Two major classes of sugar molecules are _______ (______) and _______ (_______).
Aldose (aldehyde) and ketose (ketone)
__________ is the simplest aldose sugar. The simplest ketose sugar is ________ and it is _______
- Glyceraldehyde (aldotriose)
- dihydroxyacetone (ketotriose)
What do these colors signify? How many stereoisomers are possible?
What is the caveat for long sugars
Green represents the aldehyde functional group and the chiral carbons are in red
There are four chiral stereocenters so 24 = 16
Caveat: sugars that are five or more carbons in length are not linear in nature. Rather, they are cyclical
Name the shortest aldose and ketose?
How many stereoisomers does each form?
Cyclization reaction occurs between ______ and a(n) ______ (which carbons are involved?). This results in a ________.
- aldehyde and an alcohol (C1-C5 reaction)
Right -OH groups in Fischer projections point ______ in ring form. Left -OH groups in Fischer projection, point _______ in the ring form. Draw the α and β haworth forms and statehow many stereoisomers do the haworth forms have?
How do you number rings in chair conformations
Moving away from the ring oxygen starting with 1) the anomeric carbon
The anomeric carbon is the _____ center of a sugar. How do you identify the anomeric carbon?
What is unique about carbon #6 on D-glucose
- reactive center
- The anomeric carbon will have a hydroxyl group or OH- relic bound to it and one carbon away from the embedded oxygen
- Carbon #6 is not part of the ring
Draw the chair conformations of both glucose conformations and compare stabilities and steric clashes
Sugars can have isomers known as ______ (define) (essentially, they are __________).
- epimers: differ at the positioning of the -OH group at one chiral carbon
The formation of cyclic forms of mannose and galactose follows the same reaction mechanism as _______
Draw the Fischer and Haworth diagrams of the epimers D-galactose and D-mannose. Compare the stereo isomeric count in both forms
Biophysical method: The concept and illustrative as well as graphical example of ________. What is being observed in the graph? What do the arcs and their point of convergence mean?
- Graphical illustration of mutarotation occurring in an AQUEOUS solution
- You are observing a change in the optical rotation as the sugar interconverts to its other anomer
The downward arc from 112° to 52.7° represents decreasing degree of plane polarized light rotation
The upward arc from 18.7° to 52.7° represents increasing degree of plane polarized light rotation
- Both converging on about 52.7°, a mixture of both α and β glucose so essentially, it opens up Fischer style
What are the percentages of α and β glucose in the body?
- 67 % β glucose because less steric clashes
- 33 % α glucose
The cyclization of fructose is similar to that of _______. What is the cyclic version of D-Fructose called?
- α-D-Fructofuranose and β-D-Fructofuranose
Draw both the Fischer projection of D-Fructose and the Haworth projections (There should be 3 drawings total). Also state the number of stereoisomers for each diagram
The cyclization of fructose involves ____ and the ___ ___ group and is an example of a ________ reaction (define)
- C2 and C5-OH group
- Hemiketal reaction: reaction between an alcohol functional group and a ketone function group
- **Not the same as a hemiacetal reaction that we saw prior where an alcohol reacts with an aldehyde
Define oxidized and reduced
- oxidized(1): combine or become combined chemically with oxygen (2): undergo or cause to undergo a reaction in which electrons are lost to another species
- reduced(1): combine or become combined chemically with hydrogen (2): undergo or cause to undergo a reaction in which electrons are gained by one atom from another
What are the biochemical experimental methods to determine whether a sugar is a reducing sugar or a non-reducing sugar? Illustrate each
- Tollens' or Benedict's test
How do you know if a sugar is a reducing sugar (3) vs a non-reducing sugar (2) (is there another name for these)?
- A reducing has a free -OH on its anomeric carbon
- All reducing sugars CAN mutarotate
- All reducing sugars will produce a positive Tollens' or Benedict's test result
- All non-reducing sugars (also called glycosides) CANNOT mutarotate
- Will produce a NEGATIVE Tollens' or Benedict's test result
How many types of glycosidic linkages are there? Describe each
- Two types
- Type 1: a covalent bond between a sugar (monosaccharide) and another sugar (monosaccharide)
- Type 2: a covalent bond between a sugar's anomeric carbon and an alkyl group
What categories of type 1 glycosidic bonds do we encounter? Describe each
- Two categories
- Category A: A glycosidic bond between an anomeric carbon of one sugar AND a non-anomeric carbon of another sugar
- Category B: A glycosid bond between an anomeric carbon of one sugar AND an anomeric carbon of another sugar
Characteristics of a sugar glycoside
- When an anomeric carbon of one sugar reacts with the anomeric carbon of another sugar (hence, no FREE anomeric carbonds with -OH groups)
- When the -OH group of an anomeric carbon of a sugar is chemically transformed into an -OR
When is the presence of a sugar glycoside easiest to determine? What if there are more than two sugars?
