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Carbohydrates
James R. Ketudat Cairns
Aj. Jim
Pictures from Stryer, Biochemistry (mostly)
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What are Carbohydrates?
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(CH2O)n
• Aldehydes (aldose sugars)
• Ketones (ketose sugars)
• 3 or more carbons.
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Fischer Projections
D and L isostereomers depend on the configuration
of the chiral carbon furthest from the carbonyl.
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D-Triose to
D-Hexose
L-sugars are the
mirror image of the
D-sugar.
Sugars that differ in
stereochemistry at
one position are
called epimers.
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D-Ketoses
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Carbonyl reactions with alcohols
Note: Similar reactions can occur
with amines and other nucleophiles.
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Monosaccharide cyclization
• Formation of an internal hemiacyl or hemiketal is
favorable, if it forms a 5 or 6 member ring.
• Furanose = 5 member ring
• Pyranose = 6 member ring
D-Glucopyranose
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Anomeric Configuration
• Sugars can have two anomeric
configurations for each type of ring.
– In solution, there are a mix of linear and ring
forms that depends on the stability of each.
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Sugars are not flat and can form
different puckered shapes
• Furanose envelopes are most stable.
• Pyranose chairs & boats are stable.
– Most stable depends on steric interactions.
• Axial OH tend to bump, while equatorial do not.
Ribose envelopes
Glucose chair & boat
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Pyranoses can move through many
structures, only a few are stable
B = boat
C = chair
H = half chair
S = skew boat
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Vocadlo & Davies, 2008
Glycosides
• Reaction at the anomeric
carbon (hemiacyl or
hemiketal position) form
glycosides.
– The sugar is trapped in one
anomeric configuration.
– The bond between the
sugar and aglycone is
called a glycosidic bond
– The product is a glycoside.
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Glucosides
in nature
Glucosides are
glycosides with
glucose for a sugar.
The compounds
shown are properly
called
b-D-glucopyranosides.
Ketudat Cairns & Esen, 2010,
Cell. Mol. Life Sci.
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Modified & branched
monosaccharides
• Many modified monosaccharides exist in nature.
• There are also branched monosaccharides,
– e.g. apiose
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Oligosaccharides
• If two or more monosaccharides
polymerize through glycosidic
bonds, the are
oligosaccharides.
• The number of
monosaccharides is designated
by di-, tri-, tetra-, penta-, hexa• They can be explicitly described
as shown for the common
disaccharides to the right.
• Often, they are given names like
cellobiose, cellotriose or (1,4)-bD-mannobiose to simply indicate
their size and linkage.
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Reducing & nonreducing sugars
• Sugars that have a free anomeric carbon can
undergo redox reactions with Cu2+
– (Fehling’s reagent).
• They are called reducing sugars, since they
reduce the copper,while they are oxidized.
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Redox products of sugars
• Sugars can be oxidized at the anomeric carbon
to form aldonic acids.
– E.g. D-gluconic acid, the produce of a Fehling reagent
reaction.
• Sugars can be oxidized at a primary alcohol to
form a uronic acid.
– E.g. D-glucuronic acid, D-galacturonic acid, etc.
• These carboxylic acids can form 5 or 6 member
rings, such as in L-ascorbic acid.
• Sugars can also be reduced to alditols
(polyalcohols).
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Carbohydrate Composition Analysis
• Carbohydrate sugar composition can be tested
by hydrolysis (acid or base with heat to break
glycosidic bonds), TLC, HPLC, IC or
modification and GC/MS.
– Compare to standard sugars.
– HPLC, IC and GC can potentially quantify sugars.
• Modification by acetylation, methylation or
trimethyl silanation can make sugars volatile for
GC (and acetylation can make them detectable
by UV for HPLC).
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Carbohydrate Linkage analysis
• Carbohydrate linkages can be determined by
Nuclear Magnetic Resonance, if the polymer is
not too complex.
• Methylation analysis can determine which
hydroxyls are linked.
–
–
–
–
First methylate all free hydroxyls
Then hydrolyze glycosidic bonds
Reduce and acetylate the linkage positions.
Run methyl acetyl alditols on GC/MS and compare
elution positions to standards.
