title: Interactions of recombinant mouse erythrocyte transglutaminase with membrane skeletal proteins.
abstract: Transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent gamma-glutamyl-epsilon-lysine crosslinks between glutamine (Q) acyl-donors and lysine (K) acyl-acceptors. Here, we report the cDNA cloning of a TG from mouse reticulocytes, its 4.6-kb message size and high-yield synthesis of recombinant TG in yeast cultures. Its activity was assayed by crosslinking the amine of monodansylcadaverine (DC) onto casein and inside-out vesicles of erythrocytes. The latter contain TG substrates including the anion ion exchanger (AE1) or band 3, and the crosslinking activity was the highest at physiological [GTP] and [ATP] of erythrocytes. To study individually how TG interacts with band 3 and what role P4.2, a pseudo-TG that is normally associated with band 3, may play in their interaction, recombinant cytoplasmic domain of band 3 (cdb3) and P4.2 were also cloned by polymerase chain reaction from mouse reticulocytes, expressed and affinity-purified from Escherichia coli. Enzyme-linked immunosorbent assay and Western blot analysis revealed that increasing [CaCl(2)] enhanced TG-mediated crosslinking of DC to cdb3 but decreased TG binding to cdb3. P4.2 inhibited the TG-mediated crosslinking of cdb3 but stabilized the binding of TG to cdb3 in the presence of calcium. This in vitro study suggests a relationship among TG, cdb3 and P4.2 in erythrocyte membrane during calcium influx.
authors.affiliation: Department of Bioengineering and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, 92093-0412, USA.
authors.forename: Edgar
authors.initials: E
authors.lastname: Gutierrez
authors.forename: L Amy
authors.initials: LA
authors.lastname: Sung
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011503:Transglutaminases / Q000097:blood* / Q000235:genetics / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011503:Transglutaminases