An arthropod cuticular chitin-binding protein endows injured sites with transglutaminase-dependent mesh.
In mammals, the cornified cell envelope forms beneath the plasma membrane in epithelia and provides a vital physical barrier consisting of insoluble proteins cross-linked by transglutaminase (TGase). In the horseshoe crab Tachypleus tridentatus, TGase is stored in hemocytes and secreted in response to the simulation of bacterial lipopolysaccharides. Here we characterized a TGase substrate designated as caraxin that was identified in horseshoe crab cuticle. One of the homologs, caraxin-1, possessed a unique domain structure consisting of N-and C-terminal heptad repeats and a central domain with a tandem-repeated structure of a pentapeptide. Western blotting showed the specific localization of caraxin-1 in sub-cuticular epidermis. Moreover, we identified the pentapeptide motif to be a chitin-binding unit. Analytical ultracentrifugation revealed that caraxin-1 exists as an oligomer with 310-350 kDa, which is approximately 20-mer based on the molecular mass of the monomer. The oligomers were cross-linked by TGase to form an elaborate mesh with honeycomb structures, which was electron-microscopically found to be different from the clotting mesh triggered by lipopolysaccharide-induced hemocyte exocytosis. We determined several cross-linking sites in the N-and C-terminal domains of caraxin-1. The replacements of Leu to Pro at positions 36 and 118 in caraxin-1 reduced the alpha-helix content, which destroyed the TGase-dependent mesh, thus indicating the importance of the N-and C-terminal domains for the proper mesh formation. In arthropods, TGase-dependent protein cross-linking may be involved in the initial stage of host defense at the sub-cuticular epidermis, as in the case of mammalian skin.
Matsuda
Yasuyuki
Y
Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan.
Koshiba
Takumi
T
Osaki
Tsukasa
T
Suyama
Haruka
H
Arisaka
Fumio
F
Toh
Yoshihiro
Y
Kawabata
Shun-Ichiro
S
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002686:Chitin / Q000737:chemistry*; D002942:Circular Dichroism; D005089:Exocytosis; D006434:Hemocytes / Q000378:metabolism; D006737:Horseshoe Crabs / Q000378:metabolism; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D011503:Transglutaminases / Q000737:chemistry*; D014461:Ultracentrifugation
D011994:Recombinant Proteins; D002686:Chitin; D011503:Transglutaminases