The polymorphism in the ARMS2 gene, rs10490924 was described as highly associated with both forms of AMD leading to geographic atrophy (dry form) or neo-vascularization (wet form) [12, 13]. Patients diagnosed with the wet form of AMD according to the modified version of AMD study grading system (AREDS) as described previously by Spencer et al.  were genotyped for the polymorphisms in the ARMS2 gene rs2736911, rs10490924, and del443ins54 as described [2, 12]. Genomic DNA was extracted from 10 ml whole blood cells of each patient using the PAX gene blood DNA kit (QiaGen). ARMS2 was amplified with primers (forward 5′TGTCACCACATTATGTCCC3′ or 5′TGTCACTGCATTCCCTCCTGTCAT3′ and reverse 5′GGCACCACTCCAGAATTT3′ or 5′AAGCTTCTTACCCTGACTTCCAGC3′), and the PCR products were separated by agarose gel electrophoresis, visualized under UV light and subsequently validated by bi-directional sequencing on an automated DNA sequencer (ABI/1130x, Applied Biosystems). According to the presence of the polymorphisms rs2736911, rs10490924, and del443ins54 in the ARMS2 gene, three groups of genotypes were created (homozygous without these polymorphisms (type I/I), heterozygous for rs10490924 and del443ins54 (type I/II), homozygous for rs10490924 and del443ins54 (type II/II), and homozygous for rs2736911 (type III/III).
Human donor eyes
Retinal samples of controls and AMD patients were obtained from the Center of Ophthalmology Eye Bank, University of Cologne. Retina 1: type I/I, craniocerebral injury, unknown hour postmortem, age 22. Retina 2: type I/I, intracranial bleeding, 27 h postmortem, age 82. Retina 3: type I/I, hypoxia brain damage, 4.5 h postmortem, age 53. Retina 4: type II/II, exenteratio orbitae, 8 h postmortem, age 78.
CHO-K1 Chinese ovary hamster cells (ATCC-CCL-61), pgsD-677 heparan sulfate deficient CHO cells (ATCC CRL-2244), pgsA-745 xylosyltransferase 1 deficient CHO cells (ATCC CRL-2242), THP-1 human monocytes (ATCC TIB-202), RAW264.7 Mouse leukemic macrophages (ATCC TIB-71), and native RPE cells (InnoProt) were all cultivated according to the costumer’s advise. Human T cells, monocytes, and human erythrocytes were obtained from human blood samples of healthy volunteers. Human T cells, peripheral blood mononuclear cells (PBMCs), and erythrocytes were isolated with micro beads from Miltenyi Biotech, according to the manufacturer’s protocol. Apoptosis of cells was induced by incubation of the cells with 0.4 μg/ml staurosporine for 24 h and necrosis by 1 h at 65 °C. Apoptosis was confirmed by PI and annexin V-pacific blue positive staining (Life Technologies) using flow cytometry. Human microglia cell lines (iPSdM) were generated from induced pluripotent stem (iPS) cell lines obtained by reprogramming skin fibroblasts as previously described . The cells proliferate without addition of growth factors, and they were passaged 1:3 twice a week. The microglia phenotype was confirmed by flow cytometry by expression of CD11b, CD11c, CD14, CD16, CD32, CD36, CD45, CD206, CX3CR1, and TREM2.
ARMS2 gene expression (PCR)
Cells (about 1 × 106 from each cell type) with or without stimulation by 400 ng PMA LPS for 24 h or of 10 ml whole blood were harvested and homogenized; total RNA was isolated using the PAX gene blood RNA kit (QiaGen). A 20 ng of isolated RNA was transcribed into cDNA using QuantiTect® Reverse Transcription Kit (QiaGen). cDNA was amplified using Phusion PCR Kit (New England Biolabs), and primers situated in exon 1 (5′TCGGTGGTTCCTGTGTCCTTCATT3′) and exon 2 (5′TCACCTTGCTGCAGTGTGGATGAT3′) of ARMS2 or for amplification of actin (forward 5′ACCAACTGGGACGACAT3′; reverse 5′CTAGAAGCATTTGCGGTG3′). Synthesis was performed by denaturing for 60 s at 96 °C followed by annealing for 30 s at 69 °C (ARMS2) or 60 °C (actin) and synthesis for 60 s (ARMS2) or 120 s (actin) at 72 °C for 35 cycles. Extension was performed for 480 s at 72 °C. Amplified PCR products were separated in agarose and visualized under UV light. Bands were excised, purified, and sequenced on an automated DNA sequencer (ABI/1130x, Applied Biosystems). Sequences were aligned to ARMS2 genomic sequences (NM_001099667).
