Characterization of circulating APOL1 protein complexes in African Americans
2016; Elsevier BV; Volume: 57; Issue: 1 Linguagem: Inglês
10.1194/jlr.m063453
ISSN1539-7262
AutoresAllison Weckerle, James A. Snipes, Dongmei Cheng, Abraham K. Gebre, Julie A. Reisz, Mariana Murea, Gregory S. Shelness, Gregory A. Hawkins, Cristina M. Furdui, Barry I. Freedman, John S. Parks, Lijun Ma,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoAPOL1 gene renal-risk variants are associated with nephropathy and CVD in African Americans; however, little is known about the circulating APOL1 variant proteins which reportedly bind to HDL. We examined whether APOL1 G1 and G2 renal-risk variant serum concentrations or lipoprotein distributions differed from nonrisk G0 APOL1 in African Americans without nephropathy. Serum APOL1 protein concentrations were similar regardless of APOL1 genotype. In addition, serum APOL1 protein was bound to protein complexes in two nonoverlapping peaks, herein referred to as APOL1 complex A (12.2 nm diameter) and complex B (20.0 nm diameter). Neither of these protein complexes associated with HDL or LDL. Proteomic analysis revealed that complex A was composed of APOA1, haptoglobin-related protein (HPR), and complement C3, whereas complex B contained APOA1, HPR, IgM, and fibronectin. Serum HPR was less abundant on complex B in individuals with G1 and G2 renal-risk variant genotypes, relative to G0 (P = 0.0002–0.037). These circulating complexes may play roles in HDL metabolism and susceptibility to CVD. APOL1 gene renal-risk variants are associated with nephropathy and CVD in African Americans; however, little is known about the circulating APOL1 variant proteins which reportedly bind to HDL. We examined whether APOL1 G1 and G2 renal-risk variant serum concentrations or lipoprotein distributions differed from nonrisk G0 APOL1 in African Americans without nephropathy. Serum APOL1 protein concentrations were similar regardless of APOL1 genotype. In addition, serum APOL1 protein was bound to protein complexes in two nonoverlapping peaks, herein referred to as APOL1 complex A (12.2 nm diameter) and complex B (20.0 nm diameter). Neither of these protein complexes associated with HDL or LDL. Proteomic analysis revealed that complex A was composed of APOA1, haptoglobin-related protein (HPR), and complement C3, whereas complex B contained APOA1, HPR, IgM, and fibronectin. Serum HPR was less abundant on complex B in individuals with G1 and G2 renal-risk variant genotypes, relative to G0 (P = 0.0002–0.037). These circulating complexes may play roles in HDL metabolism and susceptibility to CVD. A major breakthrough in human molecular genetics was identification of the powerful contribution of APOL1 gene G1 and G2 renal-risk variants to nondiabetic nephropathy susceptibility in African Americans (1.Genovese G. Friedman D.J. Ross M.D. Lecordier L. Uzureau P. Freedman B.I. Bowden D.W. Langefeld C.D. Oleksyk T.K. Uscinski Knob A.L. et al.Association of trypanolytic ApoL1 variants with kidney disease in African Americans.Science. 2010; 329: 841-845Crossref PubMed Scopus (1401) Google Scholar). HIV-associated nephropathy, idiopathic focal segmental glomerulosclerosis, severe lupus nephritis, sickle cell nephropathy, and hypertension-attributed nephropathy strongly associate with APOL1 G1 and G2 renal-risk variants on chromosome 22q13.1 (1.Genovese G. Friedman D.J. Ross M.D. Lecordier L. Uzureau P. Freedman B.I. Bowden D.W. Langefeld C.D. Oleksyk T.K. Uscinski Knob A.L. et al.Association of trypanolytic ApoL1 variants with kidney disease in African Americans.Science. 2010; 329: 841-845Crossref PubMed Scopus (1401) Google Scholar, 2.Tzur S. Rosset S. Shemer R. Yudkovsky G. Selig S. Tarekegn A. Bekele E. Bradman N. Wasser W.G. Behar D.M. et al.Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene.Hum. Genet. 