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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 2  |  Page : 78-83

Pan-specific antibodies as novel tools to detect valyllysine

1 Department of Reproductive Biology, NHC Key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
2 College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China

Date of Submission26-Apr-2020
Date of Decision13-May-2020
Date of Acceptance20-May-2020
Date of Web Publication26-Jun-2020

Correspondence Address:
Hua Diao
NHC Key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), School of Basic Medical Sciences, Fudan University, 2140 Xietu Road, Shanghai 200032
Zhi-Yu Shao
College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 North Renmin Road, Shanghai 201620
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.288018

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Objective: Amino acyl modification of lysine residues is an essential mechanism of nutrient sensing that regulates various biological functions including reproduction. At present, the lack of pan-specific antibodies for a recently identified lysine valylation hinders the characterization and detection of this modification. The objective of this study is to raise pan-specific antibodies that may facilitate the identification of novel expression patterns of lysine valylation.
Methods: Chicken ovalbumin was valylated as an immunogen to raise polyclonal antibodies (PcAbs) in rabbits. The population of the pan-specific antibodies recognizing valylated lysine was purified using the chemically synthesized valylated peptides consisting of random amino acids. The specificity of the antibodies was evaluated using ELISA, dot blots, Western blots, and immunohistochemistry (IHC) staining in human epididymis as well.
Results: A preliminary and simple strategy to make an anti-valylated lysine PcAb was developed. The recognition of the antibodies to valyllysine was evaluated as pan specific. This was useful for the detection of the newly identified valyl modification using ELISA, dot blots, and Western blots. The antibodies were also successfully utilized in IHC assays, which revealed novel valyllysine modification patterns in epididymis tissues of human.
Conclusions: A new antibody tool was provided for the study of lysine valylation. The novel expression patterns of valyllysine in the epididymis suggest that this modification may be involved in sperm maturation.

Keywords: Anti-Valyllysine Antibody; Epididymis; Posttranslational Modifications; Valyllysine

How to cite this article:
Wang YL, Liu MY, Li YH, Yang YT, Wang WW, Yu HG, Shao ZY, Diao H. Pan-specific antibodies as novel tools to detect valyllysine. Reprod Dev Med 2020;4:78-83

How to cite this URL:
Wang YL, Liu MY, Li YH, Yang YT, Wang WW, Yu HG, Shao ZY, Diao H. Pan-specific antibodies as novel tools to detect valyllysine. Reprod Dev Med [serial online] 2020 [cited 2020 Jul 8];4:78-83. Available from: http://www.repdevmed.org/text.asp?2020/4/2/78/288018

  Introduction Top

The increasing infertility rate in China is a growing public and economic concern, as 15%-20% of the population is affected,[1]with at least half of these cases due to infertility in males. Therefore, it is necessary to develop a better understanding of how to improve the reproductive health of males to ensure new population growth and health of the newborn population. Nutrition, metabolic molecules, and metabolic alterations are suggested to influence or indicate reproductive functions.[2],[3] Appropriate nutritional modifications and a healthy diet can improve semen quality and the natural conception rate of infertile couples.[4] Although nutrition and metabolism play a vital role in infertile patients, the mechanisms underlying the relationship between nutrition, energy metabolism, and reproductive function are poorly understood.

Glucose, fatty acids, and amino acids are three major nutrients that, through their sensors and respective signaling pathways, regulate the hypothalamus–pituitary–gonadal axis, resulting in direct and indirect effects on gamete production and health.[4] In animals, of the three metabolic pathways, amino acid metabolism potentially plays the most important role in gamete production, as adding essential amino acids or methionine under dietary restriction (DR) has been shown to increase fecundity to levels similar to those of full feeding. However, other nutrients, such as lipids or carbohydrates, had little or no effect in fecundity levels under DR.[5] Similarly, other studies have shown that nutrient supplementation with amino acids improves sperm quality and subsequently increases fertilization capacity and the number of live piglets in boars.[6] However, little work has investigated the molecular mechanisms of amino acid sensing in the male reproduction, although amino acid levels are important for diagnosis and treatment of male infertility.