Expression and purification of recombinant ARMS2
ARMS2 was recombinant expressed using the Pichia pastoris expression system. The expression vector pPICZB contained codon usage optimized full length cDNA of the ARMS2 gene coupled to a myc and 6 × histidine coding tag for purification (Life Technologies) (Additional file 1: Figure S1). P. pastoris cells (strain X33) were transformed with the recombinant expression vector, and ARMS2 expressing clones were selected via zeocin containing selection medium according to the standard protocols. Expressed His-tagged proteins were purified by Ni2+-chelate affinity chromatography from the cells [18, 19]. Cell lyses were performed in binding buffer (10 mM Na2HPO4, 10 mM NaH2PO4, 10 mM imidazol, 1 M NaCl, 2% (v/v) Nonidet P-40, 20% (v/v) glycerol; pH 7.0) together with glass beads (Roth) by FastPrep®-24 (MP Biomedicals) at 4 m/s for 60 s. Protease activity was inhibited by addition of complete EDTA-free (Roche). Protein was eluted from HisTrap™ HP column (GE Healthcare) using elution buffer (10 mM Na2HPO4, 10 mM NaH2PO4, 500 mM imidazol, 500 mM NaCl; pH 7.0). Purified ARMS2 was concentrated in storage buffer (10 mM Na2HPO4, 10 mM NaH2PO4, 500 mM NaCl; pH 7.0). In addition to the recombinant protein, three ARMS2 peptides were generated. Peptide 1 included amino acids 1 to 40, peptide 2 amino acids 41 to 70, and peptide 3 amino acids 71 to 107 (Jerini Peptide Biotechnologies). Polyclonal ARMS2 antiserum was generated by immunization of rabbits with the here described recombinant ARMS2 protein expressed in P. pastoris (Davids Biotechnologies). Generated ARMS2 antiserum (ARMS2Jena) was purified with HiTrap Protein A HP 1 ml column (GE Healthcare) and the major epitope of these antibodies determined by pepspot analysis. Peptides of ARMS2 (33 peptides of 13 amino acids with three amino acids overlap) were spotted to a membrane (Jerini Peptide Technologies), incubated with the antiserum and secondary anti-rabbit antibodies for detection. Polyclonal rabbit antiserum generated to the N-terminus of ARMS2 (ARMS2com) was purchased from CovaLab. In addition, a mouse monoclonal antibody was generated to the C-terminal peptide 3 of ARMS2 (aa 71–107 = IHTELCLPAFFSPAGTQRRFQQPQHHLTLSIIHTAAR) (ARMS2mAb) by standard methods.
Recombinant ARMS2 was separated by SDS-PAGE under reducing conditions (50 mM TRIS-HCL, 1.6% (w/v) SDS, 7% (v/v) glycerol, 8 M UREA, 4% (v/v) β-mercaptoethanol, 0.016% (w/v) bromophenol blue; pH 6.8) and visualized either by silver staining or immune-blotting using ARMS2com, ARMS2Jena antiserum, or mouse α-Penta-His monoclonal antibodies (QiaGen). Mass of whole deglycosylated recombinant ARMS2 was determined by mass spectrometry. To remove the carbohydrate side chains from recombinant ARMS2 (10 μg), the protein was incubated with 0.5 units of PNGase (Roche) for 3 h at 37 °C or the purification tag was cleaved off by enterokinase (New England Biolabs) overnight at RT and repurified with a HisTrap™ HP column.
