In vitro Cross-Linking of Recombinant Human Involucrin
- 1 October 1998
- journal article
- research article
- Published by S. Karger AG in Skin Pharmacology and Physiology
- Vol. 11 (4-5), 214-226
- https://doi.org/10.1159/000029830
Abstract
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.Keywords
This publication has 9 references indexed in Scilit:
- S100A11, S100A10, Annexin I, Desmosomal Proteins, Small Proline-rich Proteins, Plasminogen Activator Inhibitor-2, and Involucrin Are Components of the Cornified Envelope of Cultured Human Epidermal KeratinocytesJournal of Biological Chemistry, 1997
- Direct Evidence That Involucrin Is a Major Early Isopeptide Cross-linked Component of the Keratinocyte Cornified Cell EnvelopeJournal of Biological Chemistry, 1997
- The Proteins Elafin, Filaggrin, Keratin Intermediate Filaments, Loricrin, and Small Proline-rich Proteins 1 and 2 Are Isodipeptide Cross-linked Components of the Human Epidermal Cornified Cell EnvelopeJournal of Biological Chemistry, 1995
- Plasminogen Activator Inhibitor Type 2: An Intracellular Keratinocyte Differentiation Product That Is Incorporated into the Cornified EnvelopeExperimental Cell Research, 1995
- Up-Regulation of Elafin/SKALP Gene Expression in Psoriatic EpidermisJournal of Investigative Dermatology, 1994
- A Dual Role for Involucrin in the Epidermis—Ultrastructural Localization in Epidermis and Hair Follicle in Humans and Transgenic MiceJournal of Structural Biology, 1993
- Molecular Characterization and Evolution of the SPRR Family of Keratinocyte Differentiation Markers Encoding Small Proline-Rich ProteinsGenomics, 1993
- Involucrin—Structure and Role in Envelope AssemblyJournal of Investigative Dermatology, 1993
- Phosphorylated cystatin α is a natural substrate of epidermal transglutaminase for formation of skin cornified envelopeFEBS Letters, 1992