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

Abstract

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.

Keywords: Fc receptor; albumin; cellular recycling; endosome; intracellular processing; intracellular trafficking; receptor recycling.

Figure 1.

Albumin variants cellular uptake. a–c, mean fluorescence intensity (MFI) detected by flow cytometry of low (HMEC-1) and high (HMEC-1-FcRn) FcRn-expressing cells after exposure to Alexa488-labeled recombinant albumin variants. Shown are FcRn low binder (LB) (a), WT (b), and FcRn high binder (HB) (c), at pH 6.0 for 2 h followed by incubation in HBSS (pH 7.4) for 0, 1, or 2 h. MFI was normalized to cells at t = 0 h for each albumin variant. Albumin uptake in HMEC-1 (d) and HMEC-1-FcRn (e) cells after exposure to 8 μm Alexa488-labeled LB, WT, and HB recombinant albumin variants at pH 6.0 or pH 7.4 for 2 h. Cellular MFI was determined by flow cytometry and normalized to non-treated cells.

Figure 2.

Albumin variants cellular uptake in the presence of excess albumin competition. HMEC-1-FcRn cells were incubated with Alexa-594 labeled albumins WT, FcRn low binder (LB), and FcRn high binder (HB) at 8 μm in the presence of 0, 8, or 80 μm WT albumin labeled with Alexa488 at pH 6.0 for 1 h. A, representative confocal sections showing the intracellular accumulation of Alexa-594-labeled albumin with the distinct FcRn-binding affinities versus the intracellular accumulation of Alexa-488 WT albumin. B, plot of mean fluorescence intensity of Alexa-594-labeled albumin. The bars represent the mean ± S.D. of at least 10 cells. C, normalized % fluorescent inhibition whereby the Alexa594-labeled fluorescence in the absence of inhibitor is set to 100. Data are representative of at least 10 cells. Scale bar = 20 μm.

Figure 3.

Albumin variants cellular uptake and endosomal involvement. a and b, albumin cellular uptake in HMEC-1 (a) and HMEC-1-FcRn (b) cells after exposure to 8 μm 5FAM-labeled WT, FcRn low binder (LB), and FcRn high binder (HB) recombinant albumin at pH 6. 0 for 2 h. Mean fluorescence intensity (MFI) of the cells was measured by flow cytometry before and after incubation with monensin (20 μm) for 10 min.

Figure 4.

Albumin intracellular sorting. A, HMEC-1-FcRn cells expressing GFP early or late endosome markers (Rab5 and Rab7) or lysosome marker (Lamp1) were incubated with incubated with 8 μm Alexa-594-labeled WT, FcRn low, and FcRn high binder (LB, HB) albumins for 1 h at pH 6.0 before fixation. Projections were made of 3D image volumes for each fluorophore and were merged as single images (green, GFP markers; red, albumins; yellow, co-localization of albumin with markers). B, pixel co-localization was quantified as a percentage of albumin co-localized with the respective marker in the 3D structure. Co-localization with endosome markers Rab5 and Rab7 is associated with uptake and sorting, whereas co-localization with lysosome marker Lamp1 is associated with degradation. The bars represent the mean ± S.D. of at least 10 cells.

Figure 5.

Endothelial cellular recycling of albumin. A, WT, FcRn high binder (HB), and FcRn low binder (LB) albumin variants were recycled in HMEC-1-FcRn cells. HMEC-1-FcRn cells were exposed to 0.15 μm albumin at pH 6.0 for 1 h to allow cellular uptake. After thorough washing of the cells, fresh pH 7.4 medium was added in which recycled albumin was quantified by ELISA after 1 h incubation. One representative of three experiments is shown. Student's t test was used for comparison of LB and HB albumin variants to WT. ***, p < 0.001. Error bars, S.D. B, 15 independent recycling experiments with WT and HB albumin were performed as in A, and data are presented as recycling efficiency relative to WT. Error bar, S.E. C, IgG and IgY were recycled as in A and measured by respective immunoglobulin specific ELISA. One representative of two experiments is shown. BD, Below detection. Error bars, S.D.

Figure 6.

The effect of FcRn expression, pH, and albumin concentration on albumin recycling. WT, low (LB), and high FcRn binder (HB) albumin variants were recycled in HMEC-1-FcRn (A) and HMEC-1 (B) cells as described under “Experimental Procedures.” All exposure cells received either 0.15 μm or 8 μm albumin where the pH was either 6.0 or 7.4. During recycling and release of albumin pH was 7.4 in all cases. The figures show concentrations of albumin measured in supernatants, and one representative of two experiments performed is shown. Student's t test was used to compare LB and HB albumin variants to WT. ***, p < 0.001; *, p < 0.05. BD, below detection. NS, non-significant. Error bars, S.D.

Figure 7.

FcRn-dependent recycling of albumin variants conjugated to a small chemical molecule or peptide. WT, low (LB), and high FcRn binder (HB) albumin variants conjugated to Alexa Fluor 488 (A), exenatide (B), or non-conjugated were recycled in HMEC-1-FcRn cells and detected by ELISA as described in Fig. 5. Results are presented as relative recycling with WT albumin set as 100%. Error bars, S.D.