The aims of this proposal grow out of recent results from our research on protein modifying reactions occurring in the lens upon elevation of Ca2+-ion concentration. Specifically, this application reflects the continued commitment of our laboratory to devote its specialized protein chemical resources and knowledge in regard to the functioning of the endogenous cross-linking enzyme: transglutaminase for the aging of human lens and the problem of cataract formation. Subjects for investigation are grouped under the following topics: 1. Analysis of acceptor/donor relationships for the transglutaminase-catalyzed cross-linking of proteins in human and bovine lens. a. Identification of acceptor and donor protein partners in whole lens homogenates. b. Separation of the labeled acceptor and donor substrates of transglutaminase from other lens proteins. Raising of MAb's to the isolated acceptors and donors. c. Sequencing of acceptor and donor domains in the transglutaminase-reactive lens proteins. 2. Examination of oligomeric and polymeric structures formed by transglutaminase in human and bovine lens. a. New inhibitors of transglutaminase. b. Comparison of the 50K protein found in lenses of old individuals with X beta(2) generated in vitro through transglutaminase. c. Role of spectrin as a transglutaminase substrate in forming polymers. 3. Does 'oxidative stress' influence reactivities of human lens proteins with transglutaminase, and vice versa would transglutaminase action render these more susceptible to oxidative changes? 4. The human lens transglutaminase. cDNA sequence. Novel approaches for isolating the enzyme and its regulator(s). 5. Is there a turnover of N[epsilon](gamma-glutamyl)lysine in human lens?

National Institute of Health (NIH)
National Eye Institute (NEI)
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Visual Sciences A Study Section (VISA)
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Northwestern University at Chicago
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Murthy, S N; Lomasney, J W; Mak, E C et al. (1999) Interactions of G(h)/transglutaminase with phospholipase Cdelta1 and with GTP. Proc Natl Acad Sci U S A 96:11815-9
Murthy, S N; Velasco, P T; Lorand, L (1998) Properties of purified lens transglutaminase and regulation of its transamidase/crosslinking activity by GTP. Exp Eye Res 67:273-81
Lorand, L; Stern, A M; Velasco, P T (1998) Novel inhibitors against the transglutaminase-catalysed crosslinking of lens proteins. Exp Eye Res 66:531-6
Clement, S; Velasco, P T; Murthy, S N et al. (1998) The intermediate filament protein, vimentin, in the lens is a target for cross-linking by transglutaminase. J Biol Chem 273:7604-9
Parameswaran, K N; Cheng, X F; Chen, E C et al. (1997) Hydrolysis of gamma:epsilon isopeptides by cytosolic transglutaminases and by coagulation factor XIIIa. J Biol Chem 272:10311-7
Velasco, P T; Lukas, T J; Murthy, S N et al. (1997) Hierarchy of lens proteins requiring protection against heat-induced precipitation by the alpha crystallin chaperone. Exp Eye Res 65:497-505
Clement, S; Trejo-Skalli, A V; Gu, L et al. (1997) A transglutaminase-related antigen associates with keratin filaments in some mouse epidermal cells. J Invest Dermatol 109:778-82
Lorand, L (1996) Neurodegenerative diseases and transglutaminase. Proc Natl Acad Sci U S A 93:14310-3
Trejo-Skalli, A V; Velasco, P T; Murthy, S N et al. (1995) Association of a transglutaminase-related antigen with intermediate filaments. Proc Natl Acad Sci U S A 92:8940-4
Lorand, L; Velasco, P T; Murthy, S N et al. (1992) Isolation of transglutaminase-reactive sequences from complex biological systems: a prominent lysine donor sequence in bovine lens. Proc Natl Acad Sci U S A 89:11161-3

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