Molecular characterization and complement activating functional analysis of a new collectin(TfCol-11) from Trachidermus fasciatus

Dev Comp Immunol. 2020 Jan:102:103486. doi: 10.1016/j.dci.2019.103486. Epub 2019 Aug 29.

Abstract

The complement system is a crucial component of the innate immune system that links innate and adaptive immunity. CL-11, a protein similar to Mannose-binding lectin (MBL), plays significant role in the innate immune system in mammals and fish, serving as an initiator of the lectin pathway of complement activation. In this study, a CL-11 homolog (TfCol-11) was identified in roughskin sculpin (Trachidermus fasciatus), and its expression and role in immune responses were characterized. The open reading frame of TfCol-11 is 795 bp long, encoding a 264 amino acid polypeptide. The deduced amino acid sequence of this protein is highly homologous to sequences in other teleosts, and is similar to vertebrate CL-11, containing a canonical collagen-like region, a carbohydrate recognition domain, and a neck region. Recombinant TfCol-11 purified from Escherichia coli(E.coli) was able to bind to different microbes in a Ca2+-independent manner. Meanwhile, a 993 bp-long of partial MASP cDNA with a 96 bp 5' untranslated region (UTR) was also cloned from roughskin sculpin, containing 299 amino acids and consisting of three domains (CUB-EGF-CUB). qRT-PCR indicated that TfCol-11 and MASP mRNAs were predominately co-expressed in the liver. The temporal expression of TfCol-11 and MASP were both drastically up-regulated in the liver, skin, and blood by LPS challenge. Recombinant TfCol-11 purified from E.coli BL21(DE3) was able to agglutinate some bacteria in a Ca2+-dependent manner. In addition, an in vitro pull-down experiment demonstrated that TfCol-11 was able to bind to MASP, and in vivo experiments showed that TfCol-11 was associated with increased membrane attack complex (MAC) levels. It is therefore possible that TfCol-11 may plays a role in activating the complement system and in the defense against invading microorganisms in roughskin sculpin.

Keywords: Collectin-11; Complement activation; MASP; Trachidermus fasciatus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agglutination Tests
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Collectins / chemistry
  • Collectins / genetics
  • Collectins / metabolism*
  • Complement Activation*
  • Complement Membrane Attack Complex / metabolism
  • Fish Proteins / chemistry
  • Fish Proteins / genetics
  • Fish Proteins / metabolism*
  • Gene Expression Regulation
  • Immunity, Innate
  • Mannose-Binding Protein-Associated Serine Proteases / chemistry
  • Mannose-Binding Protein-Associated Serine Proteases / genetics
  • Mannose-Binding Protein-Associated Serine Proteases / metabolism
  • Open Reading Frames
  • Perciformes / immunology*
  • Phylogeny
  • Protein Domains
  • Sequence Alignment
  • Tissue Distribution

Substances

  • Collectins
  • Complement Membrane Attack Complex
  • Fish Proteins
  • Mannose-Binding Protein-Associated Serine Proteases