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Immunogenetics

, Volume 71, Issue 5–6, pp 421–432 | Cite as

Identification and characterization of the lamprey cathepsin genes

  • Dong Wang
  • Peng Su
  • Xuefeng Wang
  • Kai Liu
  • Changzhi Li
  • Xingxing Gao
  • Jiali Lu
  • Feng Sun
  • Qingwei LiEmail author
  • Yue PangEmail author
Original Article
  • 91 Downloads

Abstract

Cathepsins are key mammalian proteases that play an important role in the immune response. Several studies have revealed the versatile and critical functions of cathepsins. Here, we obtained ten kinds of cathepsin homologs and identified seven homologs with complete coding sequences. Phylogenetic analysis verified their identities and supported the classification of cathepsins into seven families, which is similar to other vertebrates. Tissue-specific expression analysis showed that all lamprey cathepsins (L-cathepsins) are present in the supraneural body (SB), kidney, gill, intestine, brain, heart, and liver, but their relative abundance varied among tissues. Additionally, we focused on the lamprey cathepsin L (L-cathepsin L) and used recombinant L-cathepsin L protein (rL-cathepsin L) to prepare anti rL-cathepsin L polyclonal antibodies, which were used to detect its distribution in lamprey tissues. The L-cathepsin L protein was primarily detected in the SB, kidney, gill, intestine, brain, and liver via western blot and immunohistochemistry assays. Importantly, quantitative real-time PCR (RT-PCR) revealed that the expression level of L-cathepsins mRNA significantly increased after exposure to three different stimuli (poly I:C, Staphylococcus aureus (S.a) and Vibro anguilarum (V.an)). This suggested that L-cathepsins may participate in defense processes. These results revealed that L-cathepsins may play key roles in the immune response to exogenous stimuli. The findings provide important information for future studies aiming to understand the molecular mechanisms underlying the immune response to pathogen invasion in lamprey.

Keywords

Lamprey Cathepsins Evolution Immune 

Notes

Funding information

This work was funded by the Chinese Major State Basic Research Development Program (973 Program; Grant2013CB835304), the Marine Public Welfare Project of the State Oceanic Administration (No.201305016), Chinese National Natural Science Foundation Grants (No. 31170353, No. 31202020, No. 31772884, and No. 31801973), Science and Technology Innovation Fund Research Project (No. 2018J12SN079), and the project of Department of Ocean and Fisheries of Liaoning Province (No. 201805).

Compliance with ethical standards

Competing interests

The authors declare that they have no conflicts of interest.

Supplementary material

251_2019_1117_Fig7_ESM.png (1 mb)
Fig. S1

Fig. 1 Expression of L-cathepsin L recombinant proteins and production, affinity purification and identification of anti- rL-cathepsin L antibodies. a Expression of rL-cathepsin L protein in E. coli BL21 (DE3). Lane 1: total protein of uninduced E. coli harboring pCold I-L-cathepsin L; Lane 2-6: total protein of induced E. coli harboring pCold I-L-cathepsin L after treatment with 0.1 mM, 0.05 mM, 0.15 mM, 0.01 mM and 0.5 mM IPTG for 24 h at 16℃; Lane 7-8: supernatant precipitation from induced E. coli BL21 (DE3) harboring pCold I-L-cathepsin L after treatment with 0.1 mM IPTG for 24 h at 16℃ after sonication. The arrow points to L-cathepsin L. b Purification of rL-cathepsin L protein by HisTrap™ affinity columns. Lane 1: supernatant from induced E. coli BL21 (DE3) harboring pCold I-L-cathepsin L after sonication; Lane 2: flow through; Lane 3: equilibrium; Lane 4-9: elution by iminazole, concentration gradient of 30, 50, 100, 200, 300 and 400 mM; M: protein marker. The arrow points to rL-cathepsin L. c The titer of anti-rL-cathepsin L polyclonal antibody by ELISA assay. The rabbit sera before immunization was used as a control. d Identification of the anti-rL-cathepsin L polyclonal antibody by SDS-PAGE. Lane 1: rabbit anti-rL-cathepsin L antisera; Lane 2: flow through; Lane 3: equilibrium; Lane 4: elution by 0.1 M glycine-HCI (pH 2.7); M: protein marker. The arrow points to the heavy chains and light chains of the antibody (PNG 1.0 MB)

251_2019_1117_MOESM1_ESM.tif (99.6 mb)
High resolution image (TIF 99.6 MB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of Life ScienceLiaoning Normal UniversityDalianChina
  2. 2.Lamprey Research CenterLiaoning Normal UniversityDalianChina

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