For immunoprecipitation, monoclonal α-ARMS2 antibody (20 μg) was loaded onto Protein G Magnetic beads (New England BioLabs) for 1 h at 4 °C in binding buffer (20 mM sodium phosphate buffer, pH 7.0). THP-1 cells (1 × 107) were lysed in 1 ml lysis buffer (150 mM NaCl, 1% NP-40, 25 nM Tris-HCl, 1 mM EDTA, 5% glycerol) containing 1 mM PMSF, centrifuged at 16,000 g for 10 min at 4 °C, added to the antibody-loaded beads and incubated on a rotating shaker overnight at 4 °C. After removal of the supernatant, the beads were washed and proteins were eluted from the beads in 30 μl of elution buffer (0.1 M glycine, pH 2.7). The eluted proteins were evaluated for the presence of ARMS2 by SDS-PAGE and mass spectrometry. To show endogenous ARMS2 expression, monocytes were isolated from fresh blood and about 1 × 106 cells were stressed by addition of H2O2 (0.001–1 mM) for 1 h. Supernatants were replaced by cell culture medium, and cells were incubated for 20 h. Cells were lysed in buffer (50 mM TRIS-HCL, 1.6% (w/v) SDS, 7% (v/v) glycerol, 8 M UREA, 4% (v/v) β-mercaptoethanol, 0.016% (w/v) bromophenol blue; pH 6.8), centrifuged and supernatants were separated by SDS PAGE, blotted to a membrane and ARMS2 detected with αARMS2Jena.
Recombinant ARMS2 or immunoprecipitated ARMS2 from THP-1 cells were separated by SDS-PAGE and stained with coomassie blue. Protein bands were excised from the gel and washed repeatedly in water and 50 mM NH4HCO3/acetonitrile 1 + 1 (v/v) for 15 min. Gel pieces in acetonitrile were dried by vacuum centrifugation. Gel pieces were soaked in 50 mM NH4HCO3 with 10 mM DTT for 45 min at 56 °C followed by alkylation (50 mM iodoacetamide in 50 mM NH4HCO3 for 30 min in the dark). Upon washing in 50 mM NH4HCO3/acetonitrile, gel pieces were incubated in trypsin digestion buffer (20 ng/μl trypsin in 25 mM NH4HCO3) overnight at 37 °C. The peptides were extracted from the gel plugs by incubation in acetonitrile/trifluoroacetic acid 1 + 1 (v/v) for 30 min at RT. Extracted peptides (1 μl) were premixed with the same volume of the matrix α-CHCA solution and spotted on a matrix-assisted laser desorption ionization (MALDI) plate. Mass was analyzed using MALDI TOF-mass spectrometer (UltrafleXtreme, Bruker Daltonics, Germany) in the reflector mode with appropriate m/z range and laser intensity. For analysis of the whole ARMS2 protein, the recombinant protein was deglycosylated and untagged as described above. Analysis of the protein, the instrument was utilized in linear mode. Data analysis was performed with current NCBInr database using Mascot search with a peptide mass tolerance of 100 ppm. The analysis was repeated three times.
Detection of endogenous ARMS2 by LSM microscopy
For detection of endogenous ARMS2 protein, monocytes of seven ARMS2 genotyped individuals (two (I/I), one (I/II), two (II/II) one (III/III)) were isolated from PBMCs derived from 20 to 40 ml human blood samples. Blood samples were diluted 1:1 in PBS and overlaid with 15 ml Ficoll. After centrifugation at 1600 rpm for 20 min, PBMCs were collected and washed in 10 ml PBS. Monocytes were isolated from PBMCs using a Percoll gradient (3 ml of PBMCs were overlaid by 6 ml of 54% Percoll) centrifuged as above, harvested, and washed in ice-cold PBS. Cells (1 × 106) were transferred into a 4-well slide chamber (Lab-Tek®) and stained for laser scanning microscopy as previously described . Briefly, isolated monocytes or THP-1 cells in chamber slides were fixed with paraformaldehyde (3% at RT for 15 min) permeabilized in Triton X-100 (0.3%) (Roth) 5 min on ice, blocked with FcR blocking reagent (Miltenyi) 10 min at 4 °C and stained with polyclonal rabbit ARMS2 antiserum (αARMS2Jena) (1:200), monoclonal ARMS2 antibodies (αARMS2mAb) (1:200) followed by incubation with Alexa-647 labeled anti-rabbit or anti-mouse (1:400) for 1 h at RT. DNA was stained with DAPI (10 μg/ml) for 15 min at RT. After washing, the samples were examined by LSM (LSM 510, Carl Zeiss, Jena).
CHO-K1 cells (~1 × 105) were incubated with 10 μg ARMS2 and properdin, either alone or together. After 1 h, cells were washed and placed on a chamber slide (Nunc). After 1 h, adherent cells were fixed with 3% paraformaldehyde for 15 min. CHO surface-bound ARMS2 was visualized using rabbit ARMS2 antiserum (1:100) and Alexa-488 conjugated secondary antibody (1:200; Life Technologies) in assay buffer (1% BSA in RPMI media). Properdin was detected by mouse monoclonal properdin antibody (1:100; QuiDel) and secondary Alexa-647 conjugated antibody (1:200; Life Technologies) in assay buffer. DAPI (Sigma) was used to stain DNA. Images were taken by LSM 710 Meta (Zeiss) and co-localization was analyzed with Zen 2009 software (Zeiss). All incubation steps were done in RPMI media at RT.
