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. 2016 Oct 20:6:33760.
doi: 10.1038/srep33760.

An inhibitory receptor of VLRB in the agnathan lamprey

Fenfang Wu  1 , Liyong Chen  2 , Yong Ren  1 , Xiaojing Yang  1 , Tongzhou Yu  1 , Bo Feng  1 , Shangwu Chen  1 , Anlong Xu  1   3
Affiliations

Affiliations

  • 1 State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory for Pharmaceutical Functional Genes, Department of Biochemistry, School of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, P. R. China.
  • 2 Guangdong Province Key Laboratory for Medical Molecular Diagnostics, China-America Cancer Research Institute, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, P. R. China.
  • 3 Beijing University of Chinese Medicine, Beijing, 100029, P. R. China.

An inhibitory receptor of VLRB in the agnathan lamprey

Fenfang Wu et al. Sci Rep. .
. 2016 Oct 20:6:33760.
doi: 10.1038/srep33760.

Authors

Fenfang Wu  1 , Liyong Chen  2 , Yong Ren  1 , Xiaojing Yang  1 , Tongzhou Yu  1 , Bo Feng  1 , Shangwu Chen  1 , Anlong Xu  1   3

Affiliations

  • 1 State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory for Pharmaceutical Functional Genes, Department of Biochemistry, School of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, P. R. China.
  • 2 Guangdong Province Key Laboratory for Medical Molecular Diagnostics, China-America Cancer Research Institute, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, P. R. China.
  • 3 Beijing University of Chinese Medicine, Beijing, 100029, P. R. China.

Abstract

Lamprey, the primitive jawless vertebrate, uses variable lymphocyte receptor (VLR) as alternative adaptive immune system instead of immunoglobulin (Ig)-based receptors used in jawed vertebrates. In the present study, we characterized a potential inhibitory receptor of VLRB from leucocytes in lamprey. It is a novel ITIM-containing IgSF protein and was therefore named as NICIP. NICIP has two Ig-like domains in extracellular region, a transmembrane domain and two classical ITIM motifs in cytoplasmic domain. It is mainly expressed on the surface of granulocytes and monocytes and can interact with VLRB. In transiently transfected HEK293T cells, it was confirmed again that it could interact with VLRB and the two phosphorylated ITIM motifs could recruit SHP-1 and SHP-2. These results imply that NICIP may play a role as a potential inhibitory receptor of VLRB and involve in negative regulation of immune response mediated by VLRB.

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Figures

Figure 1

Figure 1. Identification and prediction of the…

Figure 1. Identification and prediction of the conserved domains of NICIP in Lamprey.

( a …

Figure 1. Identification and prediction of the conserved domains of NICIP in Lamprey.
(a) Sequences and domains of NICIP. Wavy lines: Signal peptide, Double underline: Ig-like V-set, Underline: Ig-like C2-set, Character border: TM (Transmembrane) domain, Dashed underline: ITIMs. Red is key amino acid and yellow is potential tyrosine phosphorylation sites in ITIM. (b) Three dimensional structure of NICIP. Y means tyrosine in C-terminal of NICIP.
Figure 2

Figure 2. Real-time PCR analysis of NICIP …

Figure 2. Real-time PCR analysis of NICIP mRNA expressed in adult tissues.

The relative expression…

Figure 2. Real-time PCR analysis of NICIP mRNA expressed in adult tissues.
The relative expression quantity of NICIP mRNA in other tissues compared to kidney from un-stimulated lamprey was calculated. All tissues samples were performed in triplicates. **P < 0.01.
Figure 3

Figure 3. Distribution of NICIP on the…

Figure 3. Distribution of NICIP on the surface of different leukocyte subsets.

The experiments were…

Figure 3. Distribution of NICIP on the surface of different leukocyte subsets.
The experiments were done three times independently, and the representative result is shown. (a) Distribution of NICIP on the surface of leukocyte subsets by flow cytometry analysis. (b) western blotting assays. According to the amino acid sequences, the predicted molecular weight of NICIP is about 36 kDa. Lane 1, monocyte lysate. Lane 2, granulocyte lysate. Lane 3, lymphocyte lysate.
Figure 4

Figure 4. The interaction between NICIP on…

Figure 4. The interaction between NICIP on the membrane of lamprey leukocytes and free secreted-type…

Figure 4. The interaction between NICIP on the membrane of lamprey leukocytes and free secreted-type VLRB in the antisera.
(a) Membrane localization of NICIP-EGFP fusion protein in transiently transfected 293T cells. (b) The interaction of VLR and NICIP by co-immunoprecipitation analysis. Lamprey leukocytes were pre-incubated using lamprey anti-LPS antisera with or without LPS antigen. Input 1 was antisera. Input 2 was leukocytes lysate. (c) Localization of NICIP on the surface of lamprey granulocytes and monocytes (red arrow) and localization of membranous-type VLRB on the surface of lamprey lymphocytes. (d) Interaction between free secreted-type VLRB and NICIP on the surface of lamprey granulocytes and monocytes but not on lymphocytes under the condition of pre-stimulating by antisera and antigen.
Figure 5

Figure 5. Immunoprecipitation analysis of SHP-1 and…

Figure 5. Immunoprecipitation analysis of SHP-1 and SHP-2 association with NICIP.

( A ) Mutants…

Figure 5. Immunoprecipitation analysis of SHP-1 and SHP-2 association with NICIP.
(A) Mutants construction of NICIP-GFP fusion protein expression vector. Tyrosines in ITIM of NICIP are indicated by the letter Y, while tyrosines to phenylalanine mutations are indicated by F. TM, transmembrane region. (B) 293T cells of overexpressing NICIP-GFP were stimulated using lamprey anti-LPS antisera with or without LPS. Whole cell lysates were immunoprecipitated (IP) with anti-GFP polyclonal antibody, and immunoprecipitates were subjected to western blotting (WB) analysis with anti-SHP-1, anti-SHP-2 and anti-pTyr antibodies. Equal loading was verified by re-probing of the membranes with anti-GFP antibodies.
Figure 6

Figure 6. Comparison of the IgSF members…

Figure 6. Comparison of the IgSF members containing ITIM.

Domains and amino acid sequences for…

Figure 6. Comparison of the IgSF members containing ITIM.
Domains and amino acid sequences for the following proteins were cited from UniProtKB/Swiss-Prot: myeloid cell surface antigen CD33 (Human), P20138.2; leukocyte immunoglobulin-like receptor subfamily B member 4 (Gp49B, Mouse), Q64281; Allergin-1 (Human), Q7Z6M3; low affinity immunoglobulin gamma Fc region receptor II-b (CD32) (Human), P31994; paired immunoglobulin-like type 2 receptor alpha (FDF03) (Human), Q9UKJ1 and leukocyte-associated immunoglobulin-like receptor 1 (CD305) (Human), Q6GTX8.
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References

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