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Abstract: FR-PO419

Development and Application of Sequential Enrichment Approach for ApoL1 Purification

Session Information

Category: Dialysis

  • 701 Dialysis: Hemodialysis and Frequent Dialysis

Authors

  • Carty, Joshua, University of Louisville School of Medicine, Louisville, Kentucky, United States
  • Klein, Jon B., University of Louisville Kidney Disease Program, Louisville, Kentucky, United States
  • Merchant, Michael, University of Louisville School of Medicine, Louisville, Kentucky, United States
Background

Apolipoprotein L1 (ApoL1) is associated with either HDL3 or the Trypanolytic Factor (TLF) complex. The TLF complex also includes Haptoglobin-Related Protein (HRP) and Apolipoprotein A1 (ApoA1). Both human and transgenic mouse data suggest genotype is insufficient to cause renal disease. We hypothesized that circulating ApoL1 variants undergo PTM that promote renal disease progression or severity. To test address this hypothesis, a method for the selective enrichment of ApoL1 containing lipoprotein particles would be essential for ApoL1 characterization by mass spectrometry. Our objective here was to develop a affinity enrichment method that enabled selective TLF purification from patient plasma and serum samples.

Methods

Serum lipoproteins were isolated from healthy controls (HC) and from diabetic-/non-diabetic-African American (AA) dialysis patients using differential dextran-sulfate precipitation. TLF enrichment methods were based on known high affinity interaction of hemoglobin (Hb) with Hb-savaging receptors haptoglobin (Hp) and haptoglobin-related protein (Hpr). Ligands tested for TLF enrichment included Hb- agarose, hemin- (Hm-) agarose, and control agarose resins. Analysis of the enriched TLF was based on ApoL1, ApoA1, and Hpr immunoblot (IB) analysis or proteomic analyses.

Results

ApoA1, Hpr, and ApoL1 IB and proteomic studies demonstrate both Hb-agarose and Hm-agarose enrich ApoL1, ApoA1 and Hpr from serum HDL. IB data suggest enhanced enrichment of ApoL1, ApoA1 and Hpr using Hm-agarose beads compared to Hb-agarose beads. IB analysis of control, diabetic, and non-diabetic AA serum samples suggests specific enrichment of Hpr protein in AA TLF samples. Proteomic studies identified (1) a significant decrease of ApoL1 in Hm-bead TLF isolates of 2-fold for diabetic and 6-fold for non-diabetic AA dialysis patients as compared to healthy controls and (2) decreased phosphorylation of ApoL1 Ser-327 and Ser-S330 in dialysis patients samples.

Conclusion

An affinity-isolation method based on Hm-agarose was developed to enrich Hpr-containing lipoproteins from dilute solutions. This method may be parallelized into 96-well plates to enables higher throughput processing of patient samples. TLF ApoL1 abundance is decreased in non-diabetic AA ESRD patients versus diabetic AA ESRD patients versus healthy controls.

Funding

  • NIDDK Support