Glycobiology Flashcards
What are the three main components of glycobiology
Lectins Glycans and Glycoenzyems
What are glycans
Carbohydrates and sugars
What are lectins
Glycan binding protiens
What are glycan modifying enzymes
Glycotransferase and glycosidases
What are the central molecules of glycobiology
Glycans that are produced or modified by glycoenzymes and recognized by lectins
What is glycocalyx
Used to describe the carbohydrate rich zone on the cell surface
How can you recognize the glycocalyx zone
Using a variety of stains such as ruthenium red
Also its affinity for carbohydrate-binding protiens called lectins which can be labeled with a flourescent dye or another visible marker
What is glycocalyx made up of
Glycan chains of membrane glycoproteins and glycolipids and absorbed glycomolecules
What is the function of the glycocalyx
Protect cells against mechanical and chemic to keep foreign objects and other cells at a distance preventing undesirable protien-protein interacations
What else can membrane glycoproteins do
Involved in transmembrane signalling and intercellular communications
What does the glycocalyx represent
Glycans represent complex branched structures of different sizes that looks like trees
What does the different dimensions of the glycocalyx mean
That the role of the glycocalyx may be cell-speceific and be involved in regulation of different cellular responses
How many common monosaccharides are there
9
D-glucose can exist in how many forms and what are they
three different forms a linear and two different rings a pyranose and furanose
Which ring of D-glucose dominantes in biological systems
Pyranose
What are the two different forms of anomers
Anomers and steriosmers which include alpha and beta anomers depending on the positon of the hydroxol group at position one
What is it called when sugars differ only by the configuration around one carbon atom
Epimers D-manose and Dglacotse are epimers of D-glucose
What are protiens
Linear polymers containg up to several thousand amino acids linked by peptide bonds
What are nucleic acids
Linear polymers containt hundreds to millions of nucleotides by phosphodiester bonds
What are glycans
Linear or branched polymers or monsaccharides linked by glycosidic bonds
What is the glycosidic bond formed by
The anomeric carbon of one monosaccharide and a hydroxyl group of another
What is the difference between glycans and proteins and nucleic acids
The glycosidic bonds can be formed between individual monsaccharides
What is an example of different bonds between individual monosaccharides
Lactose has a different bond between glactose and glucose then glucose has with fructose as result complex branched structures are common
What is the linkage in N-linked glycans
GlcNAc B linked to the amide nitrogen of an asparagine residue in the seqeuce Asn-X-Ser or Asn-X-Thr where X is any amino acid expect proline or aspartic acid
What is the first sugar of N-glycans attached
GlcNac
What is the linkage of glycans in O-linked
Attached to the OH group of either serine or theroine resuidues
What is the most common O-glycosidic attachment
Involves the disaccharide core B-glactosyl (1-3) a- N-acetylglactoasimne
What are less common O-linked glycans
Glactose, mannose and xylose
Can O-linked glycans vary in size
YES from a single sugar residue to the long chains of up to 1000 monosaccharides units in proteoglycans
What do all N-linked glycans contain
A common core pentassacharide structre attached to asparagine but differ in temrinal elborations that extend from this core
What is glycoproteins
Glycosylate protiens with covalently linked N-glycans and O-glycans glycoprotiens are assembled through the ER-golgi pathway
What is GPI
Anchored glycoprotiens contain a specific glycan bridge between lipid anchor and the C-terminus of a protien
What is EGF/TSR
Epidmerla growth factor and thrombospondin represent membrane protiens which are glycosylated with only short unbranched carbohydrate structures
What are proteoglycans
Are glycoconjugates that have one or more glycoasminoglycans covalently attached to core protien they are linear
What are glycosphingolipids
Consists of a glycan usually attached via glucose or glactose to the terminal OH group of the lipid molecule
What are lectins
Protien that specifically recognizes and binds to glycans without catalyzing a modification of the glycan carbohydrate binding proteins other than immunoglobins that no enzymatic
What are lectins
Highly specic to individual monosaccharides which can link to other sugars in a complex glycans
What domain does lectin have
Carbohydrate-recognition domain which is respobsible for binding glycans
What do lectins function as
Multimers which allows them to bind seceral glycan molecules at a time
What happens if you add them to cell suspension
Bind cell surface glycans and make bridges between cells inducing cell aggregation
How can cell aggregation be detected
Spectrophotometrically by mesauring the light transmission of cellular suspension as the suspension of aggregated cells transmits more light than the supesnion of indivudal cells
What happens if inhibitory sugard were added
No cell aggregation was observed blocks CRD of VAA and GlcNAc blocks the CRD of WGA
What are the four types of animal lectins
- galectins - soluble glactose binding lectins
- C-type lectins transmembrane proteins with Ca depedent glycan binding specificiy
- P type lectins transmembrane proteisn with aset of immunoglobuin domains
- I typed lectins - transmembraen proteins with a set of immunoglbuil domains
What are glactins
Animal lectins that preferntially recognize B-glactoside containing glycans
Share significant sequence similarties in the CRD
Soluble multifunctional protiens
What are the three subfamiles of galectins
Proto-type with one CRD
Tandem-repeat type with two different CRDs