2010; 128: 345-350Crossref PubMed Scopus (457) Google Scholar, 3.Freedman B.I. Kopp J.B. Langefeld C.D. Genovese G. Friedman D.J. Nelson G.W. Winkler C.A. Bowden D.W. Pollak M.R. The apolipoprotein L1 (APOL1) gene and nondiabetic nephropathy in African Americans.J. Am. Soc. Nephrol. 2010; 21: 1422-1426Crossref PubMed Scopus (207) Google Scholar). Although APOL1 mRNA and APOL1 protein are present in human kidney (4.Madhavan S.M. O'Toole J.F. Konieczkowski M. Ganesan S. Bruggeman L.A. Sedor J.R. APOL1 localization in normal kidney and nondiabetic kidney disease.J. Am. Soc. Nephrol. 2011; 22: 2119-2128Crossref PubMed Scopus (190) Google Scholar, 5.Ma L. Shelness G.S. Snipes J.A. Murea M. Antinozzi P.A. Cheng D. Saleem M.A. Satchell S.C. Banas B. Mathieson P.W. et al.Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines.J. Am. Soc. Nephrol. 2015; 26: 339-348Crossref PubMed Scopus (96) Google Scholar), the major APOL1 reservoir appears to be circulating protein (5.Ma L. Shelness G.S. Snipes J.A. Murea M. Antinozzi P.A. Cheng D. Saleem M.A. Satchell S.C. Banas B. Mathieson P.W. et al.Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines.J. Am. Soc. Nephrol. 2015; 26: 339-348Crossref PubMed Scopus (96) Google Scholar, 6.Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). APOL1 was initially discovered as a minor apolipoprotein of plasma HDLs (6.Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar); however, its distribution among HDL subfractions has not been well-defined. APOL1 nephropathy variants associate with HDL subfraction concentrations (7.Gutierrez O.M. Judd S.E. Irvin M.R. Zhi D. Limdi N. Palmer N.D. Rich S.S. Sale M.M. Freedman B.I. APOL1 nephropathy risk variants are associated with altered high-density lipoprotein profiles in African Americans.Nephrol. Dial. Transplant. 2015; 272Google Scholar) and CVD risk, although controversial results have been reported with CVD (8.Ito K. Bick A.G. Flannick J. Friedman D.J. Genovese G. Parfenov M.G. Depalma S.R. Gupta N. Gabriel S.B. Taylor H.A. et al.Increased burden of cardiovascular disease in carriers of APOL1 genetic variants.Circ. Res. 2014; 114: 845-850Crossref PubMed Scopus (120) Google Scholar, 9.Detrano R. Guerci A.D. Carr J.J. Bild D.E. Burke G. Folsom A.R. Liu K. Shea S. Szklo M. Bluemke D.A. et al.Coronary calcium as a predictor of coronary events in four racial or ethnic groups.N. Engl. J. Med. 2008; 358: 1336-1345Crossref PubMed Scopus (2121) Google Scholar, 10.Langefeld C.D. Divers J. Pajewski N.M. Hawfield A.T. Reboussin D.M. Bild D.E. Kaysen G.A. Kimmel P.L. Raj D.S. Ricardo A.C. et al.Apolipoprotein L1 gene variants associate with prevalent kidney but not prevalent cardiovascular disease in the Systolic Blood Pressure Intervention Trial.Kidney Int. 2015; 87: 169-175Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 11.Freedman B.I. Langefeld C.D. Lu L. Palmer N.D. Smith S.C. Bagwell B.M. Hicks P.J. Xu J. Wagenknecht L.E. Raffield L.M. et al.APOL1 associations with nephropathy, atherosclerosis, and all-cause mortality in African Americans with type 2 diabetes.Kidney Int. 2015; 87: 176-181Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Trypanosome lytic factors (TLF1 and TLF2) contain APOL1 protein (12.Uzureau P. Uzureau S. Lecordier L. Fontaine F. Tebabi P. Homble F. Grelard A. Zhendre V. Nolan D.P. Lins L. et al.Mechanism of Trypanosoma brucei gambiense resistance to human serum.Nature. 2013; 501: 430-434Crossref PubMed Scopus (114) Google Scholar) and are minor HDL subfractions in humans that contribute to innate immunity via protection from infection, including from African trypanosomes (13.Samanovic M. Molina-Portela M.P. Chessler A.D. Burleigh B.A. Raper J. Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.PLoS Pathog. 2009; 5: e1000276Crossref PubMed Scopus (62) Google Scholar). Association was not observed between plasma APOL1 concentrations and APOL1 genotype in African Americans with treated HIV infection; plasma APOL1 levels also did not associate with the risk of HIV-associated nephropathy or chronic kidney disease (14.Bruggeman L.A. O'Toole J.F. Ross M.D. Madhavan S.M. Smurzynski M. Wu K. Bosch R.J. Gupta S. Pollak M.R. Sedor J.R. et al.Plasma apolipoprotein L1 levels do not correlate with CKD.J. Am. Soc. Nephrol. 