Chemical modulation of the chaperone function of human alphaA-crystallin.
alphaA-crystallin is abundant in the lens of the eye and acts as a molecular chaperone by preventing aggregation of denaturing proteins. We previously found that chemical modification of the guanidino group of selected arginine residues by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), makes human alphaA-crystallin a better chaperone. Here, we examined how the introduction of additional guanidino groups and modification by MGO influence the structure and chaperone function of alphaA-crystallin. alphaA-crystallin lysine residues were converted to homoarginine by guanidination with o-methylisourea (OMIU) and then modified with MGO. LC-ESI-mass spectrometry identified homoargpyrimidine and homohydroimidazolone adducts after OMIU and MGO treatment. Treatment with 0.25 M OMIU abolished most of the chaperone function. However, subsequent treatment with 1.0 mM MGO not only restored the chaperone function but increased it by approximately 40% and approximately 60% beyond that of unmodified alphaA-crystallin, as measured with citrate synthase and insulin aggregation assays, respectively. OMIU treatment reduced the surface hydrophobicity but after MGO treatment, it was approximately 39% higher than control. FRET analysis revealed that alphaA-crystallin subunit exchange rate was markedly retarded by OMIU modification, but was enhanced after MGO modification. These results indicate a pattern of loss and gain of chaperone function within the same protein that is associated with introduction of guanidino groups and their neutralization. These findings support our hypothesis that positively charged guanidino group on arginine residues keeps the chaperone function of alphaA-crystallin in check and that a metabolic alpha-dicarbonyl compound neutralizes this charge to restore and enhance chaperone function.
Biswas
Ashis
A
Department of Ophthalmology & Department of Visual Sciences, Case Western Reserve University, Cleveland, OH 44106, USA.
Lewis
Shawn
S
Wang
Benlian
B
Miyagi
Masaru
M
Santoshkumar
Puttur
P
Gangadhariah
Mahesha H
MH
Nagaraj
Ram H
RH
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism; D006709:Homoarginine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007093:Imidazoles / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry; D008782:Methylurea Compounds / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D009952:Ornithine / Q000031:analogs & derivatives / Q000032:analysis; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D011743:Pyrimidines / Q000032:analysis / Q000737:chemistry; D011765:Pyruvaldehyde / Q000737:chemistry
D000596:Amino Acids; C511246:CRYAA protein, human; D003459:Crystallins; D007093:Imidazoles; D008782:Methylurea Compounds; D018832:Molecular Chaperones; D021122:Protein Subunits; D011743:Pyrimidines; C107313:argpyrimidine; C531307:homoargpyrimidine; C531308:homohydroimidazolone; D006709:Homoarginine; C002249:O-methylisourea; D011765:Pyruvaldehyde; D009952:Ornithine