CLASS: HOUR 1 Scribe: Spencer Terry

DATE: 8-19Proof: Lauren Morris

PROFESSOR: PritchardGlycoproteinsPage1 of 5

Glycoproteins [S1]
Glycoproteins are an extremely important group of molecules. There are dozens, if not hundreds.
They are on the surfaces of all of our cells, as well as our blood serum and all of our secretions.
There are more glycoproteins than there are proteins that don’t have sugars attached.
Proteolycans are technically glycoproteins, but we tend to keep them separate.
Typically with glycoproteins, we are talking about much smaller oligosaccharide side chains.
O-linked oligosaccharides are linked through an oxygen of a serine or threonine.
N-linked are linked through a nitrogen of asparagine.
O-linked Oligosaccharide Chains [S2]
Linked through oxygen or either serine or threonine
N-acetylgalactosamine because the hydroxyl is up. If the hydroxyl is down, it would be a glucosamine.
All O linked oligosaccharides are O-linked through an extra galactosamine.
N-acetylgalactosamine often referred to as GalNAc
Mucin Glycoproteins [S3]
This is a major group of O-linked glycoproteins
These typically have loads of serines and threonines, sometimes every 3rd residue.
Oligosaccharides are attached to these serines and threonines.
There are a dozen mucin genes in people. While the genes are identical in people, the oligosaccharide part can be different.
Salivary Mucins [S4]
Provide all kinds of important functions, not going to dwell on this.
See slide.
Human Gastric Mucin [S5]
1985, discovered almost all ulcers are caused by Helicobacter pylori bacteria.
It took physicians years to realize that all they needed to do was kill the bacteria with tetracycline (antibiotic)
Half the people on the planet have Helicobacter in their stomach, but not all people have ulcers. The people that do tend to have different oligosaccharides on their gastric mucins.
Most people have terminal alpha 1,4-GlcNAc, which have antibiotic activity, therefore limiting ulcers.
ABO Blood Group Determinants [S6]
Type O is the most common, but there are loads of A and B around.
Type B have terminal galactose. Type a have terminal GalNAc. Type O have neither.
80% of people are secretors. They secrete in all their secretions glycoproteins that have their own blood group type on it. If you are type A, then in your saliva, you will have mucins that have this structure on them.
The other 20% don’t have these on their mucins.
Red Blood Cell from individuals of type…[S7]
Shortly after babies are born, they start making antibodies for the blood group types they don’t have. (type A make antibodies for type B, etc)
When you get a blood transfusion, you are getting red blood cells from someone else. If Type A gives to Type A, then there is no agglutination. However, if Type A is given to type B, then agglutination will occur and the patient likely dies.
Blood group Type O is the universal donor, because type O doesn’t have the terminal groups that are specified for by the antibodies.
An AB person is a universal acceptor. They can receive blood from anyone and not have agglutination.
The Expression of A,B, and H…[S8]
Whether you are a secretor or not depends on whether you express a certain gene (Se). It allows you to produce a fucosyl transferase.
This enzyme attaches a fucose to the structures in slide 6. If you don’t have this, then you are not a secretor.
ABH Secretor Status and Disease Susceptibility[S9]
As it turns out, there is an advantage to being a secretor.
Women that are not secretors, who are blood type B and AB, are more likely to have UTI’s. Why? Mucins coat the urinary tracts of people. Bacteria must get through this mucin layer and adhere. So, depending on the receptors on the cell and what mucins are made, some bacteria are more likely to do it to some people than others.
Men who are non-secretors are much more likely to have peptic ulcers. Depending on the nature of your mucins, it determines how susceptible you are to these Helicobacter infections.
See slide for diabetics and akylosing.
Garrett/Grisham, Biochemistry 7.34[S10]
Glycoproteins with O-linked oligosaccharides very often have serines and threonines every 3 residues or so.
When it is important for a protein to be in an extended conformation, it can be coded to be bristling with these oligosaccharides. Normally, protein will fold up in a compact form, but if it is important to be extended, then the protein can be glycosylated so it won’t fold up.
Garrett/Grisham , Biochemistry 7.35[S11]
About half of the fish in the arctic make antifreeze glycoprotein. Virtually all the fish in Antarctic waters make it.