In mammals, the target of rapamycin (TOR) is believed to act as an amino acid sensor to regulate various cellular processes. Particular amino acids, including leucine and glutamine, are essential for mTORC1 activity, via regulation on Rag GTPase, hVps34, and MAP4K3 proteins.[7],[8] Evidence suggests that branched-chain amino acids (BCAAs), comprised leucine, isoleucine, and valine, are critical nutrient signals that regulate cell growth, the cell cycle, cell survival, and autophagy, among other physiological functions.[9],[10] However, the molecular mechanisms of amino acid sensing, particularly for BCAAs, which coupling amino acid sufficiency and mTOR signal, are not fully understood. Only leucine (a BCAA) is known to regulate mTORC1 activity via leucylation on K142 of RagA and K203 of RagB.[11]

Recently, Zhao et al. reported a novel mechanism for how intracellular amino acid signals are sensed and transmitted.[12] At physiologic levels, all common l-amino acids can be sensed by aminoacyl-tRNA synthetases (ARSs). This promotes the formation of aminoacyl lysine (K-AAs) on the ε-amine of lysine (K) residues of a variety of substrates through production of reactive aminoacyl adenylates. K-AA is a novel posttranslational modification (PTM), similar to acetylation that can be removed by deacetylases, such as SIRT1 and SIRT3, via the same mechanisms as deacetylation. Although the molecular mechanisms of amino acid sensing via ARSs and dynamic regulation of K-AAs are thought to be universal, the sensing of BCAAs, besides leucine, and the modifications that they perform on substrates, have not been well characterized, especially in the male reproductive system.

As an important member of BCAAs, valine is postulated to be sensed and the signal is transmitted, in a novel form of PTMs on lysine valylation. Similar to leucylation, valylation may represent an evolutionarily-conserved modification, either as a histone PTM, or as a nonhistone protein PTM, playing key roles in the regulation of diverse cellular physiology. And the postulated mechanism can be explored with anti-valyllysine antibodies. In order to develop a better understanding of BCAA sensing, in the male reproductive system, we choose to examine the novel modification on lysine: valyllysine, identified by Zhao et al.[12] Currently, the lack of useful antibodies is a major limitation, as pan-specific antibodies are essential tools for the identification and characterization of valyllysine, for use in dot blots, Western blots, or IHC. As no published methods for convenient characterization of valyllysine have been developed, we sought to establish methods to make antigens, generate anti-valyllysine antibodies, and confirmed the use of pan-specific antibodies in detection of valylated proteins.

  Methods Top

Synthesized peptides

Peptide libraries with and without lysine valylation [Supplementary Table 1] were synthesized by GL Biochem (Shanghai, China). A cysteine residue (Cys) was added to the peptide N terminus to facilitate conjugation with carrier proteins (Bovine Serum Albumin, BSA). This conjugation reaction was performed in 0.2% glutaraldehyde solution.

Preparation of antigen

FmocVal-OH (104 mg) (Aladdin, Shanghai, China) was dissolved in 65 mL of 0.2 mol/L PB (pH 6.8) in a 100 mL beaker. 6.9 mL of 104 mmol/L EDC (Thermo Scientific, Waltham, MA, USA) was added to the beaker, followed by 7.75 mL of 174 mmol/L NHS (Thermo Scientific, Waltham, MA, USA), and incubated for 30 min in the dark, at room temperature. Next, Ovalbumin (OVA) (Sigma-Aldrich, St. Louis, MO, USA) (20 mg) in 20 mL of 0.2 mol/L PB (pH 6.8) was added to the activated FmocVal solution in a dropwise manner. The reaction mixture was stirred at room temperature overnight. The modified protein (crude product FmocVal-OVA) was purified via dialysis or ultrafiltration, the buffer was replaced with deionized water. The protein solution was collected and lyophilized under a vacuum. After complete solvent removal, in order to release of Fmoc groups, the powder was dissolved in 30 mL of 20% piperidine in DMF solution, sonicated, and incubated for 20 min at room temperature. The piperidine and other small molecules were removed using 15 mL of 10 K Da centrifugal filters with 150 mL deionized water. The concentration of valylated OVA was determined using a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) and further analyzed by SDS-PAGE. Valylated BSA was generated following the same protocol, and used to evaluate pan-specific anti-valyllysine specificity of the antibodies.

Generation of pan-specific anti-valyllysine polyclonal antibodies

Reagents for immunization of the rabbit, including complete Freund's adjuvant and incomplete Freund's adjuvant, were purchased from Sigma-Aldrich. New Zealand rabbits (female; 3 kg body weight) were purchased from Sippr-BK Laboratory Animal Co. Ltd. (Shanghai, China). All animal experiments were performed according to the animal care protocols approved by the institution.

