Nancy A. Robinson

In vitro Cross-Linking of Recombinant Human Involucrin

Human involucrin (hINV) is a constituent of the scaffolding of the cornified envelope. In the present study, we describe an in vitro model system to study the role of hINV in scaffold formation. We characterize the in vitro cross-linking of full-length (585 amino acid) recombinant hINV, rhINV(1–585). When reacted with detergent-solubilized, particulate transglutaminase type 1 (TG1) or partially purified type 2 transglutaminase (TG2), rhINV(1–585) functions as a TG substrate in a calcium-dependent manner. When the reaction is supplemented with 14C-putrescine tracer, the radiolabeled cosubstrate is incorporated into a high-molecular-weight product in a calcium-, rhINV(1–585)- and time-dependent manner. 35S-rhINV(1–585) is also cross-linked to form a high-molecular-weight product. These results suggest that rhINV(1–585) is extensively multimerized. Products having a molecular weight smaller than authentic rhINV(1–585) are also formed, providing evidence for intramolecular cross-link formation. Transmission electron microscopy of cross-linked product reveals immunoreactive large-molecular-weight loop-string-loop and branched structures. Our studies (1) show that rhINV(1–585) is a substrate for both TG1 and TG2, (2) indicate that rhINV(1–585) can be cross-linked to form macromolecular products having distinct structural features, (3) demonstrate that rhINV(1–585) forms intramolecular cross-links when hINV concentration is limiting and (4) establish that hINV possesses reactive Gln and Lys residues.

Transglutaminase Reactivity of Human Involucrin

Human involucrin (hINV) is assembled into cornified structures via formation of transglutaminase (TG)-dependent interprotein ε-(γ-glutamyl)lysine bonds. The hINV sequence includes 150 glutamine residues that could function as potential sites of cross-link formation. The present studies were designed to evaluate the extent to which hINV can function as a TG substrate under optimal conditions and in the absence of other substrates. Incubation of hINV with TG results in formation of 4–5 isopeptide bonds per hINV molecule. When the small amine donor 14C-putrescine is included in the reaction, 48 Q residues are labled. Isotope distribution and sequence analysis suggests that the 14C-putrescine-labeled sites are located throughout the protein. Our present results show that many hINV Q residues can be utilized for cross-link formation, and that hINV can be cross-linked at very high cross-link densities. These results suggest that, in vivo, factors other than hINV structure limit the number of residues used for cross-link formation.