2014; 25: 634-644Crossref PubMed Scopus (77) Google Scholar). Whether serum APOL1 protein levels and their distribution among HDL particles are associated with APOL1 genotypes in healthy individuals is unknown. Because free APOL1 protein may be taken up by podocytes in vitro (5.Ma L. Shelness G.S. Snipes J.A. Murea M. Antinozzi P.A. Cheng D. Saleem M.A. Satchell S.C. Banas B. Mathieson P.W. et al.Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines.J. Am. Soc. Nephrol. 2015; 26: 339-348Crossref PubMed Scopus (96) Google Scholar), it remains critical to determine whether serum APOL1 concentrations or the structure/composition of variant APOL1 proteins and their associated complexes are specific to the G1 and G2 renal-risk variants, relative to nonrisk G0. To address these issues, serum APOL1 protein levels and size distribution were examined in age- and gender-matched African Americans without kidney disease based on APOL1 genotype using fast protein LC (FPLC) and immunoblot analysis. Proteomics analysis was performed to compare the composition of APOL1 protein-containing complexes. These results provide novel information on APOL1 genotype-specific circulating APOL1 protein and multiprotein complexes potentially involved in human CVD and HDL metabolism. Eighty-four unrelated healthy African Americans without nephropathy (estimated glomerular filtration rate >60 ml/min/1.73 m2 and urine albumin:creatinine ratio <30 mg/g) were selected from Natural History of APOL1-Associated Nephropathy (NHAAN) participants. These participants had first degree relatives with nondiabetic etiologies of end-stage kidney disease. Among them (42 female/42 male), mean ± SD age was 42.5 ± 13.8 years in females and 43.2 ± 13.4 years in males. Serum creatinine concentration (mean ± SD) was 0.79 ± 0.15 mg/dl in females and 1.05 ± 0.13 mg/dl in males. Seven men and seven women were included in each potential genotypic group, G0/G0, G1/G0, G2/G0, G1/G1, G2/G2, and G1/G2. Significant differences were not observed for age or kidney function with respect to APOL1 genotype. The Institutional Review Board at the Wake Forest School of Medicine approved the study and all participants provided written informed consent. Descriptive clinical data of the participants are provided in Table 1.TABLE 1Serum APOL1 levels and other clinical data by APOL1 genotype in 84 African American subjects without kidney diseaseAllG0/G0G1/G0G2/G0G1/G2G1/G1G2/G2PN (M/F)84 (42/42)14 (7/7)14 (7/7)14 (7/7)14 (7/7)14 (7/7)14 (7/7)1.000Age (years)42.8 ± 13.542.5 ± 12.542.0 ± 13.643.5 ± 13.442.6 ± 14.042.3 ± 14.244.1 ± 15.70.999African ancestry (%)0.795 ± 0.0890.773 ± 0.0690.787 ± 0.1190.795 ± 0.0670.816 ± 0.0960.796 ± 0.0710.805 ± 0.1080.864Creatinine (mg/dl)0.920 ± 0.1920.934 ± 0.2070.900 ± 0.2540.861 ± 0.1830.923 ± 0.1330.988 ± 0.1640.912 ± 0.2000.256Cystatin C (mg/l)0.709 ± 0.1240.666 ± 0.1310.711 ± 0.1100.689 ± 0.1180.679 ± 0.1290.803 ± 0.1330.707 ± 0.0900.060Urine Alb:Creat (mg/g)6.30 ± 5.714.66 ± 4.475.04 ± 5.276.11 ± 5.186.44 ± 4.018.59 ± 7.556.96 ± 7.040.344APOL1 (μg/ml)20.5 ± 30.816.2 ± 13.025.0 ± 24.915.6 ± 22.117.7 ± 11.932.6 ± 64.515.8 ± 15.50.723Triglycerides (mg/dl)99.4 ± 54.5101.3 ± 77.093.8 ± 50.0110.6 ± 66.286.0 ± 34.1103.6 ± 54.9100.9 ± 39.40.881TC (mg/dl)183.1 ± 36.5179.5 ± 49.5185.8 ± 35.0186.8 ± 36.5182.1 ± 29.9175.6 ± 23.7189.3 ± 43.50.945HDL (mg/dl)50.7 ± 11.146.9 ± 10.552.7 ± 13.153.0 ± 10.851.1 ± 9.1952.5 ± 11.948.0 ± 11.20.532LDL (mg/dl)111.5 ± 32.1111.4 ± 41.7115.7 ± 34.8109.2 ± 30.5110.4 ± 22.6102.3 ± 25.3120.2 ± 37.00.864VLDL (mg/dl)19.9 ± 10.820.3 ± 15.418.7 ± 9.9122.2 ± 13.217.3 ± 6.8020.7 ± 10.920.1 ± 7.800.875P values were determined by ANOVA, representing the