Acceptor-donor relationships in the transglutaminase-mediated cross-linking of lens .beta.-crystallin subunits

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Abstract
Following the isolation of the N.epsilon.(.gamma.-glutamyl)lysine-containing polymers from human cataracts, our efforts were directed to induce such cross-links experimentally in rabbit lens, and evidence was obtained for the selective reactivities of certain .beta.-crystallin subunits in this transglutaminase-catalyzed event. In the present work, we examined the enzymatic cross-linking of purified crystallins individually (.alpha., .beta.H, .beta.L, and .gamma.) and in combinations, with particular emphasis on forming the approximately 55K dimer. This species was the primary product in the cross-linking of .beta.H-crystallins; .beta.L also reacted with transglutaminase. Neither .alpha.- nor .gamma.-crystallins formed appreciate amounts of cross-linked structures with transglutaminase. Dansylcadaverine, nown to compete against the reactive lysines of proteins in forming N.epsilon.-(.gamma.-glutamyl)lysine cross-bridges, was shown to inhibit the generation of dimeric and higher ordered oligomers from .beta.H and .beta.L. The fluorescent amine specificially laeled only two subunits in .beta.H (.apprx.29-30K and .apprx.26K) and one in .beta.L (.apprx.26K), identifying these substrates as possessing transglutaminase-reactive endo-.gamma.-glutaminyl residues. An antiserum to bovine .beta.Bp recognized th .apprx.23K subunit of rabbit .beta.-crystallins and also the .apprx.55K dimer, suggesting that the .apprx.23K protein participates as a lysine donor in generating the cross-linked dimer with transglutaminase. Inasmuch as the same antiserum reacts with a .apprx.50K material reported to appear in increasing amoutns with age in human lens, the results lend added support to the physiological significance of transglutaminase in the aging of lens.