RESUMO
Molecular and functional characterization of the natural cytotoxicity receptor (NCR) NKp44 in species other than Homo sapiens has been elusive, so far. Here, we provide complete phenotypic, molecular and functional characterization for NKp44 triggering receptor on Pan troglodytes NK cells, the closest human relative, and the analysis of NKp44-genomic locus and transcription in Macaca fascicularis. Similar to H. sapiens, NKp44 expression is detectable on chimpanzee NK cells only upon activation. However, basal NKp44 transcription is 5-fold higher in chimpanzees with lower differential increases upon cell activation compared with humans. Upon activation, an overall 12-fold lower NKp44 gene expression is observed in P. troglodytes compared with H. sapiens NK cells with only a slight reduction in NKp44 surface expression. Functional analysis of 'in vitro' activated purified NK cells confirms the NKp44 triggering potential compared with other major NCRs. These findings suggest the presence of a post-transcriptional regulation that evolved differently in H. sapiens. Analysis of cynomolgus NKp44-genomic sequence and transcription pattern showed very low levels of transcription with occurrence of out-of-frame transcripts and no surface expression. The present comparative analysis suggests that NKp44-genomic organization appears during macaque speciation, with considerable evolution of its transcriptional and post-transcriptional tuning. Thus, NKp44 may represent an NCR being only recently emerged during speciation, acquiring functional relevance only in non-human primates closest to H. sapiens.
Assuntos
Células Matadoras Naturais/metabolismo , Macaca fascicularis/imunologia , Receptor 2 Desencadeador da Citotoxicidade Natural/genética , Pan troglodytes/imunologia , Animais , Evolução Molecular , Mutação da Fase de Leitura/imunologia , Especiação Genética , Humanos , Imunidade Inata , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Receptor 2 Desencadeador da Citotoxicidade Natural/biossíntese , Filogenia , Processamento de Proteína Pós-Traducional/imunologia , Transcrição Gênica/imunologiaRESUMO
The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.