Eyes were soaked in phosphate buffered saline (PBS) containing 4% formaldehyde followed by punching of a tissue strip ranging from the optic nerve to the fovea/parafovea which was then processed for paraffin embedding. Sections of 12 μm thickness derived from 2 genotyped individuals (genotype (I/I) and (II/II)) were obtained and subjected to immunohistochemical staining. After de-paraffinization by a series of graded ethanol slides were placed in a 97 °C hot water bath in antigen retrieval buffer (0.05 M Tris buffered saline, 0.05% Tween-20, pH 9.0) for 20 min. Slides were then washed with 1% goat serum in 1xPBS-T (PBS with 0.4% Triton-X) for 10 min for two times. To prevent unspecific binding, sections were covered with blocking buffer (5% goat serum in 1xPBS-T) in a humidified chamber and incubated for 30 min at room temperature. Primary antibodies included anti-ARMS2 (1:200, rabbit polyclonal), anti-CD68 (1:200, monoclonal rat anti-mouse, AbDSerotec), and anti-CD68 (1:200, monoclonal mouse anti-human, Dako). Primary antibodies were incubated overnight at 4 °C. After the two washing steps with 1% serum PBS-T, sections were incubated with labeled secondary antibodies coupled to Alexa-488 (green) or Alexa-594 (red) (Jackson ImmunoResearch, West Grove, PA, USA). Retinal nuclei were counter-stained with DAPI and mounted in DAKO fluorescent mounting medium (Dako Deutschland GmbH, Hamburg, Germany) and analyzed on an Axioskop 2 MOT plus Apotome microscope (Zeiss, Jena, Germany).
ARMS2 binding to complement proteins
C3, C3b, C3c, C3d, iC3b, factor B, properdin (all Complement Technologies) and human serum albumin (HSA) (Nutritional Biochemicals) (each 500 ng in PBS) were coated onto MediSorp microtiter plates (Nunc) over night at 4 °C. Wells were blocked with buffer I (AppliChem) for 2 h at RT and incubated with 500 ng ARMS2 in assay buffer (2% (w/v) BSA, 0.05% (v/v) Tween-20 in PBS) for 1 h at RT. ARMS2 was detected with ARMS2 antiserum diluted 1:1000 and a secondary HRP conjugated antibody 1:2000 in CrossDown buffer (AppliChem) at 492 nm.
Properdin binding to ARMS2
Recombinant ARMS2 or HSA (each 500 ng in PBS) was coated onto MaxiSorp microtiter plates (Nunc) over night at 4 °C. Wells were then washed with PBS + 0.05% (v/v) Tween-20 and blocking was performed with blocking buffer (5% (w/v) BSA, 0.05% (v/v) Tween-20 in PBS) for 2 h at RT. After another washing step, wells were incubated with increasing amounts of properdin (12.5–100 nM) in assay buffer (2% (w/v) BSA, 0.05% (v/v) Tween-20 in PBS). After incubation for 1 h at RT, wells were washed and bound properdin was detected with a goat properdin antiserum diluted 1:2000 and a secondary horse radish peroxidate (HRP)–conjugated antibody diluted 1:2000 in assay buffer. Multiscan Ascent ELISA Reader measured absorption at 492 nm. To determine the interaction domain in ARMS2 responsible for properdin binding to ARMS2, peptide 1, peptide 2, and peptide 3 (each 500 nM) were coated to the microtiter plate, incubated with 2 μM properdin (TECOmedical GmbH) in PBS and binding of properdin was detected as described before. Primary monoclonal properdin antibody (1:1000)  and secondary anti mouse 1:2000 (Dako, Denmark) were used for detection.
The binding affinity of properdin to ARMS2 was measured using biolayer interferometry on a BLITZ system (Forte Bio). His-tagged ARMS2 was loaded onto Ni2+ NTA biosensors (Forte Bio) in assay buffer (PBS (Lonza) with 0.1% (m/v) gelatin). Loaded biosensors were blocked using assay buffer with 10 μg/ml biocytin (Sigma-Aldrich) for 60 s. After washing, the biosensors were dipped in 4-μl properdin solution with concentrations varying from 70 to 1800 nM properdin. The k
D was determined by fitting data to a 1:1 model algorithm with the BLITZ software.
