RESUMO
The leaf extracellular space contains several peptidases, most of which are of unknown function. We isolated cDNAs for two extracellular serine carboxypeptidase III genes from tobacco (Nicotiana tabacum), NtSCP1 and NtSCP2, belonging to a phylogenetic clade not yet functionally characterized in plants. NtSCP1 and NtSCP2 are orthologs derived from the two ancestors of tobacco. Reverse transcription-polymerase chain reaction analysis showed that NtSCP1 and NtSCP2 are expressed in root, stem, leaf, and flower tissues. Expression analysis of the ß-glucuronidase reporter gene fused to the NtSCP1 transcription promoter region confirmed this expression profile. Western blotting of NtSCP1 and expression of an NtSCP1-green fluorescent protein fusion protein showed that the protein is located in the extracellular space of tobacco leaves and culture cells. Purified His-tagged NtSCP1 had carboxypeptidase activity in vitro. Transgenic tobacco plants overexpressing NtSCP1 showed a reduced flower length due to a decrease in cell size. Etiolated seedlings of these transgenic plants had shorter hypocotyls. These data provide support for a role of an extracellular type III carboxypeptidase in the control of cell elongation.
Assuntos
Carboxipeptidases/metabolismo , Crescimento Celular , DNA de Plantas/metabolismo , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Carboxipeptidases/genética , Clonagem Molecular , Meios de Cultura/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , DNA de Plantas/genética , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Espaço Extracelular/genética , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Filogenia , Componentes Aéreos da Planta/genética , Componentes Aéreos da Planta/crescimento & desenvolvimento , Componentes Aéreos da Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicotiana/genética , Nicotiana/crescimento & desenvolvimentoRESUMO
Peptidases in the extracellular space might affect the integrity of recombinant proteins expressed in, and secreted from, plant cells. To identify extracellular peptidases, we recovered the leaf intercellular fluid from Nicotiana tabacum plants by an infiltration-centrifugation method. The activity of various peptidases was detected by an in vitro assay in the presence of specific inhibitors, using BSA and human serum gamma-globulin as substrates. Peptidases were detected by 1- and 2-D zymography in a polyacrylamide gel containing gelatin as substrate. Proteolytic activity was observed over a wide range of molecular masses equal to, or higher than, 45 kDa. To identify the peptidases, the extracellular proteins were digested with trypsin and analyzed by LC and MS. Seventeen peptides showing identity or similarity to predicted plant aspartic, cysteine, and serine peptidases have been identified. The extracellular localization of a cysteine peptidase aleurain homolog was also shown.
Assuntos
Nicotiana/metabolismo , Proteínas de Plantas/química , Proteômica/métodos , Cromatografia Líquida/métodos , Cisteína/química , Cisteína Endopeptidases/química , Fluoresceína-5-Isotiocianato , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Peptídeo Hidrolases/química , Folhas de Planta/metabolismo , Sensibilidade e Especificidade , Soroalbumina Bovina/química , gama-Globulinas/químicaRESUMO
The ospA gene of Borrelia burgdorferi codes for an outer membrane lipoprotein, which is a major antigen of the Lyme disease agent. Recombinant OspA vaccines tested so far were expressed in Escherichia coli. In this study, we investigated the expression of a soluble OspA protein in Nicotiana tabacum suspension cells and evaluated the secretion of OspA driven by either its own bacterial signal peptide or a plant signal peptide fused to the amino-terminal cysteine of the mature form. In both cases, the signal peptide was cleaved off and OspA secreted. During secretion, OspA was N-glycosylated. Addition of a C-terminal KDEL sequence led to retention of OspA in the endoplasmic reticulum.
