Lecture 13 Effects of glycosylationon protein structure and function
Lecture'13'Effects'of'glycosyla3on' on'protein'structure'and'func3on
Learning objects· Experimental approaches to studying theeffects of glycans on protein structure andfunction: How glycans affect protein folding andstability: The effects of glycans on interactions ofproteins
Learning'objects' • Experimental'approaches'to'studying'the' effects'of'glycans'on'protein'structure'and' func3on' • How'glycans'affect'protein'folding'and' stability' • The'effects'of'glycans'on'interac3ons'of' proteins
. In manyinstances,glycosylation directlyaffects the properties of proteins andmembranes, sometimes with importantbioogical consequences: The examples illustrate some of the ways inwhich glycoprotein functions are modulatedby glycosylation, usually by affecting theirstability or surfaceproperties
• In'many'instances,'glycosyla3on'directly' affects'the'proper3es'of'proteins'and' membranes,'some3mes'with'important' bioogical'consequences' • The'examples'illustrate'some'of'the'ways'in' which'glycoprotein'func3ons'are'modulated' by'glycosyla3on,'usually'by'affec3ng'their' stability'or'surface'proper3es
1 Various approaches can be used tostudy the effects of glycosylation
1 Various approaches can be used to study the effects of glycosylation
Altering glycosylation to determinewhat effect the alternation haseliminationof all sugarsfromall theattachmentsites on the protein. Elimination of sugars at specific attachment sites.Removalof portions of the sugar structures· N-linked sugars can be removed completely fromglycoproteins by TfOH or anhydrous HF
Altering'glycosyla3on'to'determine' what'effect'the'alterna3on'has • elimina3on'of'all'sugars'from'all'the'aEachment' sites'on'the'protein' • Elimina3on'of'sugars'at'specific'aEachment'sites' • Removal'of'por3ons'of'the'sugar'structures' • NJlinked'sugars'can'be'removed'completely'from' glycoproteins'by'TfOH'or'anhydrous'HF
drawbacks: Difficulty of ensuring that sugar release isquantitative? Damage to the protein in the form ofoccasional cleavage of thepolypeptide chain. Overall denaturation of the folded structureunderharsh conditions.Loss of biological function can't confidently beascribed to the removal of sugar
drawbacks' • Difficulty'of'ensuring'that'sugar'release'is' quan3ta3ve' • Damage'to'the'protein'in'the'form'of' occasional'cleavage'of'the'polypep3de'chain'' • Overall'denatura3on'of'the'folded'structure' under'harsh'condi3ons' • Loss'of'biological'func3on'can’t'confidently'be' ascribed'to'the'removal'of'sugar
Other methodsEg1: Peptide:N-glycanase (PNGase)Or other endoglycosidasesRemove sugars under non-denaturing conditions,Although many glycoproteins contain glycans that areinaccessible to these enzymes unless the foldedstructureisdisrupted
Eg1: Peptide:N-glycanase (PNGase) Or other endoglycosidases Remove sugars under non-denaturing conditions, Although many glycoproteins contain glycans that are inaccessible to these enzymes unless the folded structure is disrupted Other'methods
Eg 2: Inhibitors of glycosylation such as tunicamycin ispossibletobeemployedInhibit the initial step in the formation of the dolichol-linked precursor oligosaccharide to preventglycosylation in the frst placeEg 3:proteins isolated from tissues consist of mixturesof glycosylated and unglycosylated forms that can beseparated and studied
Eg 2: Inhibitors of glycosylation such as tunicamycin is possible to be employed Inhibit the initial step in the formation of the dolichollinked precursor oligosaccharide to prevent glycosylation in the first place Eg 3: proteins isolated from tissues consist of mixtures of glycosylated and unglycosylated forms that can be separated and studied
TunicamycinChangesto CoreChangestoterminal structuresorpeptide:NeuraminidaseN-glycanase1MutagenesisNeuAca2-3Galβ1-4GlcNAco1-2Manαl9Manβ1-4GIcNAcβ1-4GIcNAcβ1-AsnGInNeuAca2-3Galβ1-4GIcNAca1-2Manα1-3n1个XaaEndoglycosidasesProductionin→AlaSer/Thrmutantcells1Manα1-2ManalManalManx1-2Manal-3Manβ1-4GicNAcβ1-4GIcNAcβ1-Asn2ManlManx1-2Manal
. Simultaneously probe the role of all of theglycans attached to a protein at the same time.Other methods for specific sites:: Site-directed mutagenesis to alter glycosylationtarget sequences in proteins. Possible to study the effects of individual glycans· N-linked glycosylation at a particular site can beeliminated by changing either the asparagineattachment residue or the Serine or threonineside chain
• Simultaneously'probe'the'role'of'all'of'the' glycans'aEached'to'a'protein'at'the'same'3me' • Other'methods'for'specific'sites:' • SiteJdirected'mutagenesis'to'alter'glycosyla3on' target'sequences'in'proteins' • Possible'to'study'the'effects'of'individual'glycans • NJlinked'glycosyla3on'at'a'par3cular'site'can'be' eliminated'by'changing'either'the'asparagine' aEachment'residue'or'the'Serine'or'threonine' side'chain