 6phosphate
• Amino sugar synthesis:
1. Amino group transferred from
amide of glutamine to fructose
6-P to make glucosamine-6-P
2. Amino sugar can then be Nacetylated at amino group by
transfer of an acetyl group from
acetyl CoA
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
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
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
Glycoproteins vs. Proteoglycans
Glycoprotein
• Carb portion less monotonous does not have repeating
disaccharides
• Carbs form short highlybranched chains
Proteoglycan
• Carb portion greater in mass
than protein portion
• Carb dominates structure
• Carb forms long, linear,
unbranched chains with
repeating disaccharides (GAGs)
Glycoprotein Structure
• Sugar attached at its anomeric carbon
through a glycosidic link to protein
residue
N-linked glycoprotein
– O-linked = attached to -OH of Ser/Thr or
-OH group of hydroxylysine (collagen)
– N-linked = attached to amide N of Asn
NANA
Gal
GlcNAc
Man
Fuc
= N-acetylneuramine
= galactose
= N-acetylglucosamine
= mannose
= fucose
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
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)
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 nonreducing end
N-linked Oligosaccharide
Structure
• 2 classes of N-linked oligosaccharides:
1. High mannose - simple - only 2 GlcNAc and up to 9 Mannose
2. 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
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
N-linked Glycoprotein Synthesis
Individual sugars added to dolichol phosphate
one at a time by specific glycosyl transferases,
then oligosaccharide donated to protein.
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
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 Man6-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
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
Lysosomal Pathway
for Ganglioside
GM1 degradation
Various enzymes
may be missing in
specific lipid
storage diseases
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.
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
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 Dgalactose 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.
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 :OSO33sulfogalactosylceramide
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
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. 30.17). Type A individuals produce an Nacetylgalactosamine transferase (encoded by the A gene) that attaches Nacetylgalactosamine 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.
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
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?
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
Whole Blood separated into
• Red blood cells have A and/or B
blood
plasma and cells
antigens on surface
• 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.
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.
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
AB
Antibodies in serum
Anti-A & B
Anti-B
Anti-A
None
Frequency (in Caucasians)
45%
40%
10%
5%
Can accept blood types
O
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: http://anthro.palomar.edu/blood/Rh_system.htm