Erythrocyte Cytochrome b Reductase 1 Protein Levels and Structure in Sickle Cell Patients
Authors: Kulmacz Richard , Farzaneh Hamidreza , McElhinney Kathryn L , Rodriguez Jose M. , Thomason Nicole C. , Irajizad Ehsan , Berka Vladimir , Liu Wen , Idowu Modupe , Juneja Harinder S. 
Published: October 2019

Abstract
In humans, vitamin C (ascorbic acid) is required as substrate or cofactor in a number of important cellular processes. Patients with sickle cell disease (SCD) often are deficient in vitamin C and thus potentially adversely affected. A major determinant of the cellular availability of ascorbate is the plasma level of the vitamin. This reflects the balance between dietary supply, uptake by tissues, excretion, oxidative reactions in the plasma, and RBC dependent recycling or irreversible decomposition of oxidized ascorbate. The mechanisms for depressed plasma ascorbate levels in SCD patients have not been identified, and the effectiveness and safety of dietary ascorbate supplements have not been established. As RBC dysfunction is central to SCD, we have focused on the possibility that SCD patients' RBC membranes have an altered level, structure or function of CYBRD1, a key enzyme in recycling oxidized plasma ascorbate (Fig. 1A).
Materials and methods:
Blood samples were collected in EDTA tubes from adult SCD patients at routine ambulatory clinic visits and from healthy African American adults. Protocols were approved by institutional review. RBC were isolated by centrifugation and lysed in hypotonic buffer; membranes were isolated by centrifugation and stored at -80 C.
Electrophoretic and western blot analyses: RBC membrane proteins were separated by SDS-PAGE, stained with Coomassie Blue and analyzed by densitometry to quantitate total membrane protein (standard: BSA). Alternatively, separated proteins were transferred to nitrocellulose or PVDF and probed with antibodies against CYBRD1. Immunoreactive bands were visualized colorimetrically and quantitated by densitometry (standard: recombinant human CYBRD1 (rCYBRD1)).
Mass spectroscopic analyses: CYBRD1 was extracted from RBC membranes with dodecyl maltoside, and further enriched by immunoprecipitation. The immunopurified mixture was separated by SDS-PAGE and stained with Coomassie Blue. Gel pieces containing the CYBRD1 monomers were subjected to in-gel digestion and MS analysis of the tryptic peptides.
Results and conclusions:
Detailed dose-response analyses found unsuspected positively cooperative behavior between 0 and ~2 ng CYBRD1 in both rCYBRD1 and RBC membrane samples. This invalidated calculations of RBC CYBRD1 content based on linear response to rCYBRD1. However, selected RBC membrane samples were routinely included as quality controls on many blots. Further, the dose-response curves for RBC membrane samples had a consistent shape that fit a simple saturable model with an x-axis offset parameter. It was thus possible to estimate the CYBRD1 content for 36 of the RBC samples using one of them (V7) as reference (Fig. 1B). This permitted our ongoing comparison of CYBRD1 content in patients and controls, and in SCD subtypes.
RBC CYBRD1 content in 4 homozygous SCD subjects sampled at 5 regular clinic visits changed little over 7-9 months (averages: 0.32 ± 0.05; 0.25 ± 0.05; 0.41 ± 0.04; and 0.37 ± 0.04 ng CYBRD1/µg RBC membrane protein). This suggests that patients' RBC CYBRD1 contents are relatively stable despite the short RBC half-life, and that a single sampling is sufficient for cross sectional studies.
There was no significant difference in CYBRD1 level among ambulatory HbSC (N=6) and HbSS (N=8) patientsand healthy controls (N=8)(Fig. 1C, top). However, there were notable differences in CYBRD1 protein modification, with significantly less modification in CYBRD1 from HbSS patients than either HbSC patients or healthy controls (Fig. 1C, bottom).
Similar isoform banding patterns are seen for patient and control CYBRD1 with antibodies against either the N- and C-terminal peptides (Fig. 1D). Retention of epitopes at both ends of CYBRD1 in the isoforms argues against differential proteolysis being involved. The mass spectrometric analyses found extensive oxidative modification of multiple methionine residues, but similar patterns were seen in RBC CYBRD1 from an SCD patient and a healthy control. Phosphorylation was found at multiple sites in residues 200-285 of CYBRD1 from both patient and control, but the fraction phosphorylated appeared much too low to account for the large proportion of CYBRD1 in individual isoforms. Thus, the structural basis for the isoforms' differential gel mobility remains to be identified.
Support: American Heart Association, 16GRNT29170013 (R. Kulmacz); NIH 5T35DK007676 (B. Kone)

Disclosures
No relevant conflicts of interest to declare.