Immunization of the rabbits and polyclonal antibody (PcAb) production were performed as described previously.[13] Two New Zealand rabbits were immunized with valylated OVA, and after four doses of immunization, the antiserum was collected. The antiserum with high ELISA titer was used for enriching anti-valyllysine antibody. The pan anti-K-Val antibody was enriched using Sepharose 4B beads (GE Healthcare Life Sciences, Chicago, IL, USA) conjugated with BSA-P1 (val). The sera were centrifuged at 10,000 ×g to remove sedimentation. About 10 mL of sera were incubated for 3 h with 2 mL of the BSA-P1 (val)-conjugated Sepharose 4B beads in a column. The beads were then sequentially washed with 40 mL of 0.1 mol/L Tris-HCl (pH 7.6, containing 0.5 mol/L NaCl) and 2 mL of 0.5 mol/L NaCl. The antibodies bound to the beads were eluted with 0.1 mol/L glycine (pH 2.5) and immediately neutralized with 0.1 mol/L NaHCO3(pH 8.3, in 30% glycerol). The antibodies were dialyzed in cold PBS overnight. Both the dot-blot assay and Western blotting were performed to assess the quality of the purified antibodies.

ELISA and dot-blot assays

An indirect ELISA was used to characterize the specificity of the PcAbs. Microtiter plates were coated with 1 mg/liter antigen dilutions (valylated OVA, BSA, valylated BSA, BSA-P1 conjugate, BSA-P1 (Val), BSA-P2, and BSA-P2 (Val). Primary antibodies derived from antiserum were serially diluted; the secondary antibody used was HRP-conjugated goat anti-rabbit IgG (1:1,000, Sigma-Aldrich, St. Louis, MO, USA). The peroxidase substrate, 3,3', 5, 5'-tetramethylbenzidine (TMB), was used to induce a colored reaction product and was measured at 450 nm using a microtiter plate spectrophotometer.

Synthesized peptides were dotted onto a nitrocellulose membrane and dried. Nonspecific binding was blocked by incubating the membrane in 1% gelatin in TBS for 2 h at 37°C. The nitrocellulose membrane was incubated with primary antibody (1:1,000 dilution for PcAbs) dissolved in 1% gelatin in TBS at 4°C overnight, washed with TBS-Tween 20, and incubated with goat anti-rabbit IgG conjugated with HRP (1:3,000 dilution, Sigma-Aldrich, St. Louis, MO, USA). After washing three times with TBS-Tween 20, the signals were visualized with the ECL reagent and recorded using a Tanon image system (Tanon, Shanghai, China).

Western blots

Ten-week-old mouse testes tissues, from C57/BL6 mice, were lysed in radio-immunoprecipitation assay buffer (50 mmol/L Tris [pH 7.4], 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors. After centrifuging at 4°C for 10 min, the protein extract was separated using SDS-PAGE (4%–20% acrylamide gel) and transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were incubated for 2 h at room temperature in TBS containing 5% nonfat milk powder and then incubated with primary antibody. For anti-valyllysine antibody incubation, polyclonal anti-valyllysine antibody was diluted in 1 × NET overnight at 4°C with or without competitive agent, valylated BSA. After four washes in TBS-Tween 20, the membranes were incubated with goat anti-rabbit IgG-HRP antibody for 1 h at room temperature. ECL reagents (Millipore, Burlington, MA, USA) and a Tanon image system were utilized to detect proteins on the blots.

Immunohistochemistry of epididymis via anti-valyllysine antibody

Immunohistochemistry (IHC) was performed using the indirect enzyme-labeled antibody method, as described previously.[14] Briefly, paraffin sections of the epididymis were deparaffinized with toluene and dehydrated in serially graded ethanol solutions. Antigen retrieval was performed by heating the sections in 10 mmol/L sodium citrate solution (pH 6.0) using a microwave oven for 10 min. Endogenous peroxidases were inactivated with 3% H2O2 and nonspecific adhesion sites were blocked using PBS containing 5% BSA for 30 min at 37°C. Sections were subsequently incubated overnight at 4°C with rabbit polyclonal primary antibodies. After washes in PBS, sections were incubated with secondary antibody HRP-Goat anti-rabbit IgG at 37°C for 1 h. The signal was visualized using 3,3-diaminobenzidine (DAB) (Boster, Wuhan, China) and all sections were counterstained with hematoxylin. As a negative control, normal rabbit IgG was used at the same concentration, instead of using primary antibodies, in every experiment.

  Results Top

The generation of valylated protein antigen

There are currently no protocols to date that describe the development and production of a valylated protein antigen. Therefore, we followed a common chemical reaction typically utilized to modify proteins. The two-step reaction coupled carboxylates to the ε-amino group of lysine and arginine residues on OVA or BSA. OVA and BSA contain 31 and 79 positively charged residues (lysine and arginine), respectively.