difference across APOL1 genotypes. P values were calculated for log transformed serum creatinine, cystatin C, APOL1, triglycerides, TC, HDL, LDL, VLDL, and urine albumin/creatinine ratio (Urine Alb:Creat) due to the skewness of raw data from normal distribution. The P value for serum creatinine level by APOL1 genotype was additionally adjusted for gender. Data are presented as the mean ± SD. Open table in a new tab P values were determined by ANOVA, representing the difference across APOL1 genotypes. P values were calculated for log transformed serum creatinine, cystatin C, APOL1, triglycerides, TC, HDL, LDL, VLDL, and urine albumin/creatinine ratio (Urine Alb:Creat) due to the skewness of raw data from normal distribution. The P value for serum creatinine level by APOL1 genotype was additionally adjusted for gender. Data are presented as the mean ± SD. One microliter of sera from the 84 study participants was separated by 4–20% SDS-PAGE, transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), and blocked for 1 h at room temperature with TBS containing 1% skim milk powder and 0.1% Tween 20 (TBST). Blots were incubated overnight at 4°C with a monoclonal anti-APOL1 antibody (1:1,000; Epitomics, 3245-1). Membranes were washed three times in TBST and incubated for 1 h in blocking buffer with HRP-conjugated anti-rabbit IgG (1:20,000; Jackson Immuno-Research, West Grove, PA). Bound antibodies were visualized using ECL (Super Signal West Pico; Thermo Pierce, Rockford, IL) and recorded on X-ray film. Bands were scanned and densities quantitated using ImageJ software (http://rsbweb.nih.gov/ij/). As a standard, an APOL1-maltose binding protein (MBP) fusion protein previously generated in our lab (15.Cheng D. Weckerle A. Yu Y. Ma L. Zhu X. Murea M. Freedman B.I. Parks J.S. Shelness G.S. Biogenesis and cytotoxicity of APOL1 renal-risk variant proteins in hepatocytes and hepatoma cells.J. Lipid Res. 2015; 56: 1583-1593Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) was loaded at varying concentrations. One hundred fifty microliters of sera from four healthy individuals homozygous for APOL1 G0, four homozygous for G1, and four homozygous for G2 variants were injected into a Superose 6 analytical column (GE Healthcare Life Sciences, Pittsburg, PA) for FPLC fractionation. Fractions 17–48 were collected at a flow rate of 0.4 ml/min. A total cholesterol (TC) enzymatic assay was performed to examine TC distribution among samples. Fractions 22–46 (sizes ranging from ∼7 to 20 nm or ∼66 to 1,240 kDa, as determined by nondenaturing gradient gel electrophoresis) were analyzed by immunoblot and probed with APOL1, APOAI, and APOB antibodies, as described below. One microliter of sera from four healthy individuals homozygous for APOL1 G0, four homozygous for G1, and four homozygous for G2 variants was electrophoresed in 4–20% native gels (BioRad Ready Gel® Tris-HCl gel) for 900 V/h and transferred to polyvinylidene fluoride membranes at 35 V for 22 h at 4°C. Membranes were blocked in 5% nonfat dry milk/0.1% TBST at room temperature for at least 1 h. Anti-APOL1 (Epitomics, 1:1,000), anti-APOA1 (Meridian Life Science, 1:1,000), and anti-APOB (Academy Bio-Medical Co., 1:1,000) primary antibodies were incubated with membranes overnight at 4°C. Membranes were washed three times with 0.1% TBST, incubated with anti-rabbit (GE Healthcare, 1:15,000) or anti-goat (Santa Cruz Biotechnology, 1:6,000) HRP-conjugated secondary antibodies for 2 h, and washed three times with 0.1% TBST. SuperSignal® West Pico chemiluminescent substrate was added and membranes were visualized using a FUJIFILM LAS-3000 imager. Twenty microliters of plasma from four healthy individuals homozygous for APOL1 G0, four homozygous for G1, and four homozygous for G2 variants were brought to a final volume of 1 ml with addition of normal saline and adjusted to d = 1.225 g/ml with solid KBr. Samples were overlaid with an additional 1 ml d = 1.225 