Seawater is -2 degrees Celsius. The fish makes several types of these glycoproteins where every 3rd residue is a threonine.
As seen in the slide, an N-acetylgalactosamine and galactosyl is attached to the threonine. This keeps the fish from freezing solid.
O-linked oligosaccharides are synthesized post-translationally [S12]
How are O-linked Oligosaccharides made?
The key phrase here is that they are synthesized post-translationally. This means that the whole protein chain will have been synthesized, then you start adding the sugars to it one at a time. (illustrated on slide)
There is a protein with a serine residue, then GalNAc transferase adds a GalNAc, then others are added.
The whole point is it occurs on a protein that has already been synthesized.
When we get to N-linked oligosaccharides, it’s just the opposite. They will be added not one at a time, but in block to a incomplete protein chain.
N-Linked Oligosaccharide chains [S13]
In slide, an asparagine residue from a protein is shown. Notice that instead of GalNAc, we have GlcNAc
Also, instead of being alpha linked, this is beta linked. This is how all N-linked oligosaccharides are linked.
This bond is alkali resistant. If you stirred up the glycoprotein in 10 N sodium hydroxyl, the bond would not break. However, if the same was done to O linked, the bond would break.
No Title - structures [S14]
These are typical structures of N linked oligosaccharides. There are hundreds of different possibilities.
The one thing they must all have in common is that there are two GlcNAc’s here; a mannose that is branched, more mannoses, and very often they are decorated later with fucose or GlcNAc.
Sa stands for saliac acid.
No title - structures
Don’t worry about learning these structures.
The point of this slides is to see that there are many different arrangements, but there are certain things in common (mainly 2 GlcNAc’s and branching mannose
No title - structures
Different tissues and different people often have different N-linked oligosaccharides on glycoproteins.
Dolichol Phosphate
N-linked oligosaccharides are all made on this big lipid carrier protein.
This is a so-called isoprene unit. Isoprene is an organic unit used in making rubber.
The point is, this is a big, big unit ( sometimes 13 units, sometimes 23).
It is a gigantic lipid that has a phosphate attached to it (called Dolichol phosphate). N-linked oligosaccharides are built up on it.
Biosynthesis of Oligosaccharides and Glycoproteins
This is ER. Dolichol phosphate at step 1. Notice the phosphate is on the cytosolic side of the ER.
So at step one, add N-acetylglucosamine phosphate (UDP – N-acetylglucosamine) to the dolichol phosphate, so you add both the phosphate and the N-acetylglucosamine (GlcNAc).
Step 2 – Again use UDP- GlcNAc, but this time, you are only adding the GlcNAc. So now you have 2 GlcNAc’s attached (outside in the cytoplasm).
Then 5 different mannosyl transferases, one at a time, will add a mannose residue.
Then translocase (which is also called flippase). A flippase has the ability to flip the structure from the cytosol into the ER. This only works once the 5 mannoses have been added.
More mannoses are then added, but GDP mannoses are no longer used directly. What happens first is there is a dolichol phosphate in the cytosol that is charged with a mannose residue by using GDP mannose, and then another type of flippase will flip this mannose phosphate part into the interior of the ER.
The dolichol phosphate mannose then adds a phosphate to the structure with 5 mannoses. This process is repeated until the structure has 9 mannoses.
This same trick is used to add glucose residues. The first step is charging a dolichol phosphate with a glucose using UDP-glucose, and another type of flippase flips it in. This then adds the glucose residues. 3 are added, resulting in a 14 unit structure.
Then eventually, this 14 unit structure is added to growing polypeptide chains being extruded into the ER. The more of the polypeptide extruded into the ER, the more of these N-linked chains will be transferred from the dolichol phosphate.
No title – cell diagram
Rough ER is rough because ribosomes are studding the ER.
Some of the vesicles budding off the ER will move through the cytoplasm and fuse with the Cis-golgi, with various processing occurring. Then more will bud off and fuse with the medial Golgi. Then more will bud off and fuse with the trans Golgi. Then more will bud off and fuse with the plasma membrane.
Soluble glycoproteins – vesicles will fuse with the membrane and open up, dumping these into the bloodstream.
