Lecture 13 - N-glycosylation (golgi)

Question 1

How are high mannose and complex N-glycans formed?

Answer 1

HIgh mannose N-glycans result from lack of trimming of ER N-glycans

Complex oligosaccharides result from complex golgi modification of the N-glycans.

Question 2

What is the use of endoglycosidase H?

Answer 2

endoglycosidase H is found in bacteria determines the type of n-glycan found on newly synthesized glycoproteins. This is used by researchers.

Question 3

When is the N-glycan considered endo H resistant?

Answer 3

Once second glcnac is added to the glycoprotein the endo H is no longer able to cleave the sugar group from the glycoprotein.

Question 4

What happens to N-glycans at each compartment of the golgi?

Answer 4

mannoses are removed at cis golgi.

middle golgi has initiation of complex antennae by addition of first glcnac residues.

trans golgi contains addition of final sugar residues to form the complex antennae.

Question 5

What does Endo H resistance mean?

Answer 5

Endo H resistance indicates that the second glcnac residue has been added and that the glycoprotein is now in the medial golgi

Question 6

What are lactosamine antennae?

Answer 6

GlcNAc-Gal are known as a lactosamine antennae

Question 7

What is the function of lactosamine antennae?

Answer 7

lactosamine antennae may be further modified with a variety of diifferent sugars.

Question 8

What happens to high mannose residues in yeast cells?

Answer 8

In yeast high mannose residues are modified with more mannose.

Question 9

What happens to mice when GlcNAc transferase 1 is knocked out?

Answer 9

Knocking out gene for GlcNAc transferase 1 results in high mannose oligosaccharide and is embryonically lethal.

Question 10

What is the result of removing the second GlcNAc from the N-glycan?

Answer 10

Knocking out GlcNAc transferase 2 results in a hybrid antennae. As a result normal complex N-glycans are not made and the mice die early in life after being born.

Question 11

How is the assembly of O and N -glycans regulated if there is no template that builds it?

Answer 11

The enzyme specificity ensures that a certain shape is always built.

The enzymes that act to modify the N-glycans are localized within separate stacks based on the sequence of carbohydrate adition required.

Protein backbone of glycoproteins influences the carbohydrate processing.

Question 12

What is the difference between O-linked and N-linked glycosylation?

Answer 12

O-linked glycosylation the polypeptide links directly to the GalNAc via a serine or a threonine residue.

O-glycans are not synthesized first then transferred but rather are built 1 by 1.

Question 13

What are some ways of studying glycosylation functions?

Answer 13

Chemical/Enzymatic cleavage of glycans

Prevention of initial glycosylation

Studying glycosylation mutants

Question 14

What are some roles of glycans?

Answer 14

Folding of proteins

Stabilization of mature protein conformation

Facilitate interactions with lectin chaperones

Interaction with transport cargo receptors (glyco code can be read by other proteins)

Question 15

What is ERGIC-53?

Answer 15

ERGIC-53 is the major marker of ER-Golgi-Intermediate compartment and continually recycles through the ER.

It is involved in regulation of exit of some glycoproteins from the ER.

Question 16

What is the evidence that ERGIC-53 is essential for some glycoproteins’ secretion?

Answer 16

Analysis of patients with natural mutations in ERGIC-53 has resulted in defects in coagulation factors 5 and 8.

Expression of non-recycling forms of ERGIC-53 results in limited secretion.

Chemical cross-linking experiments -allowed identification of interacting cargo.

Question 17

What are the steps in ERGIC-53 function?

Answer 17

1) Oligosaccharide binds to ERGIC-53.
2) ERGIC-53 + glycoproteins are incorporated onto COP2 vesicles by interactions with the double phenylalanine motif within the membrane.
3) ERGIC-53 follows COP1 via interactions with the double lysine motif.
* Proteins transported by this method are soluble and often folded.

Question 18

What is the glycan modification required for targetting to the lysosome?

Answer 18

A terminal mannose residue with a phosphorylation of the 6th carbon is required for lysosomal targeting.

This sorting event occurs in the trans-golgi network.

Question 19

How does the mannose-6-phosphate containing glycoprotein exit the golgi?

Answer 19

In the golgi newly synthesized signal s produced. In the trans golgi network a specific receptor can bind the mannose-6-phosphate. The tail of the mannose 6 phosphate receptor interacts with clathrin and an adaptor which acts in the formation of the clathrin buds and is transported to the lysosomes.

Question 20

How is the protein released into the lysosome and how is the phosphate cleaved?

Answer 20

Low pH results in disassembly of the proteins from their carriers. Low pH also results in cleavage of phosphate from the mannose-6-phostphate.

Question 21

What is the variant of clathrin at the TGN responsible for localization of the lysosomal hydrolases?

Answer 21

Variant of clathrin at TGN is the adaptor ap-1. This is different to the cell surface protein.

Question 22

How is the mannose-6-phosphate added to the glycoprotein destined for secretion into the lysosome?

Answer 22

Addition is a 2 step process and requires a high mannose n-glycan and is also dependent on the particular protein itself. The protein expresses a signal patch which fits onto a GlcNAc phosphotransferase. This enzyme also fits a UDP-glcnac which is transferred along with a phosphate group to the end of the oligosaccharide chain.

Question 23

What is the result of defect in M6P synthesis?

Answer 23

Defect in synthesis of M6P results in disease. This is because the protein destined for th lysosome ends up being secreted. This occurs in I-cell disease which is due to a defect in the sorting of lysosomal hydrolases. (lysosomal cells can be made but they get secreted) They lack GlcNAc-1-P transferase activity. This results in neurological disease due to accumulation of waste products.

Question 24

How can oligosaccharides be used to clear aging cells or proteins?

Answer 24

Oligosaccharide can be used as a timer of age and so when the terminal sialic acid is lost the underlying galactose residue can interact with hepatocytes or immune cells to be cleared from the body.

Question 25

How can glycans be used by phagocytes to enter site of inflammation?

Answer 25

Migration of phagocytic white blood cells from blood into the tissues is regulated by interaction of immune cell receptors with exposed e-selectins. Expression of e-selectins is done in response to inflammation signals.

Question 26

What is the function of sialyl-Lewis X oligosaccharide?

Answer 26

Receptor present on the surface of the phagocytes.

Question 27

What is the cause of Leucocyte adhesion deficiency?

Answer 27

The inability to synthesise siacyl-Lewis X oligosaccharide making it difficult for neutrophils to interact with selectin receptors and in turn an inability to respond to inflammation.

Patients with this condition have an increased susceptibility to bacterial infection.

Question 28

Why is there a lack of siacyl-Lewis X oligosaccharide in people with leucocyte adhesion deficiency?

Answer 28

deficiency in addition of fucose to oligosaccharides.