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Lecture 9 Endocytosis and Exocytosis:
Reasons Why Glycosylation is Important
Somethings about glucose trimming at the Golgi
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So glycosylation plays an important role in
folding
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Third, when you have glucose sugars, they tend to bind to lectins (they are sugar binding protein, it is a
whole class of proteins that bind to sugar units)
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The sugar unit could become part of the
structure important for the interaction for cell to cell recognition
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Having
the membrane proteins glycosylated plays a role in cell to cell recognition
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The fifth is receptor specificity: it
is talking about ligand(anything that can bind to a receptor) receptor specificity
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It means that without this type of
glycosylation there is no recognition, no binding
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You can measure the insulin biological activity
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This means that when this insulin passes through the
Golgi there are specific glycosylation sugars attached to it, unless this happens it will not have its full
biological activity
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You purify
the hell out of it to make sure you can get as much insulin as possible
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Well, it is hard to produce a lot of these bovine insulin
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An example is bovine
insulin and also a lot of enzymes that are used to prevent heart attached
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I have a friend of mine who owned the biggest pharmocutocal company in Taiwan
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He found out that the urokinase of this horse was the
most active form that he had ever seen
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You can use engineering methods to make the recombinant urokinase more active
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So this was his first product
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In the early part of genetic engineering Ecoli was used for insulin
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So you grow a lot of this recombinant insulin and then isolate it from the

fermentation
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Early on they had a problem, they had a toxicity problem
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Sometimes the Ecoli will produce small toxic factors
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But they overcame this
and started to screen more and more strains of E
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But
there is a problem that recombinant insulin from the E
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More protein into your body to get the same activity as the
biological and the more stuff you put into your system it’s not good
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coli and it could not reach the activity of the biological form
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So afterwards, they found out that the protein sequence of the
insulin is the same as the natural sequence, but because it was not glycosylated it is not as active, so
they started to use Yeast
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What is Antigenicity? All of you know how an antibody is produced? You produce it by injecting an
antigen into an animal and the animal responds by producing antibodies if the protein is foreign to it
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) Affinity: it binds to the antigen very tightly 2
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If you have a protein that is properly glycosylated it has the function of producing an antibody that has
higher affinity to the antigen
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All these
criteria I have given you is to emphasize the importance of glycosylation in proteins
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Where does it start? It all starts in the ER
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It is called N-linked because the oligioscharide is linked to the
Asparagine of a growing polypeptide
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This product that is formed in the ER
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The membrane has a lipid called
Dolichol and if you have a growing peptide near by with the requisite asparagines residue, followed by
another amino acid and then by serine or threonine; You have another enzyme in the membrane called
oligiosaccharyl transferase that will transfer this whole oligiosaccharide complex to the Asparagine of
the growing polyptide
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With the
soluble protein having this oligiosccharide it is very important for its proper folding
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With the protein and the oligiosaccharide linkage, we mentioned that it starts at the ER, but eventually
it forms two very important products
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They both come from the original oligiosccharide that was attached to
the Dolichol in the ER membrane
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You have two Nactylglucosamine that is linked to the protein, 3-mannone, 3-galactose, 3-NaNa
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It is a sequence of enzymes that acts
sepecifically in the ER
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Then folllowes two more
removal of glucose by glucosidase II
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You
will have an intermediate (check slide) and it will go into the cis Golgi area and there the trimming
continues
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There is another enzyme called N-acetylglucosamine
transerase 1 and it will add one N-actylglucosamine next to the mannose
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At this point you have sugar additions that are all added with UDP
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So then you have this complete molecule
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The short name is endo H (endoglycosidase)
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Once passing this point, the bonds becomes
protected so the endo-H can no longer remove the oligiocomplex
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When secreted it will give protection to
the lining of the Gut
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So from
the start where you have 9 Mannose in the oligiosaccharide to the point where you have 4 mannose in
the olgiosccharide, these oligiosccharide intermediates are sensitive to being cleaved by endo-H
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Overall I also mention that there is also an Olinked glycosylation
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EXPLAINATION OF LOCALIZATION OF ACTIVITY IN GOLGI
Final point to review is, make sure that you know the two models of the maturation of the Golgi
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Then you have N-actylglucosamine added, then the middle
region and later when Na-Na is added
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Well the cis
region has one group of enzymes, the trans region has another group of enzymes
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In all this glucose trimming process, one step follows the next step
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So if all your sacks have all the enzymes that
can complete glucose trimming, it really doesn’t matter when we talk about the regionalizatioin of the
Golgi
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The enzymes are still here, but there is no reason to trim the
mannose
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COP1/COP 2

FOCUSING ON CLATHRIN COATED VESSICLES

We talked about Cop1 and cop 2
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Now we will add another type that is most studied that is the
most important, called clathrin coated vesicle
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Where do you find clathrin coated vesicle? Where are they formed? First they are formed at the
trans Golgi
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But on the otherhand you have clatherin coated vesicles that undergo endocytosis at the
plasma membrane
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The secretory vesicle
will decoat and fuse with other organelles
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It has an important protein that has three
large polypeptides and three small polypeptides
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This
protein is found on the cytosolic side of the secretory vesicle
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It can either be hexagons or pentagons, it depends on how they are put together
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When a secretory vesicle is formed, say in the plasma membrane
and you have endocytosis and you have clathrin coat
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