Ethics statement.
All experiments with Thoroughbred horses were performed according to the standards specified in the guidelines provided by the Animal Care and Use Committee of the Tokyo University of Agriculture and Technology, as well as those in both the Japan Racing Association (JRA, Shiga, Japan) and Shadai Stallion Station (SSS, Hokkaido, Japan) guidelines for sample collection from horses. Blood collection from horses was carried out in accordance with guidelines and regulations of Tokyo University of Agriculture and Technology, JRA, and SSS. All methods are reported in accordance with ARRIVE guidelines. The entire study was approved by the Animal Care and Use Committee of the Tokyo University of Agriculture and Technology (approval nos. 30-104, R02-46, R03-50, and R04-85).
Polymorphism analysis of eHRG.
Blood samples were collected from the jugular vein of 1,700 Thoroughbred horses (936 males, 78 geldings, and 686 females, aged 2–28 years, mean age ± SD = 4.0 ± 2.4) using heparinized tubes during annual laboratory tests for monitoring infectious diseases at the Ritto Training Center of the JRA and SSS from 2019–2022. The samples were stored at 4°C, and leukocytes were collected within a couple of days.
eHRG polymorphisms were analyzed using genomic DNA purified from horse leukocytes. Briefly, blood was centrifuged at 1,500 rpm for 15 min at 4°C, and the plasma and blood cells were separated. Leukocytes isolated from buffy coats were used for DNA analysis, and plasma was stored at –80°C until protein analysis. DNA was extracted from leukocytes in the buffy coat using Maxwell® 16 Blood DNA Purification (Promega, Madison, WI, UAS) or Maxwell®︎ RSC Tissue DNA Kit (Promega), according to the manufacturer’s protocol. Extracted DNA samples were used for polymerase chain reaction (PCR) with GoTaq® Master Mix (Promega) and a pair of primers (Forward: 5′-ACTCTGGTCGGCATGAGCATA-3′, Revers: 5′-TTTGTGTTTATTACTGGTCACATT-3′). The PCR products were separated using 2% agarose gel electrophoresis and visualized with ethidium bromide. Allele frequencies were estimated using the gene-counting method, and Hardy–Weinberg equilibrium was performed using allele frequencies.
Cloning of eHRG.
The eHRG sequence was analyzed using liver samples from Thoroughbred horses obtained from Aizu Chikusan Co., Ltd. (Fukushima, Japan), as described previously. Briefly, total RNA was isolated from liver samples using ISOGEN (Nippon Gene, Tokyo, Japan), and cDNA was synthesized using random hexamer primers and PrimeScript Reverse Transcriptase (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. The cDNA of eHRG was amplified using a pair of primers: 5′-ATAGAAGTCTCTGCTGTGGTACA-3′ (sense primer) and 5′-TTTGTGTTTATTACTGGTCACATT-3′ (antisense primer). The PCR products were inserted into a pGEM-T TA cloning vector (Promegas) followed by PCR reactions using a BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies, Grand Island, NY, USA) with M13-forward, M13-reverse, or sequencing primer 5′-TTGAGGATACCGAGCTCTACA-3′ (sense primer). The PCR products were analyzed using an ABI Avant-3100 sequencer (Life Technologies), according to the manufacturer's instructions.
Sequencing of the deletion site within HRR of eHRG.
Amplified PCR products were separated by 2% agarose gel electrophoresis and each migrated band was cut and extracted. The extracted products were inserted into a pGEM-T TA cloning vector, followed by PCR using the BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies) with the primer 5′-TAGAAGCTCACTCTTGAACGT-3′. The PCR products were analyzed using an ABI Avant-3100 sequencer (Life Technologies), according to the manufacturer's instructions.
Detection of eHRG protein with different genotypes by western blotting.