Chimeric with one CRD linked to a non lectin N terminal domain
Why are galectins important
Both inside and outside the cell that perform different functions
What is the CRD structure
Beta sheets
Beta sandwich with two sheets formed by six beta strands on concave side and five on the convex side
What does the HL-60 differeninate into
Eosinophil like cells
Neutrophil- like cells
Macrophage like cells depending on the type of stimuli
How HL-60 cell line maintained
It is a suspension cell line
Orignated froma a female patient with acute promyelocytic lukemia
Expresses 6 gallectins
What different stressors were used in extracellular stress stimuli
Conventional cellular stress exopixa CoCl1
Tunicamycin for ER stress
What did the study include after glactein expression and adhesion charactersitics of HS-60
manose glactose and sialiac acid and GlvNAc residues are available on the cell surface for cell contancts. The glacteins didnt change the prefrence to the binding of lectins in stress but changed the expression of it
What are the four categories of galectin genes are recognize in the context of cellular stress responses
Stress inducible
Stress repressed
Stress reistant
Undetectable
How can oxidative stress be induced
Menadione which creates superoxide radiacal
What do HL-60 overexpress in oxidative stress and what does it repre
NCF1 gene and HMOX1 and 1, 3 and 10 and 9 was downrefgulates biological signficance network remodeling in conjucation with cellular stress responses
What is glycosyltransferase
An enzyme that catalyze transfer of a sugar from a sugar nucleotide dononr to substrate these enzymes are responsible for the synthesis of most glycoproteins and other glycoconjugates
What are glycosidases
Enzymes that catalyze hyrdolysis of glycosidic bonds in glycans: endoglycosidases catalyze the cleavage of internal glycosidic linkage while exoglycosidases cleave a monosaccharide from the terminal end of a glycan
What is glycan-degrading enzymes
Type of glycosidases that destroy large pollysaccharides
What does OGT do
Catalyze the transfer of a GlcNAc sugar from the donor substrate UDP-GLcNAx
WHere does GlcNAc bind
Hydrozyl groups of serine and theronine residues
What is OGA
Cataylzes the hydrolysis of this sugar modification and removes O-GlcNAc
Where does O-GlcNAcylation happens
Inside of cells in cytoplasm nucleus and mitochondria
How is UDP-GlcNAC produced
Is an OGT substrate through the hexosamine biosynthetic pathways
What is the rate liminting enzyme in Hexosamine biosynthetic pathway
GFAT
What is the pathway of hexosamine
Fru-6P to GlcN-6P through GFAT
GlcN-6P then produces UDP-GcNaC
This can also be recycled through the salvage pathway which converts GlcNAc back into GlcNAc-6P that can be utilized by the pathway
What competes with GlcNAcylation
Competes with phosphorylation because both are types of posttranslational modification that target Ser/Ther
What complex induces phosphorylation
OGA and Kinase
What complex induces glycoprotien
OGT and phosphotase
Where is O-GlcNAcylated most common
Cytsol and Nucleus less in endosome and peroxisomes
Where are the most O-GlcNacylated protiens and lowest
Body parts
Most is in the brain and liver
Least is bones saliva gall bladder and urine
What happens when there is elevated O-GlcNA
Disease
Contirbutes directly to glucose toxicity in diabetes and cancer
What does decreased O-GlcNacylation
Alzheimers disease and parkinsons disease tau protien carries O-GlcNAc residueds that compete with key phosphorylation residued and prevents it aggreations
What happens to global O-GlcNAc levels
They are elveated in response to cellular stress in all cases of they observed signficant increase in intensitniy of bands
How do you run the immunodot blot assay
Immobilize cellular protiens then incubate the membrane with primary and secondary antibodies
Does not require protein elctrophoresis
Intensinity of dot is proportional to the levels in cells
How is the efficnancy confirmed
Increasing or decreasing levels of O-GlcNaC in cells treated with OGT inhibitor and OGA inhibitos
What was the finding in cellular differentiation
Levels of O-GlcNAc significantly decrease in differentiated cells versus progenitor cells
What else can O-GlcNAc regulate
Protien traffcking and turnover they are less transportable, more stable and usually not secreted from cells
What do regulatory fuctions of glacteins depend on
Localization of glactins in cells
O-GlcNAc promote intracellular accumulation and where low levels of O-GlcNAc promote galectin secretion can be applied to glactein 3 and 9
What is LGALS12 for
Highest RNA expression in adipose tissue, breast and bone marrow
What is LGALS16
Highest RNA expression in normal placenta retina and brain tissues
What is the hypothesis of cell culture models to study tissue specefic galectins
Increased when cells become more specalized and decreased in cancer might act as tumor supressers and used different cell lines
What is the N1 phenotype
Showed increased activity of LGALS12 gene and behaved in a way that fights tumors and promtoes inflammation
What is the N2 phenotype
Showed decreased activity of LGALS12 and behavied in a way that supports tumor growth and reduces inflammation
What happend with LGALS16
When cells were treated with 8-BR-cAMP that promtoes the cells to mature into trophoblast like cells LGALS216 activity increased significantly CGB confrimed this
What is the role of LGALS16 in trophoblastic differentiation of JEG-3 cells?
Using CRISPR/Cas9 to knock out (KO) the LGALS16 gene in JEG-3 cells, researchers showed that these KO cells had:
Lower response to 8-Br-cAMP, as seen in reduced CGB expression (both gene and protein levels).
No changes in growth rate when treated with 8-Br-cAMP, unlike control cells.
Conclusion: LGALS16 is essential for trophoblastic differentiation of JEG-3 cells.
