Mass spectrum_L5 Flashcards
Factors affecting glycosylation
(1) Protein sequence: whether the protein contains specific motif and tandom repeats of threonine/serine
(2) Sugar metabolism: generate the basic metabolites for the biosynthetic precursors in the glycosylation
(3) Expression of glycosyltransferases: the ratio of glycosyltransferase and glycosylhydrolase impacts the glycosylation rate
(4) Competition between glycosyltransferases
(5) Physiological status: diet/stress/nutrition/pH/oxygen level/disease state
(6) Cell and tissue specific glycosylation leads to the individual speficity
Why Glycosylate?
physical improvement:
(1) Solubility: increased as a result of hydrophilic glycan chains, useful in serum and blood
(2) Stability: inhibit the activity of protease
chemical improvement:
(3) Conformation: adding the glycan orientates the receptor away from the plasma membrane to avoid the influence of ligand interaction
(4) Organizational and barrier function on the membrane e.g. muscines secreted to prevent pathogen invasion
(5) Cell-Cell and Cell-Matrix recognition: lead to functionally important event —- recognition
Definition of lectins
Lectins are proteins of non-immunoglobulin nature capable of specific recognition and reversible binding to carbohydrate moieties of complex carbohydrates without altering the covalent structure of any of the recognized glycosyl ligands.
- exclude the enzymes, e.g. glucotransferase
- cannot be antibodies, e.g. antibody against the carbohydrates on the pathogen surface
Carbohydrate Recognition Domains (CRDs) of Lectins?
(1) Usually found in shallow indentations on surfaces of lectins, the specific site for the lectin recognition, CRDs bind to the glucose-/galactose-type arrangment
(2) glycan binding modes include chelation with divalent cations especially Ca2+, hydrogen-bonding with sugar OH and amide groups(exaplain why the hydroxyl amino acid is heavy glycosylation site), van der Waals’ interactions with sugar hydrophobic faces (e.g. with galactose) and ionic interactions (e.g. with carboxylate of sialic acids), hydoxyl or acetyl group on the monosaccharides are the main players involved in the interaction with amino acid side chain.
(3) The glycan ligand is usually one to four residues in size
(4) Binding is low affinity but high specificity but multivalency can result in tight binding despite low affinity
- low affinity: the interaction between the lectin and carbohydrate is weak
- high specificity: certain orientation for the monosaccharide
- multivalency: the lectin contains multiple carbohydrate recognition domains so that the overall affinity can be improved, grouping the lectin on the cell surface can achieve the same effect.
Classification of important animal lectins and the examples falling into each category
family ligand example
C-type various selectin, hapatic lectin
I-type(siglec) sialic acid
and O-glycan CD22
galectins b-galactosides GL
P-type Man-6-P CI-MPR
C-type receptors are associated with calcium ions in coordination with different glycan sequences, C-type receptors are found particularly abundant in the immune cells for immune response and regulation
haptic lectins are resulted from multimerisation of CBDs from different lectins
selectin has multiple CBDs binding specifically to the lysozymal proteins for transporting from Golgi body to lysomes
galectins are good at cross-linking the glycoproteins and dictating the function, as well as intracellular functions
b-galactosides contains glycans, such as N-glycans and O-glycans.
introduce influenza viruses
(1) RNA virus
(2) 4 types A, B, C, D: A and B are the most common ones with A most frequently observed in the human viral studies
(3) A most virulent
(4) 18 serotypes of HA, 11 serotypes of NA
(5) the two surface proteins are fundamental to the infection processes in the influenza
(6) many virus hijack the sugar-lectin interaction events to promote the infection processes
* HA: hemagglutinin
* NA: neuraminidase
Influenza infection
(i) Attachment: HA is a lectin, a carbohydrate-binding protein on the viral cell surface, binds to the receptor of specific sequence for the virus to internalise and initiate the infection
(ii) Endocytosis
(iii) Replication
(iv) Budding: new viron particles bud out from the infected cell, however, this can still be problematic, since the newly snthesized HA can be sticked with the cell surface carbohydrates, therefore NA comes along and cleaves the sialic acid as a protease, NA may even help to break the mucus(glycoprotein barrier) on the respiratory tract or on the cell surface
(v) Release
Haemagglutinin receptors difference between human and avian?
The natutal reserviour of virus is not human, but birds, as the virus targets the 2,3-linked sialic acid which is not present in the human HA receptor. Some mutated virus are able to attach onto the 1,6-linked sialic acid, therefore, human can be potentially infected. Pig can act as an intermediate transmitter to carry virus from avians to humans since pigs’ gene codes not only for the 2,3-linked but also the 1,6-linked sialic acid. Gene assortment can happen inside the pig body and the viral function is altered slightly.
Tamiflu and Relenza (neuraminidase inhibitors) targets?
Haemagglutinin trimer is not a good drug target due to the reason that the lectin binding is relatively light and shallow, the binding strength is relatively weak.
In compared with the haemagglutinin trimer, neuraminidase monomer interaction is much stronger as a result of deep binding pocket.
Many interaction forces are involved between the sialic acid and neuraminidase, for instance, the sialic acid backbone is rich of hydroxyl groups which benefits the hydrogen bond formation with the charged side chains in neuraminidase, the active site interaction involves a range of interactions, as well as other important packing events.
critisize on Tamiflu?
Tamiflu resembles the structure of sialic acid which bonded with the protein at Glu276, the Tamiflu changes the glycerol to hydrophobic chains for hydrophobic interaction, thus, Glu276 polar bond is not maintained and the nearby Arg224 forms non-polar interaction with the altered chemical group, the proteolytic cleavage by NA is prevented. Glycan-lectin recognition is key to cell-cell communication.
What is glycomics and how does it work?
(1) define the sequence of glycan in particular system e.g. isolated carbohydrate, single red blood cell, serum and blood sample
(2) Determining the glycan repertoire in cell, tissues organs etc as a first step to defining functions. Prior knowledge of glycan biosynthetic pathways is essential.
define Glycomics Screening Strategy
(1) biological sample from tissue or organ or individual
(2) glycoproteins, separated from glycolipids, undergo carboxymethylation protelysis and C18 chromotgraphy, consequently, enzymatic approach PNGase F isolates the N-glycan from the population and O-glycans by reductive elimination
glycolipids undergo chloroform/methanol/water extraction, followed by ceramidase digestion and butanol extraction
(3) end-product from the two lines will be processed with permethylation and C18 chromatography, in the end, mass spectrometry, such as MALDI, MS/MS and linkage analysis
How to identify the permethylated monomers?
combining the information of the mass spectrum and the monomer, in addition to taking account the reducing and non-reducing ends of the monosaccharides and charge carried.
The organ glycosylation mapping can be plotted, but how to solve the problem arised from the same mass hexasose?
The biological knowledge, such as the metabolic pathway needs to be in combination with the mass spectrometer interpretation.
The organ glycosylation mapping can be plotted, but how to solve the problem arised from the same mass hexasose?
The biological knowledge, such as the metabolic pathway needs to be in combination with the mass spectrometer interpretation.