To study the effect of each genotype on protein production, eHRG in the plasma of each genotype was detected using western blotting. Heparinized Thoroughbred blood was centrifuged at 1,500 rpm for 15 min at 4°C and the collected plasma was analyzed by western blotting as described previously using murine anti-HPRG monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA; G-10) and secondary antibody anti-mouse IgG, HRP-linked secondary antibody (Cell Signaling Technology, Beverly, MA, USA). The primary antibody is a mouse monoclonal antibody, whose epitope is part of the human HRG sequence (25 amino acids in N-terminal domain 1, 96% homology to horse). Positive bands were visualized with an LAS-4000 (Fuji Film, Tokyo, Japan)20. The molecular weight of each band visualized by western blotting was calculated using ImageJ with reference to the technical brief supplied by Bio-Rad Laboratories Inc. (Bio-Rad Laboratories, Hercules, CA, USA).
Preparation of rabbit polyclonal anti-eHRG antibody.
An eHRG-specific antibody was produced in rabbits by immunization with a synthetic 22 amino-acid peptide (PRHSEERGPGKRH) from PRR2 of the eHRG sequence. Rabbit polyclonal anti-eHRG antibody was purified from the serum using antigen affinity purification. Immunoreactivity was confirmed by immunoblotting with purified eHRG20.
Competitive enzyme-linked immunosorbent assay (ELISA).
Plasma eHRG concentrations were determined using an in-house competitive ELISA. Briefly, a 96-well polystyrene plate (Cat. no. 9018, Corning, NY, USA) was precoated with 100 μL of 0.5 μg/mL purified eHRG in phosphate buffered saline (PBS) and incubated overnight at 4°C. After incubation, the plate was washed three times with wash buffer (0.05% Tween-20 in PBS). Nonspecific binding was blocked with 300 μL blocking buffer (1% bovine serum albumin in 0.05% Tween-20 in PBS) for 1 h at room temperature. The blocking buffer was discarded, and each well was washed three times with wash buffer. After washing, 100 μL of 20-fold diluted equine plasma from each genotype (n=20) and 2 μg/mL polyclonal rabbit anti-eHRG antibody was added to each well and incubated for 1 h at room temperature. Purified eHRG (0–100 μg/mL) was used as the standard. The standards and samples were diluted with blocking buffer containing a complete protease inhibitor cocktail (Roche, Basel, Switzerland). Subsequently, the plate was washed with wash buffer, followed by the addition of 100 μL biotinylated anti-rabbit IgG (Cell Signaling Technology, MA, USA) in blocking buffer and incubation for 1 h at room temperature. After incubation, each well was washed thrice with wash buffer, and 100 μL piece streptavidin poly-HRP (Thermo Fisher Scientific) was added and incubated for 30 min at room temperature. Then, the plate was washed with wash buffer followed by the addition of 100 μL TMB Substrate OptEIA (BD, NJ, USA), and color development was stopped by the addition of 100 μL 0.5N H2SO4. The absorbance was measured at 450 nm, and a standard curve was generated following the calculation of plasma eHRG concentration.
HRG purification from equine plasma.
eHRG purification was performed using a previously described method, with slight modifications13,20,21. Briefly, the collected equine plasma was incubated with Ni Sepharose 6 Fast Flow (GE Healthcare, Piscataway, NJ, USA) in the column for 10 min at room temperature. After incubation, the sepharose was washed with 50 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl and 20 mM imidazole and the column was washed with 50 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl and 100 mM imidazole. After the second wash, the binding protein with sepharose was eluted with 50 mM Tris-HCl (pH 7.5) containing 0.5 M NaCl and 300 mM imidazole. The eluted solution was further purified using an Amicon Ultra 0.5 mL 30 kDa centrifugation filter (Millipore, Bedford, MA, USA). The protein yield in plasma and collected samples was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Ni-binding protein was expressed as weight per mg of total plasma protein.
Statistics.
Dunnett’s multiple comparison was performed using Graph Pad Prism 9 Version 9.4.1 for the statistical analysis of the data, and p values < 0.05 were considered statistically significant.