Valine is one of the inert side chain amino acids, of which the α-amino group should be protected. Therefore, we used 9-Fluorenylmethyl Chloroformate (Fmoc) to block the amino groups on valine to prevent polymerization of the amino acid, forcing the reaction between carboxylates on the Fmoc-Valine and amines (lysine and arginine) on the carrier, to proceed. Using this method, antigen protein OVA and BSA were modified with activated FmocVal reagents and became unprotected. Valylated proteins have a higher molecular weight as compared with non-modified proteins [Figure 1] in SDS-PAGE analysis. This apparent molecular shift is the same as that estimated by the addition of valyl groups to the ε-amino group of lysine and arginine residues.
Figure 1: SDS-PAGE analysis of the antigens OVA and BSA. Un-valylated and valylated OVA and BSA proteins were resolved in an 8% SDS-PAGE gel and silver stained. M.W. indicates molecular weight of the protein ladders. M: Marker; OVA: Ovalbumin.

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Generation and evaluation of pan-valyllysine polyclonal antibodies

As the valyl group is small (117 Da) and weakly antigenic, we used a chemically valylated carrier protein, Val-OVA, as the immunogen and the valylated lysine peptides as affinity ligand to specifically isolate the anti-K-Val antibody. Another valylated conjugate, BSA-P1 (Val), was utilized to examine the titer of PcAbs. To evaluate the selectivity of PcAbs in the context of flanking amino acid motifs of Lys (Val), we used BSA, OVA (Val), BSA (Val), and BSA-conjugated peptide libraries (BSA-P1, BSA-P1 [Val], BSA-P2, and BSA-P2 [Val]) as coating antigens respectively, in the indirect ELISA assays.

All of the PcAbs recognized valylated BSA rather than BSA [Figure 2]a. These findings demonstrate that there was no cross-reactivity with carrier protein BSA. Similarly, both random peptides BSA-P1 (Val) and designated peptides BSA-P2 (Val) were recognized with high specificity [Figure 2]a. The P1 (Val) peptide library consists of peptides with conserved Lys (Val) and random flanking amino acid residues, whereas the P2 (Val) library peptide has a designated motif, CAGYDVEK (Val) NNSRIK. There were similar binding activities of PcAbs to P1 (Val) and P2 (Val), which confirmed that the conserved Lys (Val) residue, rather than other motifs, was targeted by the antibodies.
Figure 2: Specificity of the polyclonal antibody for N-ϵ-valyllysine. (a) Reactivity of the antibody with bound BSA, valylated OVA, valylated BSA, and BSA-peptide conjugates [Supplementary Table 1] was examined using ELISA. (b) Various concentrations of the synthesized random peptide libraries carrying lysine and modified lysine were dotted on a nitrocellulose membrane and detected using an anti-valylated lysine polyclonal antibody. (c) Western blot analysis of the protein extract of the mouse testis using polyclonal anti-valyllysine antibodies at a 1:4,000 dilution. During incubation of the primary antibodies with the PVDF membranes, 400 μg/mL valylated BSA was added in lane 2, whereas the same concentration of BSA was added in lane 1. GAPDH was used as a loading control.

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As lysine can undergo diverse modifications besides valylation, we also confirmed the selectivity of the antibodies to Lys (Val) using a dot blot. The results of the dot blot confirmed that the PcAbs could selectively recognize peptides of the P1 (Val) library, rather than those of unmodified lysine, leucyllysine, glutaminyllysine, glutamicyllysine, asparaginyllysine, or isoleucyllysine libraries [Figure 2]b. The PcAb recognized minimal P1 (Val) peptide of at least 10 ng/dot [Figure 2]b. These findings show that the PcAb would likely be suitable for Western blot assays. The selectivity of the PcAbs in Western blotting was further examined by addition of either a competitor, protein-valylated BSA, or BSA, during the incubation of the primary antibody with the membranes. Competitive valylated BSA, but not BSA alone, inhibited the recognition of the primary antibodies in the Western blotting assays of mouse testis protein extract [Figure 2]c.

Immunohistochemical utilization of antibodies in profiling valylation in the mammalian epididymis

As it is important to determine the intracellular and intercellular localization of a novel PTM, we used IHC staining to test the efficacy of the anti-K-Val antibody. [Figure 3] shows extensive valyllysine signals in the epithelial cells of the epididymal caput and corpus, and weak one in the epididymal cauda. In the caput [Figure 3]a epididymidis, the positive signals are located in the columnar cells and on the luminal sperm. In the corpus [Figure 3]b region, valyllysine signals are distributed in the columnar cells and myoid cells, while in the cauda, signals are only in the myoid cells [Figure 3]c. A representative staining with normal IgG is shown in [Figure 3]d.
Figure 3: Immunohistochemical staining of valylated proteins in paraffin-embedded sections of human epididymis tissues using anti-valyllysine antibodies. (a) The caput of the epididymis. (b) The corpus of epididymis. (c) The cauda of the epididymis. (d) Represents the negative control in which an adjacent section was incubated with normal rabbit IgG rather than anti-valyllysine antibodies.

