LYS-17 IS THE AMINE-DONOR SUBSTRATE SITE FOR TRANSGLUTAMINASE IN BETA-A3-CRYSTALLIN

The bovine lens protein betaA3-crystallin has recently been shown to b e an amine-donor (Lys) substrate for tissue-type transglutaminase, usi ng a newly developed amine-acceptor hexapeptide as a probe (Groenen, P . J. T. A., Seccia, M., Smulders, R. H. P. H., Gravela, E., Cheeseman, K. H., Bloemendal, H., and de Jong, W. W. (1993) Biochem. J. 295, 399 -404). In the present study, the reactive amine-donor site has been id entified by site-directed mutagenesis of the putative substrate lysine . The mutation Lys-17 --> Arg abolishes the substrate capacity. This r esidue, located in the N-terminal extension of the polypeptide, thus a cts as the sole amine-donor substrate in betaA3-crystallin. Our findin g reinforces the notion that, in the crystallins, all amine-donor as w ell as amine-acceptor substrate sites reside in the N- or C-terminal a rms. Transglutaminase-mediated cross-linking of betaA3-crystallin also gives rise to a betaA3 dimer, presumably due to the fact that Lys-17 can be cross-linked to the previously established Gln-7 or Gln-8 amine -acceptor site.