RESUMO
Bacterial IgA1 proteases share the ability to cleave human IgA1 at the hinge region. Nature has developed this trait along at least five independent evolutionary lineages. To obtain further insight into the phylogeny and function of IgA1 proteases, the nucleotide sequence of the iga gene that encodes the IgA1 protease was determined from two Streptococcus mitis strains and one Gemella haemolysans strain. Heterologous expression in Escherichia coli confirmed that the genes encode human IgA1-cleaving activity. IgA1 proteases from Streptococcus and G. haemolysans shared structural features, including a motif typical for zinc-dependent metalloproteases of clan MA(E) family M26 and an N-terminal signal sequence followed by an LPXTG cell-wall-anchor motif and two putative membrane-spanning domains. In addition, they all harboured a repeat region preceding the active site of the protease. In the streptococcal IgA1 proteases, a G5 domain, which has been suggested to bind N-acetylglucosamine, was identified. Conservation of these structures in otherwise diverse proteases suggests that they are essential to the biological function of the enzyme. The phylogenetic distribution of homologous iga genes and conservation of gene order in the iga gene region in different Streptococcus species, combined with the sequence homologies, strongly suggest that the iga gene is more ancient in Streptococcus than in G. haemolysans, and therefore that the IgA1 protease gene was transferred from Streptococcus to G. haemolysans.
Assuntos
Transferência Genética Horizontal , Serina Endopeptidases/genética , Staphylococcaceae/genética , Streptococcus mitis/genética , Sequência de Bases , Southern Blotting , Escherichia coli/genética , Dados de Sequência Molecular , Filogenia , Serina Endopeptidases/químicaRESUMO
We established a transient human joining (J)-chain gene expression system in the baby hamster kidney (BHK) cell. The J-chain was detected as a 29-kDa single band on Western blotting. Immunofluorescent staining of the transfectant revealed an exclusive localization of the J-chain in the endoplasmic reticulum (ER). Intracellular transport experiment revealed that incubating conditions favorable for vesicular stomatitis virus glycoprotein (VSV-G) transport did not allow the J-chain to exit from the ER. Analysis of glycosylation status of the J-chain in the transfectant was examined by tunicamycin treatment, endoglycosidase H digestion, and also by treatment with brefeldin A. It was found that an N-glycosylation consensus site of the J-chain was functional, and intracellular J-chain was endoglycosidase H sensitive. These results indicate that, in the absence of any immunoglobulin molecules, J-chain localizes exclusively in the ER. We also tested whether the J-chain could be exported from the ER by perturbing the Ca2+ concentration in the ER. Cultivation of the J-chain transfectant in the presence of ionomycin resulted in the time-dependent secretion of the J-chain. The secreted J-chain was modified by the Golgi resident glycosylation enzymes, indicating that the secreted J-chain passed through the normal exocytic pathway.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Cadeias J de Imunoglobulina/metabolismo , Animais , Brefeldina A/farmacologia , Células Cultivadas , Cricetinae , Retículo Endoplasmático/efeitos dos fármacos , Glicosilação , Humanos , Cadeias J de Imunoglobulina/efeitos dos fármacos , Cadeias J de Imunoglobulina/genética , Ionomicina/farmacologia , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Transporte Proteico/efeitos dos fármacos , Fatores de Tempo , Transfecção , Tunicamicina/farmacologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild-type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild-type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist.