Probing the transglutaminase-mediated, posttranslational modification of proteins during development

Abstract

Sphaerechinus granularis eggs were fertilized in seawater in the presence of 0.2 mM dansylcadaverine, and development was allowed to take place with this compound in the medium. gamma-Glutamyldansylcadaverine, indicative of the utilization of the amine tracer by intrinsic transglutaminase, was isolated from the embryonic proteins, and identity of the product with the chemically synthesized gamma-glutamyl derivative of dansylcadaverine was confirmed. Covalent labeling of proteins occurring during development was examined by means of electrophoresis in NaDodSO4, followed by immunoblotting with an antibody that specifically recognized the dansyl hapten. There was an increase in the total uptake of the tracer at an essentially constant rate with each cell division, from 2- to 8- and 64-cell stages. Moreover, multiple protein labeling was evident in all specimens. The described concept of studying posttranslational modifications in vivo by transglutaminase through detection of the haptenic or specific ligand recognizable group of an incorporated small amine substrate will undoubtedly be of general utility for probing the functions of this family of enzymes in other cell types as well.

Cloning and expression of chicken erythrocyte transglutaminase.

“…One of the subunits (designated A) of the factor XIII zymogen (fibrin-stabilizing factor) circulating in human plasma also belongs to this class of gene products (19,20). Transglutaminase was shown to become activated in sea urchin eggs soon after fertilization (21,22) and in A431 epidermal carcinoma cells exposed to epidermal growth factor (23), and it is expressed in induced murine erythroleukemia cells well before the appearance of hemoglobin (24). This paper deals with the identification, isolation, and sequence analysis of cDNAs encoding chicken red blood cell transglutaminase* and shows that the mRNA for this protein is present only in very low amounts in embryonic erythroid cells or in retrovirally transformed erythroid progenitor cells but increases significantly during erythroid cell maturation.…”

Sorting-out of acceptor-donor relationships in the transglutaminase-catalyzed cross-linking of crystallins by the enzyme-directed labeling of potential sites.

“…First we focused on dansylated derivatives because, in addition to fluorescence detection, methodologies are already in place for immunoblotting and also for the eventual isolation of the tracercarrying sequences with anti-dansyl antibodies (2,3). The present report describes our findings with two dansylconjugated (Dns) peptides, Dns-Pro-Gly-Gly-Gln-Gln-IleVal and Dns-Ala-Gln-Gln-Ile-Val, and demonstrates their usefulness in sorting out the potential acceptor and donor subunits for the cross-linking of crystallins catalyzed by the intrinsic transglutaminase in lens homogenates.…”

Identification of glycinin in vivo as a polyamine-conjugated protein via a γ-glutamyl linkage

“…The homogenate was centrifuged at 15 000 rev.\min at 4 mC for 15 min and the supernatant was assayed for enzyme activity. The γ-glutamylamine cyclotransferase activity was measured by a modified method of Cariello et al [30]. The assay mixture, containing 50 µl of plant extract and 5 µl of 1 mM γglutamyldansylcadaverine (5 nmol), was brought to a volume of 100 µl with 10 mM potassium phosphate buffer, pH 7.2, containing 10 mM dithiothreitol and incubated for 30 min at 37 mC.…”