Recruitment of C3b by ARMS2 bound properdin
Properdin (100 nM) was bound to immobilized recombinant ARMS2 as described before. Wells were washed with assay buffer (2% (w/v) BSA, 0.05% (v/v) Tween-20 in PBS) and incubated with increasing amounts of C3b (4–12 nM) in assay buffer for 1 h at RT. Unbound C3b was removed by additional wash steps, and binding was detected at 492 nm by mouse C3d monoclonal antibody (1:2000, Quidel) and secondary HRP-conjugated antibody (1:2000) in assay buffer.
ARMS2 (500 nM) was incubated with 20% NHS in Mg2+EGTA buffer (1% (w/v) BSA, 20 mM HEPES, 144 mM NaCl, 7 mM MgCl2, 10 mM EGTA, pH 7.4) for 15 min at 37 °C. The generated Ba product was analyzed at 540 nm using a Ba MicroVUE ELISA Kit (QuiDel).
All fluoresence-activated cell sorting (FACS) analyses were performed with a LSR II flow cytometer (BD Science); 10,000 cells were evaluated and data were calculated using FlowJo Software (Tree Star).
CHO-K1, pgsD-677, pgsA-745 (each 1 × 106), native RPE (1 × 105), human erythrocytes (each 1 × 107) as well as isolated naive T cells (1 × 106) were incubated with increasing amounts of ARMS2 (10–500 nM) in assay buffer (1% (w/v) BSA in PBS) for 1 h on ice. ARMS2 binding was detected with rabbit ARMS2 antiserum (1:200) and an Alexa-647 conjugated antibody (Life Technologies) (1:400). Similarly, factor H (10–500 nM, Comptech) was bound to CHO-K1 cells.
Heparan sulfate competition assay
Recombinant ARMS2 (500 nM) was incubated with 1 × 107 heparin coated beads (GE Healthcare) and increasing amounts of heparan sulfate (0–12 mM, Sigma) in assay buffer (1% (w/v) BSA in PBS) for 1 h on ice. ARMS2 binding was detected with rabbit ARMS2 antiserum 1:200 and Alexa-647 conjugated antibodies (Life Technologies) 1:400. Factor H (500 nM, Comptech) was incubated with the beads and detected with factor H antiserum (Comptech).
Complement activation assays were performed as previously described . CHO-K1 and pgsA-745 (each 1 × 106) were incubated in 20% NHS, or heat-inactivated 20% NHS (hiNHS) (30 min at 56 °C) forms healthy human donors in Mg2+EGTA buffer (1% (w/v) BSA, 20 mM HEPES, 144 mM NaCl, 7 mM MgCl2, 10 mM EGTA, pH 7.4) for 45 min at 37 °C. C3b deposition was determined by flow cytometry using mouse α-C3b monoclonal antibody (FitzGerald) diluted 1:100 and secondary Alexa-647 conjugated antibodies (Life Technologies) diluted 1:200 in Mg2+EGTA buffer for 1 h on ice. Deposition was normalized and NHS alone defined as 100%. Influence of ARMS2, factor H (CompTech), or HSA (Nutritional Biochemicals) on C3b deposition was evaluated, after preincubation of either cells (1 h on ice) or serum (15 min at 37 °C) with increasing amounts of each protein (100 to 750 nM).
CHO-K1 cells (1 × 106) were preincubated with 500 nM ARMS2 for 1 h on ice. Washed cells were incubated either in 20% NHS diluted with Mg2+EGTA buffer (1% (w/v) BSA, 20 mM HEPES, 144 mM NaCl, 7 mM MgCl2, 10 mM EGTA, pH 7.4) or EDTA buffer (1% (w/v) BSA, 20 mM HEPES, 144 mM NaCl, 10 mM EDTA, pH 7.4) for 45 min at 37 °C or with 100 nM purified properdin (CompTech) in PBS containing 1% BSA for 1 h at RT. Properdin deposition was analyzed by flow cytometry using a properdin monoclonal antibody (QuiDel) (1:200) followed by an Alexa-647 conjugated antibody (Life Technologies) (1:400).
Significant differences between two groups were analyzed using the Student’s two-tailed t test. Values of *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant.