An Arthropod Cuticular Chitin-binding Protein Endows Injured Sites with Transglutaminase-dependent Mesh

Yasuyuki Matsuda,Takumi Koshiba,Tsukasa Osaki,Haruka Suyama,Fumio Arisaka,Yoshihiro Toh,Shun-Ichiro Kawabata

doi:10.1074/jbc.m705731200

Abstract

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.

Highlights

Protein cross-linking involved in blood coagulation, skin-barrier formation, and other important biological processes, by forming an isopeptide bond between Lys and Gln residues to form the cross-linking of the ⑀-(␥-glutamyl)lysine bond [1,2,3,4,5,6,7,8]
Stimulation by LPS prompts exocytosis through a GTP-binding protein-mediating signaling pathway, which triggers the secretion of granular components, including serine protease zymogens, involved in hemolymph coagulation, a clottable protein known as coagulogen, protease inhibitors, lectins, and antimicrobial peptides [17,18,19,20,21]
We extensively determined the sequences of horseshoe crab cuticular chitin-binding proteins and grouped these proteins into classes based on their approximate isoelectric points and predominant amino acid compositions [16]

Summary

Introduction

Protein cross-linking involved in blood coagulation, skin-barrier formation, and other important biological processes, by forming an isopeptide bond between Lys and Gln residues to form the cross-linking of the ⑀-(␥-glutamyl)lysine bond [1,2,3,4,5,6,7,8]. To determine Lys residues that are susceptible to cross-linking, a biotin-labeled peptide containing a Gln residue was synthesized as a probe; biotin-DEQAAL was synthesized based on the sequence corresponding to Asp112– Leu117 of caraxin-1 with an amino acid replacement of Lys to Ala at position 115.

Results

Conclusion