Brain transglutaminase: in vitro crosslinking of human neurofilament proteins into insoluble polymers

Abstract

The accumulation in aged human neurons of insoluble, high molecular weight filamentous polymers apparently linked by nondisulfide covalent bonds led us to examine human brain for the presence of transglutaminase (EC 2.3.2.13) and endogenous protein substrates for this crosslinking enzyme. We demonstrate the presence in brain of a transamidating enzyme that can covalently crosslink brain proteins into insoluble polymers in vitro by forming gamma-glutamyl-epsilon-lysine intermolecular bridges. Brain transglutaminase is Ca2+ dependent, has an electrophoretic mobility similar to that of erythrocyte transglutaminase, and is active in human postmortem brain from aged normal individuals and patients with Alzheimer disease (senile dementia). Brain neurofilament fractions incubated in the presence of transglutaminase, Ca2+, and the fluorescent amine dansylcadaverine form a fluorescent, nondisulfide-bonded insoluble polymer; this process is associated with a decrease in the amount of soluble neurofilament polypeptides in the preparation. Electron microscopy of the polymeric material reveals an extensive network of connecting filaments, which can be immunostained with various neurofilament antisera. Cystamine, an inhibitor of transglutaminase, prevents the neurofilament crosslinking. Glial filaments and myelin basic protein can also serve as substrates of brain transglutaminase in vitro. Although Alzheimer disease-type paired helical filaments are not formed under the specific in vitro conditions employed, the data suggest one possible mechanism for the covalent crosslinking of filaments into insoluble polymers during human neuronal aging.