If it is a membrane bound protein, it will be bound inside this vesicle, and when the vesicle fuses with the membrane, the glycoproteins will then naturally be on the outside of the plasma membrane.
Golgi Complex is a sorting center
Turns out the 14 unit structure must be processed further before it ends up on a final glycoprotein that ends up being secreted.
After the 14 unit structure is transferred to the growing polypeptide chain, there is an enzyme in the ER that cleaves off the first glucose, then another enzyme cleaves off 2 more glucoses.
An enzyme then cleaves off a mannose unit. This unit is then bundled off into a vesicle that fuses with the cis golgi apparatus. 2 things can then happen: 1) UDP-GlcNAc could add a GlcNAc phosphate to a mannose. (Done twice). Then there is an enzyme that cleaves off the GlcNAc, leaving the phosphates. 2) Generally for most glycoproteins, enzymes cleave off more mannose residues. When 5 mannoses remain, it is packed in a vesicle and shipped to medial golgi, where more processing occurs. You can add GlcNAc for example, or cleave off more of the mannoses, then add more GlcNAc.
Then at the end of that stage, packed into more vesicles and sent to trans golgi, where more processing occurs. You can then add galactose residues and finally add sialic acid residues. Then it can finally leave the golgi.
The Initial Steps in the Biosynthesis
The interesting thing is, this mechanism is how all people, rats, mice, cucumbers, pine trees, mushrooms, etc. make the 14 unit structure. It is highly conserved in the ER. However, once it reaches the Golgi, the processing is not highly conserved.
There are hundreds of types of N-linked oligosaccharides, and that diversity is a result of different processing in the Golgi. What is made in the ER is essentially identical in all animals and plants (not bacteria).

One of the most important functions
Why add glucoses early on and then remove them? It is essential for getting the glycoprotein to fold up correctly.
Diagram –
This diagram is more detailed than I want.
Main message is that in the ER, the first two glucoses are taken off this 14 sugar unit. It then binds to one of these chaperone proteins (calreticulin or calnexin). If it is folded correctly, the last glucose will be cleaved off and the structure will move into a vesicle and be secreted. If it is not folded correctly, then glucose gets added back to it and the enzyme involved in this will have the ability to reduce disulfide bonds, thereby opening it back up and giving it another chance to fold. It then goes through this process again to check folding.
Calnexin and Calreticulin
This is showing the same thing.
Calnexin and Calreticulin
This is showing the same thing.
Mannose-6-Phoshate Targets an Enzyme to the Lysosome
Remember when I was going through this processing in the golgi, there was a side branch where the GlcNAc-phosphates were added to the mannoses, and then the GlcNAc was taken off, leaving the mannose. That is illustrated here.
An enzyme called a phosphotransferase adds this GlcNAc-phosphate to a mannose at the 6th position.
Some people are born are lacking the phosphotransferase. These people turn out to have many problems, including mental retardation. Under electron microscopes, it was seen that these people’s cells were filled with dark looking inclusions (dark because they are electron dense).
When glycoproteins need to be directed to the lysosome for degradation, they are tagged with a phosphate on the mannose. If you don’t have these degradative enzymes in the lysosome, then you can’t break down all these things and they build up, and you get these inclusions, which is why it is called I-cell disease.
Often, these genetic diseases provide clues to biochemists about how things work inside the cell.
Figure 7.43
Most glycoproteins that stay in the blood have sialic acid residues as their terminal sugar. Sialic acid is also called N-acetylneuraminic acid. There are low levels of an enzyme, neuraminidase, that cleave off sialic acid in the blood. This enzyme is in most of the cells as well. However, the levels are too low to rapidly take off the sialic acid. When you get a freshly made glycoprotein in the cell, it will be loaded with sialic acid residues, but after it has been around for a long time, these residues will gradually be cleaved off.
There is a protein, asialoglycoprotein receptor, on the surface of liver cells that recognizes terminal galactose residues, which become terminal after cleaving of sialic acid. It then binds to it and pulls the glycoproteins out of the cell in order to break them down. It is basically a mechanism for removing old glycoproteins.
HIV Glycosylation
Many viruses, like the one that causes AIDS, are bristling with glycoproteins on their surface. Those proteins are important for infectivity, and are targets of attempts to make vaccines against these viruses.