- Easiest to determine when you have two sugars (anomeric-anomeric glycosidic linkage)
- If there are more than two sugars, look at the sequence in pairs, paying close attention to the last pair (pair on edge should be anomeric-anomeric)
Anomeric carbons can take ____ conformations, name each.
- two conformations
- β (or equatorial) or α (axial)
Steps to determining glycosidic bond linkages (explain)
- 2 processes:
- 1) Determine if the glycosidic bond in question is between an anomeric carbon and a non-anomeric carbon
- 2) If YES, need to write it as α1-2 or β1-2
- Determine if the glycosidic bond in question is between an anomeric carbon and another anomeric carbon
- If YES, need to write as α1-α1 or β1-β1
- ONLY anomeric carbons need to be specified using α or β AND the anomeric carbon #
- Non-anomeric carbons ONLY need to be specified with a #
What are the two principal disaccharides state both coloquial and systematic names. Draw both (please note alpha/beta symbols will not be provided on the exam)
1) Maltose aka α-D-glucopyranosyl (1→4) α-D-glucopyranose
2) Lactose aka β-D-galactopyranosyl (1→4) α-D-glucopyranose)
Maltose is a ______ sugar meaning it has a _____ _____ group on its anomeric carbon (blue circle). Glycosidic bond involves a _____ glycosidic linkage. How many stereoisomers does Maltose have (HAWORTH)
- reducing sugar
- free -OH group
- α1-4 glycosidic linkage (Does not ever change)
- 210 = 1024 stereoisomers (10 total chiral centers; 5 on each sugar)
How are disaccharides formed?
The result of 2 hemiacetals reacting to form an acetal
Lactose is a ______ sugar, it has a _____ _____ group on its anomeric carbon. What does its glycosidic bond consist of? How many stereoisomers does it have?
- reducing sugar
- free -OH group
- It glycosidic bond involves ONE anomeric carbon, the β1-4 glycosidic linkage and a non-anomeric carbon
- 210 = 1024 (10 total chiral centers; five on each sugar)
How many anomeric carbons are in an α-conformation?
How many anomeric carbons are in a β-conformation?
Determine the precise number of glycosidic linkages
Will it mutarotate?
- The black arrows
- Yes there is a free anomeric carbon
- Advice: If you wish, you may use the last pair concpet
Determine the glycosidic linkage
Is this an example of a glycoside?
Is this a reducing sugar?
- Black arrows
- Yes it is, the anomeric carbons have reacted with each other
- No, because there are no free -OH groups on anomeric carbons. Both anomeric carbons are being used to create a glycosidic linkage
Determine the glycosidic linkage
How many anomeric carbons are in the α conformation?
How many anomeric carbons are in the β conformation
- black arrows 1 = α1-2; 2 = α1-2; 3 = β1-4
- 3 are in the α conformation
- 1 is in the β conformation
For the following Carbohydrate names, state:
1) Building unit
2) Types of glycosidic linkage(s) and branching
3) Function: energy storage or structural support
4) Whether it is present in plants or animals
Identify the main chain and branch points
How do amylopectin, amylose and cellulose compare with glycogen with regard to main chain and branchpoint?
- amyloecptin (plants): shares almost exactly the same main chain and branchpoint as glycogen
- amylose (plants): similar to glycogen in main chain but has no branching
- Cellulose (plants): has a similar main chain to that of glycogen and amylopectin
Eukaryotic N-linked glycosylation results in _______. Besides serving a purely ______ need, what is the OTHER functional significance of sugars in our bodies?
- In eukaryotic cells, through a process known as N-glycosylation, sugars are used to monitor the quality of protein folding in an organelle known as the endoplasmic reticulum (ER). Sugars can also eliminate improperly folded proteins in the ER.
What is N-linked protein glycosylation?
The complex addition of sugars to proteins by glycosyltransferases
Define Glycoprotein and state where it takes place
- Glycoprotein: A protein that contains covalently linked sugars
- Location: Endoplasmic reticulum (ER)
What is a glycosylation motif? And which amino acid(s) does it target?
- Short stretch of amino acid WITHIN a protein that is to be glycosylated.
- *The motifs exist for a variety of functional purposes
- ...NXS/T... (side chain of N gets glycosylated)
What is the functional significance of N-linked glycosylation?
Guides and monitors proteins through distinct stages of protein folding in the endoplasmic reticulum
How do you calculate the probability of finding a glycosylation motif? What does that probability mean?