g/ml KBr solution and ultracentrifuged in a TLA 120.2 rotor at 100,000 rpm for 4 h at 15°C. Following ultracentrifugation, top (0.5 ml) and bottom (1.5 ml) fractions were collected. Top (20 μl) and bottom (60 μl) fractions were then precipitated with TCA and subjected to 12% SDS-PAGE followed by APOL1 and APOA1 immunoblot analysis, as described. One microliter of plasma was electrophoresed on 0.7% agarose gels at 100 mA for 2 h. Gels were transferred to a polyvinylidene fluoride membrane by pressure blotting for 2 h. Following transfer, membranes were blocked with 5% nonfat dry milk/0.1% TBST for 1 h at room temperature, and incubated with either rabbit anti-APOL1 (Sigma-Aldrich, 1:1,000) or goat anti-APOA1 (Meridian Life Science, 1:1,000) antibodies overnight at 4°C. Membranes were washed three times with 0.1% TBST, incubated with anti-rabbit (GE Healthcare, 1:15,000) or anti-goat (Santa Cruz Biotechnology, 1:6,000) HRP-conjugated secondary antibodies for 2 h, and washed three times with 0.1% TBST. SuperSignal® West Pico chemiluminescent substrate was added and membranes were visualized using a FUJIFILM LAS-3000 imager. Plasma (1.5 ml) from four healthy individuals homozygous for APOL1 G0, four homozygous for G1, and four homozygous for G2 variants was injected into a Superose 6 preparative column (GE Healthcare) for FPLC fractionation. Fractions were collected at a flow rate of 1 ml/min. A TC assay was performed to examine distributions among samples. Combined peak FPLC fractions for complex B (fractions 43–47) and complex A (fractions 58–62) were concentrated 10-fold with Amicon Ultra-4 MWCO 10 kDa centrifugal filter units (Millipore, Billerica, MA). Ten microliters of glycerol were added to 50 μl of concentrated FPLC APOL1 complex A and complex B samples, 15 μl of which were loaded onto a Criterion pH 3–10 isoelectric focusing (IEF) gel and electrophoresed at 100 V for 1 h, 250 V for 1 h, and then 500 V for 30 min. The gel was transferred at 100 V for 1 h in 0.7% acetic acid. The primary antibody (Epitomics anti-APOL1) was diluted 1:3,000 and the secondary antibody (Jackson Immuno-Research goat anti-rabbit IgG) was diluted 1:20,000 for immunoblot analysis. An N-terminal APOL1 (1-199 amino acids)-MBP fusion protein was obtained, as previously reported (5.Ma L. Shelness G.S. Snipes J.A. Murea M. Antinozzi P.A. Cheng D. Saleem M.A. Satchell S.C. Banas B. Mathieson P.W. et al.Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines.J. Am. Soc. Nephrol. 2015; 26: 339-348Crossref PubMed Scopus (96) Google Scholar, 15.Cheng D. Weckerle A. Yu Y. Ma L. Zhu X. Murea M. Freedman B.I. Parks J.S. Shelness G.S. Biogenesis and cytotoxicity of APOL1 renal-risk variant proteins in hepatocytes and hepatoma cells.J. Lipid Res. 2015; 56: 1583-1593Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). cDNA corresponding to APOL1 (amino acid residues 13-130 of the APOL1 reference sequence) was generated by PCR and cloned into the pMAL-C5E vector (New England Biolabs) to generate a MBP-APOL1 fusion protein in Escherichia coli. Primer sequences used for PCR were: forward, 5′-AAG GTA CCG GAG GAA GCT GGA GCG AGG-3′ reverse, 5′-ACC GTC GAC TCA CCT TCT TAT GTT ATC CTC-3′. A polyclonal antibody against this fusion protein was raised in a rabbit (Lampire Biological Labs, Pipersville, PA). Lampire anti-APOL1 IgG was purified with a Melon Gel IgG purification kit (Thermo) according to the manufacturer's instructions. Twenty microliters of complex A and 200 μl of complex B APOL1 peak fractions, determined by FPLC, were diluted to 1 ml with PBS in a 1.5 ml centrifuge tube. Rabbit anti-APOL1 antibody (50 μl; Lampire) was then added. The antibody-immunoprecipitate mixture was incubated overnight at 4°C by gentle mixing on a shaker. Sequential incubation at 4°C was applied for 1 h after addition of 40 μl protein A agarose slurry (Invitrogen, Grand Island, NY). The tube was centrifuged again at 1,000–3,000 g for 