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  Discussion Top

Little work has investigated the mechanisms and physiological roles of valine sensing. In the current study, we used pan-specific anti-valyllysine antibodies to identify novel valylated protein and valylation sites. We developed a preliminary method to generate antigen and an anti-valyllysine PcAb, which was effective for use in ELISA, IHC, and immunoblotting of valylated proteins. To our knowledge, this is the first report about the generation of both a valyl protein antigen and pan-specific antibody.

The protocols for both antigen and antibody preparation require improvements to increase yield, as a low titer, approximately 1:32,000, is obtained 8 weeks after rabbit immunization. There are also high levels of batch-to-batch variation in PcAbs, which would lead to difficulties in using antibodies for the enrichment of valylated peptides for LC-MS/MS analysis.

Previous reports have suggested that aminoacyl-tRNA synthetase (ARS) may specifically catalyze lysine valylation (K-Val) formation.[12] However, largescale production of the antigen is not possible, due to the limits of synthetase production. The enzymatic method may lead to better substrate selectivity than chemical methods. However, the enzymatic method also has steric hindrance, resulting in low modification efficiency to OVA/BSA. We chose to use a chemical modification to obtain higher modification efficiency of protein antigens for immunization and improved the selectivity of antibodies by affinity purification with synthesized peptides. Both selectivity and productivity of the present reactions must be increased due to the limited serum titers and production of antibodies.

In the protocol described in this study, our methods were based on the following events: (1) EDC reacts with the carboxyl group of Fmoc-Val-OH and forms an unstable O-acylisourea intermediate; (2) in the presence of a carbodiimide, such as EDC, carboxylates (-COOH) may react to NHS, resulting in a semi-stable NHS ester; and (3) this ester may then react with ε-amine of lysine residues to form amide crosslinks.[15] 9-Fluorenylmethyl Chloroformate (Fmoc) was used to protect the α-amino group and the Fmoc group was removed using 20% piperidine in DMF solution, when the cross-linking reactions were complete. The activation of Fmoc-Val-OH is an essential step in this process, which may determine the modification efficiency and selectivity of the antigen preparation, as well as the final quality of the antibody. Future studies should work to improve the activation of Fmoc-Val-OH in the future.

The IHC assays using the antibody developed in this study to stain epididymis tissue showed intensive valyllysine expression, with region specific patterns. These findings indicate that valine and valylation may be involved in male reproduction. The caput segment is characterized by a narrow luminal diameter, and both the luminal diameter and the sperm concentration increase distally within the corpus and cauda epididymis. The specialized luminal microenvironment of the caput and corpus promotes the sequential maturation of spermatozoa and cauda epididymis, for subsequent storage.[16] We observed that valylated proteins were highly abundant in the caput and corpus of the normal human epididymis, which indicates that valylation of lysine residues, may play a role in sperm maturation and fertility. Although we observed distinct valyllysine patterns, further studies are needed to confirm these findings, as staining may differ due to batch-to-batch variation, the relatively low titer we achieved, and the PcAb. In order to identify the mechanisms of valine sensing and the physiological roles of this process in the male reproductive system, future studies should further optimize pan-specific and site-specific valyllysine antibodies of high quality, which is applicable to protein valylation studies in other tissues and organisms.

To our knowledge, this is the first report that reveals the existence of valyllysine in the male reproductive system, using pan-specific valyllysine antibodies. This pan-specific antibody is useful for determining how valine is sensed and signaled through valyl-tRNA synthetase to regulate reproduction. Functions of valyllysine in other organs or tissues are worth further investigating. Future studies should also extend these methods to investigate other BCAAs.[17]

Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.


We acknowledge Dr. Yonglian Zhang (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, CAS) for providing tissue chips. We thank Dr. Shimin Zhao of Life Sciences and Institutes of Biomedical Sciences of Fudan University for technical help.

Financial support and sponsorship

This work was supported by the Innovation_oriented Science and Technology Gran (No. CX2017_1) from NHC Key Laboratory of Reproduction Regulation.

Conflicts of interest

There are no conflicts of interest.

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