title: A spectroscopic study of glycated bovine alpha-crystallin: investigation of flexibility of the C-terminal extension, chaperone activity and evidence for diglycation.
abstract: The effect of glycating the C-terminal extensions of alpha-crystallin on their flexibility was investigated. In the course of the study the reaction sites were identified and double glycation of single lysine residues was found. Alpha-crystallin was incubated until approximately one mole of the sugar had reacted per subunit of the crystallin. The reaction sites were investigated by mass spectrometry and H NMR spectroscopy, and were found to be principally in the short and flexible C-terminal extensions. The chaperone ability of alpha-crystallin was unaffected by this limited glycation. There was little effect on the flexibility of the C-terminal extensions. This result supports the view that the flexibility of the C-terminal extensions of alpha-crystallin is important for chaperone activity. As alpha-crystallin consists of a mixture of unmodified and phosphorylated subunits, a detailed investigation was undertaken of the reaction of galactose with peptides comprising the C-terminal extensions of alphaA- and alphaB-crystallin. The alphaA peptide was incubated with galactose until 0.79 mole of sugar was bound per mole of peptide and the alphaB peptide reacted until 2.2 moles of galactose had been incorporated. The purified glycated peptides were examined by NMR and mass spectrometry to identify glycation site(s), and the effect of glycation on the conformation of the peptides. For both peptides, it was found that extensive glycation of the constituent lysine residues occurred. The addition of two galactose molecules to some lysine residues of the peptides was also noted. This diglycation was confirmed in control experiments with N-acetyl-lysine.
authors.affiliation: Nuffield Laboratory of Ophthalmology, University of Oxford, UK.
authors.forename: R
authors.initials: R
authors.lastname: Blakytny
authors.forename: J A
authors.initials: JA
authors.lastname: Carver
authors.forename: J J
authors.initials: JJ
authors.lastname: Harding
authors.forename: G W
authors.initials: GW
authors.lastname: Kilby
authors.forename: M M
authors.initials: MM
authors.lastname: Sheil
mesh_terms: D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D006031:Glycosylation; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry
chemical_list: D003459:Crystallins; D018832:Molecular Chaperones; D010446:Peptide Fragments; C002315:N(alpha)-acetyllysine; D008239:Lysine; D005690:Galactose