– Does not tell anomeric configuration, just linkage.
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Methylization analysis chemistry
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Carbohydrate sequencing
• Can see the loss of sugars (hexose, pentose,
etc.) by mass spectrometry (MS)
• Can see fragmentation of sugars in MS
spectrum.
• Can use specific enzymes to cut off sugars one
at a time and look at mass differences.
– E.g. neuraminidase to cut off sialic acid,
– a-mannosidase to cut off a-linked mannosyl
residues.
– These enzymes are called glycosidases or glycoside
hydrolases (GH).
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MS sequencing of N-linked
polysaccharide.
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Positive ion MALDI-TOF mass spectra of
derivatized N-linked glycans from bovine fetuin
Derivatized with MeI
Derivatized with methanol/DMT-MM
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Polysaccharides are important
structural and storage molecules
• Polysaccharides can be grouped by the kinds of
monosaccharides they contain
– Glucans contain glucose
– Mannans contain mannose
– Arabinoxyloglucans contain arabinose, xylose and
glucose.
• Cellulose, a b-glucan is the most abundant
polymer on earth.
– Chitin/chitosan, a similar structural polysaccharide is
also very abundant.
• Starch and glycogen represent storage
polysaccharides
– Alpha-linked glucose polymers
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Comparison of Cellulose with
Glycogen and Starch
• Cellulose is a straight chain, made from alternating
orientations of b-1,4-linked glucosyl residues.
• Starch and glycogen are coiled a-1,4-linked glucosyl
polymers.
Amylose coil
www.agrana.com/en/1761.asp
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Glycogen vs. Starch (Amylopectin)
• Glycogen and starch (amylopectin) differ in how
many 1,6-linked branches they contain.
– Glycogen has an a-1,6-linkage every approx. 8-14
residues.
– Starch has a-1,6-linked branches every approx. 24-30
a-1,4-linked residues.
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Cellulose in cell wall structure
• Cellulose fibers are semicrystalline due to
regular hydrogen bonding
• Therefore, they are hard to break down.
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Plant cell wall polysaccharides
Abcbodybuilding.com
• In plant cell walls, the cellulose fibers are linked
with hemicellulose (other polysaccharides) and
lignin (polyphenolic plastic).
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Other structural polysaccharides
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dalwoo.tripod.com/structure.htm
Complex Carbohydrates
• Complex carbohydrates are complexes of
carbohydrates with other macromolecules
– Glycoproteins – found in all domains of life
– Proteoglycans
– Peptidoglycans (bacterial cell walls)
– Glycolipids
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Types of Eukaryotic glycoproteins
• Cytosolic: single Nacetylglucosamine residues on Ser
or Thr hydroxyls.
– Likely a regulatory function, like
phosphorylation or acetylation.
• Secreted:
– N-linked: bound to asparagine (Asn, N)
• Initial core oligosaccharide added in ER.
– O-linked: bound to hydroxyl groups
(Ser, Thr, HyPro, HyLys).
• Mucin-like added in Golgi
• Alpha-mannose linked started in ER
• Others
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Secretory pathway
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Adding of monosaccharides to
molecules
• Glycosyl transferases
transfer sugars from
nucleotidyl glycosides
to other molecules in
nature.
• In the lab, we can also
use glycosidases to
reverse hydrolyze or
transfer glycoside sugars.
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Synthesis of core oligosaccharides
for N-linked glycosylation
• A core oligosaccharide is synthesized on dolichol in the
ER membrane for transfer to a glycoprotein Asn in the
N-X-S/T sequence.
Cytosol
ER matrix
Dolichol phosphate
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Core oligosaccharide addition to
proteins
• The core oligosaccharide is added to proteins in
the ER.
• The three Glc residues must be cleaved off
before the protein can leave the ER.
– Glucose-binding lectins prevent proteins from
escaping the ER unfolded.
– The alpha-glucosidases that cut off the Glc will not cut
off the last until the protein is folded.
– If the last Glc is not removed, glc transferase adds
another to retain the protein in the ER.
– Abnormal O-mannosylation marks for them to be
removed from the cycle and degraded.
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Calnexin, Glucosidase & Glucosyltransferase
ensure secretory protein folding.
