RESUMO
A unique family of genes encoding serine endoproteases related to the Saccharomyces cerevisiae serine endoprotease kexin was identified in Pneumocystis carinii. Unlike previously described serine endoprotease genes that are single copies, multiple copies of the P. carinii serine endoprotease are distributed throughout the genome. The proteins predicted by these variant genes demonstrate sequence variability, but they retain the conserved active sites associated with endoprotease activity. The serine endoprotease was localized to the organism surface by immunohistochemical and immunofluorescence studies and to the electron lucent layer of the cyst wall by immunoelectron microscopy. The findings of multiple copies of the serine endoprotease gene in the P. carinii genome, and its localization to the cell surface, suggest that this protease plays an important role in the biology of P. carinii and that antigenic variation of the surface-expressed serine endoprotease may be a strategy for immune evasion. P. carinii serine endoprotease provides a novel target for chemotherapeutic and immune-based approaches to the treatment of P. carinii pneumonia.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Membrana/genética , Família Multigênica , Pneumocystis/genética , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Variação Antigênica , Furões/microbiologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/imunologia , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Camundongos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Pneumocystis/imunologia , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/biossíntese , Serina Endopeptidases/imunologia , Subtilisinas/genéticaRESUMO
The major surface glycoprotein (MSG) is an abundant, immunodominant protein on the surface of the opportunistic pathogen Pneumocystis carinii. The current study identified two novel variant MSG (vMSG) gene families in rat P. carinii that are closely related to but distinct from MSG. These gene families encode proteins of approximately 90 kDa (v1MSG) and approximately 115 kDa (v2MSG). Compared with MSG, v1MSG is characterized by a deletion near the carboxyl terminus. The predicted v1MSG and v2MSG proteins are highly homologous to MSG at the carboxyl, but not the amino, terminus. Like MSG, they are cysteine-rich. Approximately 10% of the apparent molecular weight is due to N-linked glycosylation. Southern blotting studies demonstrated that, like MSG, v1MSG and v2MSG are the products of multicopy gene families. However, unlike MSG, each vMSG gene encodes a signal peptide, suggesting that the regulation of vMSG is different from that of MSG.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Variação Genética , Glicoproteínas de Membrana/genética , Família Multigênica , Pneumocystis/genética , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , DNA Fúngico/genética , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Glicosilação , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Peso Molecular , Sondas de Oligonucleotídeos/genética , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
To facilitate studies of Pneumocystis carinii infection in humans, we undertook to better characterize and to express the major surface glycoprotein (MSG) of human P. carinii, an important protein in host-pathogen interactions. Seven MSG genes were cloned from a single isolate by PCR or genomic library screening and were sequenced. The predicted proteins, like rat MSGs, were closely related but unique variants, with a high level of conservation among cysteine residues. A conserved immunodominant region (of approximately 100 amino acids) near the carboxy terminus was expressed at high levels in Escherichia coli and used in Western blot studies. All 49 of the serum samples, which were taken from healthy controls as well as from patients with and without P. carinii pneumonia, were reactive with this peptide by Western blotting, supporting the hypothesis that most adult humans have been infected with P. carinii at some point. This recombinant MSG fragment, which is the first human P. carinii antigen available in large quantities, may be a useful reagent for investigating the epidemiology of P. carinii infection in humans.