Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.
Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.
Roquemore
E P
EP
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Dell
A
A
Morris
H R
HR
Panico
M
M
Reason
A J
AJ
Savoy
L A
LA
Wistow
G J
GJ
Zigler
J S
JS
Earles
B J
BJ
Hart
G W
GW
D000117:Acetylglucosamine / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001717:Birds; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000302:isolation & purification / Q000378:metabolism*; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D051381:Rats; D012694:Serine / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014357:Trypsin / Q000737:chemistry
D003459:Crystallins; D012694:Serine; D000469:Alkaline Phosphatase; D014357:Trypsin; D003488:Cyanogen Bromide; D000117:Acetylglucosamine