Wild-type Albumin Research Articles

Direct demonstration of a neonatal Fc receptor (FcRn)-driven endosomal sorting pathway for cellular recycling of albumin

Albumin is the most abundant plasma protein involved in the transport of many compounds, such as fatty acids, bilirubin, and heme. The endothelial cellular neonatal Fc receptor (FcRn) has been suggested to play a central role in maintaining high albumin plasma levels through a cellular recycling pathway. However, direct mapping of this process is still lacking. This work presents the use of wild-type and engineered recombinant albumins with either decreased or increased FcRn affinity in combination with a low or high FcRn-expressing endothelium cell line to clearly define the FcRn involvement, intracellular pathway, and kinetics of albumin trafficking by flow cytometry, quantitative confocal microscopy, and an albumin-recycling assay. We found that cellular albumin internalization was proportional to FcRn expression and albumin-binding affinity. Albumin accumulation in early endosomes was independent of FcRn-binding affinity, but differences in FcRn-binding affinities significantly affected the albumin distribution between late endosomes and lysosomes. Unlike albumin with low FcRn-binding affinity, albumin with high FcRn-binding affinity was directed less to the lysosomes, suggestive of FcRn-directed albumin salvage from lysosomal degradation. Furthermore, the amount of recycled albumin in cell culture media corresponded to FcRn-binding affinity, with a ∼3.3-fold increase after 1 h for the high FcRn-binding albumin variant compared with wild-type albumin. Together, these findings uncover an FcRn-dependent endosomal cellular-sorting pathway that has great importance in describing fundamental mechanisms of intracellular albumin recycling and the possibility to tune albumin-based therapeutic effects by FcRn-binding affinity.

A mass-spectroscopic method for measuring des-Leu albumin — A novel marker for chronic pancreatitis

ObjectivesChronic pancreatitis is a progressive inflammatory disease leading to pancreatic insufficiency. The diagnosis of chronic pancreatitis is challenging, especially in early disease and the current tests have low sensitivity, may be invasive or have limited availability. We previously identified a truncated form of albumin lacking the C-terminal leucine, des-Leu albumin, which was present at high concentration in pancreatitis. We have developed a liquid-chromatography tandem-mass spectrometry (LC–MS/MS) method for measuring this peptide and make some preliminary observations on patient samples. MethodsSerum samples from patients with established pancreatitis and controls were obtained. Diluted serum samples or prepared standards were digested with trypsin. Aliquots of the digest were separated on a reversed-phase column using water:acetonitrile:formic acid mobile-phase with tandem-mass spectrometry detection. Percentage composition of des-Leu albumin was determined from a response curve. ResultsThe C-terminal peptide, LVAASQAALG- of des-Leu albumin was identified by m/z 901→725, wild type albumin by m/z 1014→825. Additional fragments were monitored as internal reference for digestion and sample integrity. Inter-assay imprecision was estimated at 10%. The percentage composition of des-Leu albumin segregated with the diagnosis of established pancreatitis with median levels of des-Leu albumin of 68% in patients compared to 5% in controls. ConclusionsDes-Leu albumin is a promising novel biomarker for chronic pancreatitis. It allowed clear discrimination of patients with pancreatitis from controls and its long half-life may facilitate monitoring of disease activity. The method described could readily be undertaken in modern clinical chemistry laboratories and will form the basis for further study.

Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumindomain III (R-III) and albumindomain I-RBP-albuminIII (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti-fibrotic drug.