Influenza hemaglutinin
Many other glycoproteins have components on them that specifically react with glycoproteins on our cell surfaces. Flu virus has a hemaglutinin that binds to sialic acid residues of glycoproteins on our cells.
Viruses exploit the glycoproteins on the surfaces of our cells.
Glycosylation of Surface Moities of Cancer Cells
People have known for years that when cells become transformed, tumor antigens are expressed. Tumor antigens are almost invariably altered glycosylation patterns on a normal glycoprotein.
Enzymic Deffects in Degradation of Asn-GlcNAc
We must be able to break down glycoproteins. Otherwise, they will build and there will be a problem
This is a diagram of a typical N-linked oligosaccharides.
For example of breaking down glycoproteins, there is an enzyme, fucosidase, that is responsible for cleaving the Fucose (position 8). If you lack this enzyme, you will have a disease (retardation in this case). Just look at the other examples.
Collagen Glycosylation
This is one of the minor types of glycosylation.
Lysine residues can be post-translationally modified ( oxidized) to put a hydroxyl group on them. When this occurs, there is then an enzyme that will add a galactose to it. A glucose is then added to the galactose. This can be quite extensive in collagens (up to 12%). This is important for collagen fibril assembly.
People that can’t do this make defective collagen fibrils.
O-GlcNAc Glycosylation
In the nuclei of our cells, most of the soluble proteins will undergo O-GlcNAc glycosylation. That’s a GlcNAc added to a serine or threonine of the protein. This can be dynamic.
The level of GlcNAc added can be very high or very low depending on what the cell is doing (dividing, preparing to make proteins). There is an enzyme that takes off these GlcNAc’s. When it does take it off, there is another enzyme that can add phosphate to the serine or threonine. So, constantly either GlcNAc’s or phosphates are constantly being added or removed. This is critical in regulation of the cell’s activity.
Glycosylphosphatidylinositol (GPI) Anchors
Many proteins are attached to the surface of the cell by these long leashes. It may be important to keep a protein near the cell without imbedding it in the membrane.
If we analyze the GPI anchor (structure in slide), this is what it is: 3 carbons are part of the glycerol (on the right side). There are two long chain fatty acids attached to the glycerol. These 2 chains will be imbedded in the membrane.
The glycerol is then attached to the inositol via phosphate.
Inositol is attached to 4 sugars (GlcNAc, mannose, mannose, mannose). These sugars are then attached to a phosphoethanolamine. It is the Phosphoethanolamine that is attached to the protein via an amide linkage.
This structure is flexible, so when a cell needs to have a protein kept near its surface, this is how it is often done. Many proteins linked in this way are often involved defenses against pathogens (bacteria or viruses). Some bacteria make an enzyme that breaks this linkage to wipe out the defense.
Lectins
Lectins are proteins from many sources that specifically bind sugar units.
For example, there is a lectin in green peas that binds alpha-mannose units. Others bind beta-mannose, fucose, galactose, etc.
Biochemists use these all the time. They are very useful. Can have an immobilized lectin on a column and wash everything through. The glycoproteins bind, therefore purifying the sample.
Selectins
In our blood, most of the cells are whishing along at the same speed.
In this tissues, this can be watched using a video camera.
Most white cells zoom along at the same speed as the red cells. However, around 15% don’t move that fast. The semi-adhere to the capillary endothelial cells and roll along. If there is inflammation somewhere, these rolling neutrophils will stop and interact with integrins. The net effect is that the neutrophils will push between the cells of the capillary endothelial and get to the site of inflammation (this process is extravasation)
The way they roll is that both on the endothelial cell and on the leukocytes there are selectins and ligands for selectins.
Selectins
This is what I just said. Skipped.
Selectin Chart
This particular structure (Sialyl Lewis X determinant) is the ligand. Selectins bind to this.
Drug companies are very interested in this because all kinds of human diseases involve inflammation and selectins are involved in many of them
Sugar Slide
People think sugars are all really hydrophilic and only bind hydrophilic things, but sugars have a hydrophobic side and will bind to proteins or enzymes that have these highly hydrophobic tryptophan residues.
Structure
Skipped this slide.

[End 47:31 mins]