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Cloning and characterization of CD300d, a novel member of the human CD300 family of immune receptors.

ABSTRACT: Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human peripheral blood mononuclear cells, and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking in any known signaling motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. CD300d forms complexes with the CD300 family members, with the exception of CD300c. Contrary to other activating members of the CD300 family of receptors, surface expression of CD300d in COS-7-transfected cells required the presence of an immunoreceptor tyrosine-based activating motif-bearing adaptor (Fc?R?). Accordingly, we found that CD300d was able to recruit Fc?R?. Unexpectedly, we could not detect CD300d on the surface of cells expressing Fc?R?, suggesting the existence of unknown mechanisms regulating the trafficking of this molecule. The presence of other CD300 molecules also did not modify the intracellular expression of CD300d. In fact, the presence of CD300d decreased the levels of surface expression of CD300f but not CD300c. Our data suggest that the function of CD300d would be related to the regulation of the expression of other CD300 molecules and the composition of CD300 complexes on the cell surface.

SUBMITTER: Comas-Casellas E

PROVIDER: S-EPMC3322970 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Cloning and characterization of CD300d, a novel member of the human CD300 family of immune receptors.

Comas-Casellas Emma E Martínez-Barriocanal Águeda Á Miró Francesc F Ejarque-Ortiz Aroa A Schwartz Simo S Martín Margarita M Sayós Joan J

The Journal of biological chemistry 20120130 13

Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human peripheral blood mononuclear cells, and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking in any known signaling motif, but ther ...[more]

PMID: 22291008

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Project description:BACKGROUNDSchistosome infection typically induces a polarized Th2 type host immune response. As egg antigen molecules play key roles in this immunoregulatory process, clarifying their functions in schistosomiasis would facilitate the development of vaccine and immunotherapeutic methods. Schistosoma japonicum (Sj) CP1412 (GenBank: AY57074.1) has been identified as a new member of the RNase T2 family with immune regulatory functions.METHODSThe expression plasmid Sj CP1412-pET28a was constructed and transformed into bacteria for production of recombinant Sj CP1412 protein (rSj CP1412) via IPTG induction. The RNase activity of Sj CP1412 was predicted by bioinformatic analysis and confirmed by digesting the yeast tRNA with rSj CP1412.C57BL/6j mice were immunized with rSj CP1412, and its immune regulatory effects in vivo and in vitro were investigated. Meanwhile, the relationship between the RNase activity of Sj CP1412 and its immune regulation was observed.RESULTSSj CP1412 was confirmed as a novel RNase T2 family protein with RNase activity. Immunoblotting and RT-PCR analyses demonstrated Sj CP1412 as a protein exclusively secreted/excreted from eggs, but not cercariae and adult worms. Stimulating RAW264.7 macrophages with rSj CP1412 raised the expression of CD206, Arg-1 and IL-10, which are related to M2 type macrophage differentiation. Stimulating dendritic cells (DCs) with rSjCP1412 failed to induce their maturation, and the recombinant protein also inhibited LPS-stimulated DC maturation. Depletion of Sj CP1412 from soluble egg antigen (SEA) impaired the ability of SEA to induce M2 type polarization of RAW264.7 macrophages. Immunizing mice with rSj CP1412 induced high antibody titers, increased serum IL-4 and TGF-? levels and splenic CD4?+?CD25?+?Foxp3 + T cells, downregulated serum IFN-? levels and alleviated the egg granuloma pathology of schistosome infection. In vitro stimulation by rSj CP1412 significantly increased CD4?+?CD25?+?Foxp3?+?T cell numbers in splenocytes of healthy mice. The rSj CP1412 protein with RNase activity inactivated by DEPC failed to induce M2 surface marker CD206 expression in RAW264.7 macrophages.CONCLUSIONSThe Sj CP1412 protein expressed specifically in S. japonicum eggs is a novel member of the RNase T2 family. Similar to Omega-1 of Schistosoma mansoni, the Sj CP1412 protein drives polarization of the host Th2 immune response, which is dependent on its RNase activity. These data provide new evidence towards understanding the immune regulatory role of RNase T2 family proteins during schistosome infection.

Dataset Information

Cloning and characterization of CD300d, a novel member of the human CD300 family of immune receptors.

Publications

Cloning and characterization of CD300d, a novel member of the human CD300 family of immune receptors.

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