RESUMO
The specific binding of adenosine deaminase to the multifunctional membrane glycoprotein dipeptidyl peptidase IV is thought to be immunologically relevant for certain regulatory and co-stimulatory processes. In this study we present the 3D structure of the complete CD26-ADA complex obtained by single particle cryo-EM at 22A resolution. ADA binding occurs at the outer edges of the beta-propeller of CD26. Docking calculations of available CD26 and ADA crystal data into the obtained EM density map revealed that the ADA-binding site is stretched across CD26 beta-propeller blades 4 and 5 involving the outermost distal hydrophobic amino acids L294 and V341 but not T440 and K441 as suggested by antibody binding. Though the docking of the ADA orientation appears less significant due to the lack of distinct surface features, non-ambiguous conclusions can be drawn in the combination with earlier indirect non-imaging methods affirming the crucial role of the ADA alpha2-helix for binding.
Assuntos
Adenosina Desaminase/química , Microscopia Crioeletrônica/métodos , Dipeptidil Peptidase 4/química , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Sítios de Ligação , Western Blotting , Bovinos , Linhagem Celular , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM. CD26(-/-) mice display an apparently normal phenotype. However, in their spleen lymphocyte population the percentage of CD4(+) T cells is lower, and that of NK cells is higher, than that in CD26(+/+) mice. In their peripheral blood, CD26(-/-) mice present a conspicuously decreased proportion of CD4(+) NKT lymphocytes. In vitro, the PWM-stimulated IL-4 production was decreased by 60-80% in the supernatants of spleen lymphocytes of CD26(-/-) mice compared to that of CD26(+/+) mice, whereas levels of IL-10 and IFN-gamma were increased. No significant differences were found in the production of IL-2, IL-5, IL-6 and IL-13 between knockout and wild-type mice. After immunization of mice with PWM in vivo, serum levels of total IgG, IgG1, IgG2a and IgE were markedly lower in CD26(-/-) mice than those in CD26(+/+) mice, while no difference was found in IgM production. Further analysis of cytokine levels in vivo revealed a reduced IL-4, IL-2 and delayed IFN-gamma production in sera of CD26(-/-) mice upon immunization with PWM. These results indicate that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells.
Assuntos
Dipeptidil Peptidase 4/fisiologia , Switching de Imunoglobulina/fisiologia , Imunoglobulinas/sangue , Ativação Linfocitária/efeitos dos fármacos , Linfocinas/metabolismo , Mitógenos de Phytolacca americana/farmacologia , Animais , Linfócitos T CD4-Positivos/imunologia , Concanavalina A/farmacologia , Dipeptidil Peptidase 4/genética , Feminino , Imunização , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Interferon gama/biossíntese , Interferon gama/sangue , Interferon gama/metabolismo , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Interleucinas/biossíntese , Interleucinas/sangue , Interleucinas/metabolismo , Células Matadoras Naturais/classificação , Células Matadoras Naturais/imunologia , Linfocinas/biossíntese , Linfocinas/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organismos Livres de Patógenos Específicos , Baço/citologia , Baço/imunologiaRESUMO
The human dipeptidyl peptidase IV/CD26 (DPPIV/CD26) is a multifunctional type-II membrane bound glycoprotein. As a receptor of collagen I and fibronectin it mediates cell-cell and cell-matrix adhesion, and by interacting with extracellular adenosine deaminase and CD45 it is involved in regulatory and costimulatory events in the immune system. DPPIV/CD26 has a very distinct substrate specificity, and is potentially capable of truncating many cytokines, chemokines, and peptide hormones. In this study, we describe the overexpression, purification, and characterization of human DPPIV/CD26 in Spodoptera frugiperda (Sf9) cells, using the baculovirus system. Overexpression of DPPIV/CD26 was confirmed by measurement of its peptidase specificity, SDS-PAGE, and Western blot analyses. Expression rates were between 6.4 and 17.6 mg protein per liter suspension culture (1.5 x 10(9)cells). The N-linked oligosaccharide composition was examined and compared with that of mammalian cell-expressed DPPIV/CD26. Two-step purification by immunoaffinity chromatography and size-exclusion fast protein liquid chromatography (SE-FPLC) led to highly stable protein with significant peptidase activity. A subsequent gel filtration step on a Superdex 200 column yielded 2mg homogeneous dimeric DPPIV/CD26 (per liter insect cell culture) for crystallographic studies. Protein homogeneity was confirmed by silver staining of non-denaturating PAGE gels and by MALDI-TOF analysis of tryptic peptides.
