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.
LaCelle
P T
PT
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, USA.
Lambert
A
A
Ekambaram
M C
MC
Robinson
N A
NA
Eckert
R L
RL
D001419:Bacteria / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004817:Epidermis / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007120:Immunochemistry; D015603:Keratinocytes / Q000187:drug effects / Q000648:ultrastructure; D008854:Microscopy, Electron; D008970:Molecular Weight; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011700:Putrescine / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013462:Sulfur Radioisotopes; D011503:Transglutaminases / Q000378:metabolism
D003432:Cross-Linking Reagents; D011498:Protein Precursors; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C032253:involucrin; D011503:Transglutaminases; D011700:Putrescine