2 min at 4°C, the supernatant removed, and the bead mixture washed three times using PBS. After washing, 20 μl 2× SDS-PAGE loading buffer was added to the bead-antibody mixture. The mixture was heated at 95°C for 5 min, the beads were spun down, and the supernatant kept for immunoblot analysis with mouse anti-APOL1 antibody (Novus), goat anti-APOA1, mouse anti-haptoglobin-related protein (HPR) (Abcam), and other antibodies against candidate partners of APOL1 complexes (antibody details in supplementary Table 1). Concentrated complex A and B fractions (see the IEF electrophoresis section above) were analyzed by non-denaturing gradient gel electrophoresis (NDGGE) and IEF. Gels were stained with SYPRO® Ruby (Invitrogen) overnight. After destaining, bands were excised from the gel and subjected to proteomics analysis. In-gel digestion was performed with standard reduction (dithiothreitol) and alkylation (iodoacetamide) followed by proteolysis using MS grade trypsin (Pierce) in 50 mM NH4HCO3 overnight at 37°C. Peptides were extracted from the gel, concentrated, and analyzed on a Dionex UltiMate 3000 splitless nanoLC system coupled to a Thermo Orbitrap Velos Pro high resolution mass spectrometer. LC solvents were as follows: buffers A (98% water, 2% MeCN, 0.1% formic acid) and B (80% MeCN, 20% water, 0.1% formic acid). Peptides were loaded onto a nano trap C18 column (Acclaim PepMap 100, 100 μm × 2 cm, 5 μm) with a flow of 5 μl/min of 100% buffer A for 5 min, then separated using an analytical nano C18 column (Acclaim PepMap RSLC C18, 75 μm × 15 cm, 2 μm) with a gradient of 2–85% B over 160 min and a flow rate of 300 nl/min. The columns were held at 35°C. Following separation, peptides were introduced to the mass spectrometer via positive nanospray with the following settings: spray voltage 1.9 kV, capillary temperature 200°C. The mass spectrometer was operated in data-dependent top 10 mode using Xcalibur 2.1 (Thermo). MS spectra were acquired over the range of m/z 150–2,000 at a resolution of 60,000. Precursor ions were fragmented using collision-induced dissociation (normalized collision energy of 35%, activation time 10 ms) and fragment ions detected in the linear ion trap. Acquired raw data were processed and searched using Proteome Discoverer version 1.4 with the Mascot search engine and the SwissProt human proteomic database. Search parameters allowed for two missed trypsin cleavages, a precursor mass tolerance of 10 ppm, and fragment mass tolerance of 0.8 Da. N-terminal acetylation, cysteine carbamidomethylation, and methionine mono-oxidation were selected as variable modifications. Identities of proteins within the complex were filtered at a false discovery rate of 0.01. Serum samples from 84 age- and sex-matched NHAAN participants who had first-degree relatives with nondiabetic kidney disease were analyzed by immunoblot to determine whether circulating APOL1 concentrations were associated with APOL1 renal-risk variant genotypes in African Americans without kidney disease. Seven male and seven female NHAAN participants in each genotype group (G0/G0, G1/G0, G2/G0, G1/G2, G1/G1, and G2/G2) were analyzed. Consistent with previous reports (6.Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), two bands were seen, reflecting the two APOL1 isoforms (39 kDa and 42 kDa) present in the circulation. Although APOL1 concentrations varied among individuals, effects were genotype-independent (Fig. 1). Regardless of APOL1 genotype, mean serum APOL1 concentrations were ∼15 μg/ml compared with an APOL1-MBP fusion protein standard of known concentration (Fig. 1). Mean serum APOL1 concentrations for each genotype are shown in Table 1. To examine potential differences in size distribution among APOL1-containing particles, NDGGE fractionation and immunoblot were performed on serum samples from African Americans lacking nephropathy with different APOL1 renal-risk variant genotypes. Based upon APOA1 