- Just a probability calculation:
- Position 1 = asparagine, position 2 = any amino acid EXCEPT proline, position 3 = serine or threonine
- ∴ the probability of finding ONE glycosylation motif in a protein:
- (1/20) * (19/20) * (2/20) * 100% = 0.475%
It means that at random, there is LESS than half-percent chance of finding a glycosylation motif within ANY given protein
1) What is the probability of finding TWO glycosylation motifs?
2) What is the probability of finding THREE glycosylation motifs?
- 1) (0.475%) * (0.475%) = 0.226%
- 2) (0.475%) * (0.475%) * (0.475%) = 0.107%
Below you are provided with a partial sequence of a protein known as TRPM and a study investigating the potential glycosylation of this protein was reported in November 8, 2013 issue of The journal of Biological Chemistry.
Does this sequence contain a glycosylation motif?
How many glucose, N-acetylglucosamine and mannose molecules are in an N-linked Glycan structure?
A cell-biological perspective on the secretory pathway and connecting it with glycosylation and protein folding in the ER
Define or state the role of: Signal sequence, Sec61, SRP, SRP receptor.
- Signal sequence: Hydrophobic sequence at front of secretory proteins
- Sec61: protein channel that allows passing of nascent or newly formed polypeptides
- SRP: Signal recognition particle protein
- SRP receptor: receptor for binding SRP
3 characteristics of the signal peptide
- 1) ~20 residues long at the very N-terminus
- 2) Very hydrophobic
- 3) predominantly aliphatic residues (leucine and alanine)
The figure displays N linked glycosylation and the degradation of glycosylated proteins (7-story)
- Proteins that enter the endoplasmic reticulum (ER) are often modified by the addition of a GlcNAc2-Man9-Glc3 glycan to the side chain nitrogen of Asn residues in the consensus Asn-X-Ser/Thr motif.
- First, the translocon-associated oligosaccharlyl transferase (OST) complex co-translationally transfers GlcNAc2-Man9-Glc3 glycans from dolichol to substrate proteins
- Next glucosidase-I and glucosidase-II sequentially remove two terminal glucoses, generating monoglucosylated substrates that are recognized by calnexin and calreticulin through their carbohydrate-binding globular domains (calreticulin is a soluble protein and is not shown).
- The interaction with calnexin and calreticulin facilitates folding.
- ERP57, a protein disulphide isomerase homologue that is associated with the arm domain of calnexin and calreticulin, catalyses disulphide bond formation
- Following release from the calnexin-calreticulin cycle, the final glucose is trimmed by glucosidase-II.
- If glycoproteins have adopted their native conformations, they can be demannosylated (denoted by the use of parentheses around the mannoses) by ER mannosidases I and II (ER man-1 and man-II) and exit the ER
What occurs in Route 4A and 4B? What happens after proteins exit the ER?
- Route 4A: Properly folded proteins exit or leave the ER
- Route 4B: Proteins that are NOT folding properly are tagged or quarantined for degradation
- After proteins exit the ER, they move into the golgi apparatus
State the function and abbreviation if applicable for the following enzymes:
- Glucosyltransferase: aka UGGT (UDP-Glucose Glycoprotein Glycosyltransferase in the last picture) functions to ADD glucose
- Glucosidase: functions to REMOVE glucose
- Mannosidase: aka ER Man functions to REMOVE mannose
What is the function of the ______ complex Calnexin-calreticulin?
- chaperone complex
- Function: helps to fold proteins inside the ER together with ERP57
Dolichol is a ______ molecule, what are its functions?
- lipid or fat molecule
- Function: It is an ER lipid that holds onto sugars prior to transferring them to the protein. Dolichol uses the help of an enzyme called OST (oligosaccharyltransferase) to transfer sugars
What is ERAD? When does it occur?
- It is a collective terms used for endoplasmic-reticulum associated degradation.
- This takes place when secretory proteins fail to fold properly and are destined to be degraded
What is ERP57
- ERP57: an enzyme whose function is to form disulfide bonds within secretory proteins within the ER
- **Recall: that disulfide bonds are formed between the side chains of TWO cysteine residues
Explain the four meaningful sugar-based codes that can be extracted that guide a secretory protein in its passage through the endoplasmic reticulum and their meaning (please note code #s are arbitrary)
- Code 1 → 3 glucose present at edge of glycan
- Meaning: protein just entered the endoplasmic reticulum, and will undergo folding soon
- Code 2 → 1 glucose present at edge of the glycan
- Meaning: protein is about to enter the folding pathway
- Code 3 → No glucose present at edge of glycan AND exactly ONE mannose is removed
- Meaning: protein is folded and it can leave the endoplasmic reticulum
- Code 4 → No glucose present at edge of glycan AND a LOT/SOME of mannoses are removed:
- Meaning: protein does NOT fold and it is degrraded through the ubiquitin-proteosome pathway