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C-type lectins like Calnexin use Ca
to bind sugars
• Lectins are proteins that bind specific sugars
• C-type lectins are animal lectins that use bind
calcium to help bind the sugar.
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Glycosylation is further modified in
the Golgi apparatus
• In the Golgi glycosidases cut off more of the core
oligosaccharides.
• Glycosyl transferases add other sugars after the trimming.
• The exact carbohydrate varies with the type of organism,
cell and protein.
• Variation in the amount of carbohydrate added to one
protein: microheterogeneity.
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Elastase, a simple glycoprotein
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Phosphorylation and sulfonation
also happen in the Golgi
• Phosphomannose is important for
sorting of several glycolipid &
proteoglycan degrading enzymes to
the lysosome.
• Lack of the enzymes to transfer the
phosphate to mannose results in I-cell
disease, where inclusions of
undigested glycolipids and
proteoglycan develop.
• First GlcNAc-Phosphate is added to
the Mannose 6-hydroxyl, then GlcNAc
is cut off.
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O-linked glycoproteins
• Secreted O-linked
glycoproteins can have from
one to thousands of sugars
added.
• Dystroglycans and some
other proteins have alpha-OMan added in ER.
• The first sugar added is
usually GalNAc or Gal in
mucin-like glycosylation in
the Golgi apparatus.
• Further sugars added in the
Golgi.
• The sugars added can
contain important information,
such as the blood group.
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Glycosamino Glycans
• Glycosamino glycans are carbohydrates that are
usually bound to proteoglycans.
• Play important roles in connective tissues.
• Also called mucopolysaccharides.
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Proteoglycans
• Core proteins can have many times their weight
in glycosaminoglycan carbohydrate attached.
• They hydrate and form a compressible
component to give cushioning to joints and
related tissues.
• They also form a part of the intracellular matrix
between cells.
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Jeffrey & Watt bjr.bjrjournals.org www.histo-moleculaire.com/siteconj/images/037...
Bacterial Peptidoglycans
• Peptidoglycans are major components of bacterial cell
walls
– Thick coating on outside of Gram positive bacteria
– Thinner layer between membranes of Gram negative bacteria
– Cut by Lysozyme.
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Gram positive bacteria cell wall structure
Peptidoglycan cell walls
Staphylococcus aureus peptidoglycan
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Glycolipids:
Glycosphingolipids
Glycoglycerol lipids:
Plant galactolipids
Animal PtdGlc (below)
Cholesterol glucoside
Ishibashi et al., 2013
Glycolipids
Wennekes et al., 2009,
Angew. Chem. Int. Ed. 48,
8848-8869
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Glycosphingolipid synthesis & catabolism
Wennekes et al., 2009,
Angew. Chem. Int. Ed. 48,
8848-8869
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Carbohydrate Active proteins
• Carbohydrate binding proteins/domains
– Carbohydrate binding modules (CBM) can bind
simple sugars or more extensive regions.
– Lectins bind simple sugars.
• Carbohydrate Active Enzymes (CAZy)
– Glycoside Hydrolases (GH, glycosidases)
• Transglycosidases (TG) catalyze transfer rather than
hydrolysis.
– Glycosyl Transferases (GT)
– Polysaccharide Lyases- nonhydrolytic cleavage of
glycosyl linkages
– Carbohydrate Esterases
– Other carbohydrate modifying enzymes
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Viral Carbohydrate-Active Proteins
• The flu virus strains are
distinguished by forms of
carbohydrate active proteins.
• Hemaglutanin (H in virus
name) binds to sialic acid on
cell surface to invade.
• Neuraminidase (N in virus
name) cuts off the sialic acid
to free the virus, once inside
the cell.
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Neuraminidase
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Summary
• Carbohydrates and carbohydrate-active proteins
play critical roles in living organisms.
• Carbohydrates can be analyzed by a variety of
chemical, chromatographic and spectrometric
methods.
• Carbohydrate structures and functions are
determined by the monosaccharides present and
their linkages and modifications.
• Complex carbohydrates contain carbohydrates
linked to other biomacromolecues (proteins,
lipids)
• Carbohydrates & complex carbohydrates are
synthesized by a network of glycosyl transferases
and transporters.
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