and APOB immunodetection, the vast majority of APOL1-containing particles appeared to distribute between typical HDL- and LDL-sized particles, irrespective of genotype (Fig. 2). The predominant band was ∼12 nm in diameter (500 kDa molecular mass), with a smaller APOL1-containing particle band near the LDL migration position (20 nm diameter; 1,000 kDa molecular mass). Little, if any, serum APOL1 protein was in the unbound region of the gel ( 90% of APOL1 protein was found in the d > 1.225 gm/ml (bottom) fraction, whereas >90% of APOA1 was in the d < 1.225 gm/ml (top) fraction, suggesting that APOL1 was not as strongly associated with HDL as APOA1 (Fig. 4A). This finding was independent of APOL1 genotype. Moreover, ultracentrifugation or presence of high salt (i.e., 1.225 gm/ml KBr) did not impact the FPLC elution position of APOL1, suggesting that APOL1 was in a large complex that did not contain sufficient lipid to allow floatation at d < 1.225 gm/ml (Fig. 4B). Another method (agarose gel electrophoresis) was used that separates lipoproteins primarily by charge, rather than size, to determine whether APOL1 comigrated with HDL. Serum APOL1 migrated in the α/pre-α position characteristic of lipid-free APOA1, LDL, and VLDL, not in the α position where HDL particles migrate (Fig. 4C). This also suggests that serum APOL1 is not bound to HDL particles. Immunoblot analysis of d > 1.225 gm/ml (bottom) serum fractions subsequently separated by NDGGE demonstrated that APOL1 in complexes A and B exhibited similar size distributions as in serum that was not subjected to ultracentrifugation. Combined results using three different lipoprotein separation procedures that rely on size, density, and charge support the conclusion that APOL1 is likely a component of multiprotein complexes similar in size to large HDL and large LDL particles. To determine whether there was a difference in the size of these complexes in African Americans with different APOL1 genotypes, a two-step fractionation of sera was used involving FPLC followed by NDGGE for serum samples (1.5 ml) from six individuals with different APOL1 genotypes (G0/G0, G1/G1, and G2/G2; N = 2 per genotype). APOL1 immunoblot of NDGGE gels demonstrated that complex A (∼12 nm diameter) was composed of three distinct size particles, defined as complexes A-α, A-α, and A-γ, whereas only a single band was apparent for complex B (∼20 nm diameter) (Fig. 5A). Concentrated complex A and complex B were separated by IEF based on isoelectric point (pI). Complex A separated into three bands with distinct pI values (assigned as A-1, A-2, and A-3 from top to bottom) and complex B remained as one band (Fig. 5B). Bands were carefully excised from gels for protein identification via MS. The distinct size components of complex A (α, α, and γ) detected by NDGGE were consistent with A-1, A-2, and A-3 on the IEF gel, respectively. Hereafter, these complexes are referred to as A-α, A-α, and A-γ, respectively. Table 2 lists the candidate proteins that were identified by MS from complex A and complex B (on both NDGGE and IEF gels) in six healthy African Americans. Immunoprecipitation of APOL1 complex A and complex B using a rabbit anti-APOL1 antibody directed at the N terminus of APOL1 was performed using 12 additional serum samples from African Americans with different APOL1 genotypes (G0/G0, G1/G1, and G2/G2; N = 4 per genotype). The anti-APOL1 immunoprecipitates were next examined by immunoblot analysis using antibodies targeting proteins that were identified by MS. APOL1, HPR, and APOA1 appeared on both complex A and complex B; however, complement C3 was unique to APOL1 complex A (Fig. 6B). Fibronectin appeared as a unique polypeptide on APOL1 complex B (Fig. 6C), in addition to IgM (17.Thomson R. Genovese G. Canon C. Kovacsics D. Higgins M.K. Carrington M. Winkler C.A. Kopp J. Rotimi C. Adeyemo A. et al.Evolution o
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