Characterization and functional studies of three mammalian cytochromes b561.

摘要

Cytochromes b561 (Cyts b561) are a poorly characterized family of transmembrane proteins found in most eukaryotic cells. Previous studies suggest that two mammalian Cyts b561 are involved in ascorbate (Asc) regeneration and ferric (Fe3+) reduction respectively. Among five mammalian Cyts b561, three evolutionarily closely related Cyts b561, chromaffin granule Cyt b561 (CGCytb), duodenal Cyt b561 (DCytb) and lysosomal Cyt b561 (LCytb), were studied in this dissertation. This study covers the tissue distribution, subcellular localization, posttranslational modification, biochemical characterization, and functional aspects of these three Cyts b561.;Mouse CGCytb, DCytb and LCytb were heterologously expressed in a yeast Delta fre1Deltafre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity, to study their ability to reduce Fe3+. Expressing each of these Cyts b561 was able to rescue the growth defect of the Deltafre1Deltafre2 mutant cells grown in iron-deficient conditions. In addition, each Cyt b561 showed significant Asc-dependent ferrireductase activities. Site-directed mutagenesis of LCytb was conducted to identify amino acids which are indispensable for its ferrireductase activity.;Humans have lost the ability to synthesize Asc, and therefore efficient Asc regeneration systems are needed. We identified the presence of DCytb in human, but not mouse, erythrocyte membranes by Western blot, characteristic absorption spectra and kinetic studies. We provided various lines of evidence to support that DCytb is involved in extracellular Asc recycling of human erythrocytes. The presence of DCytb in erythrocytes was found to be related with the species' ability to synthesize Asc. Human erythrocytes showed higher Asc-dependent FeCN reductase activity and ability to preserve extracellular Asc than mouse erythrocytes.;In the last chapter, LCytb, a newly described isoform of Asc-reducible Cyts b561, is described. LCytb is mainly expressed in the lung, spleen, thymus, intestine and testis. Immunofluorescence study suggests that LCytb is localized in the membranes of late endosomes and lysosomes. We also demonstrate that N-glycosylation of N185 of LCytb is not essential for the lysosomal localization, but provides protection to LCytb from the proteolytic lysosomal environment.