A proteolytic method for evaluating O-GlcNAcylation on proteins of similar molecular weight to antibody heavy chain after immunoprecipitation

Abstract

Investigating a protein of interest that runs at the same molecular weight as antibody heavy chain is a frequent deterrent to its evaluation by immunoprecipitation. Methods of minimizing the detection of the immunoprecipitating antibody are available. However, these still present a barrier to evaluating if intracellular proteins are modified by the O-GlcNAc post-translation protein modification due to interfering glycosylation on antibodies. IdeZ protease specifically cleaves antibody at the hinge region, allowing collapse of the antibody fragments to 25 kDa after denaturation. Thus, this proteolytic method uniquely allows evaluation of O-GlcNAcylation of proteins of interest formerly obscured by antibody heavy chain.

Graphical Abstract

In immunoprecipitation (IP) reactions, an antibody against the protein of interest is added to the cell lysate in order to enrich the protein of interest from the thousands of other proteins in a cell. In this way, the presence of post-translational protein modifications or protein-protein interactions may be observed or compared between different treatments. Of course, when the protein of interest is precipitated, the antibody is precipitated along with it. After denaturation of the protein-antibody complex, the antibody heavy and light chains form prominent bands on the Western blot at ~50 and ~25 kDa respectively. Thus, a common impediment to performing immunoprecipitation is an inability to see proteins of interest that run in the 40–60 kDa molecular weight range due to strong signal from the 50 kDa antibody heavy chain running at a similar molecular weight on Western blot. Initial attempts aimed at working around these limitations included simply using different species of antibody for immunoprecipitation and the primary antibody during the Western blot to reduce cross-reactivity. Others did not add denaturing reagents in order to maintain the antibody molecular weight at 150 kDa. However, antibody bands are frequently seen even with use of different species and lack of denaturation often leads to incomplete release of the protein of interest, leading to muddied and inaccurate results. This led to the development of more sophisticated reagents, including light or heavy chain specific antibodies labeled with enzymatic (i.e. horseradish peroxidase (HRP)) or fluorescent tags [1], HRP-conjugated reagent capable of binding non-denatured primary antibody only [2], or HRP-conjugated Protein A or G beads [3]. These methods do allow visualization of proteins of interest and protein-protein interactions by binding differentiating aspects of the primary antibodies while excluding the denatured heavy and light chain used for immunoprecipitation. However, the heavy chain remains present and its detection is simply minimized. Because of this, none of these methods also allow for detection of post-translation glycosylation on the protein of interest, since antibodies are also heavily glycosylated [4]. Thus, determining if a protein of interest is glycosylated by immunoprecipitation is still problematic if the protein runs at the same molecular weight of antibody bands. Ideally, one could visualize a protein of interest, any interacting proteins, and determine its glycosylation status with a single immunoprecipitation method. Here we describe a novel method of proteolytically digesting immunoprecipitating antibody to 25 kDa fragments in a single, quick step, allowing visualization of proteins of interest—and importantly—their post-translational protein glycosylation modifications, which were previously unable to be visualized using current immunoprecipitation techniques.

While protein glycosylation is traditionally thought to occur on secreted proteins, glycosylation of intracellular proteins occurs ubiquitously in the form of O-linked N- acetyl-glucosamine (O-GlcNAc) [5]. O-GlcNAcylation is the addition of a single N- acetyl-glucosamine moiety on serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins [6]. Thousands of intracellular proteins are modified by O- GlcNAc, which can affect protein stability, localization, and activity [6]. Of note, the two enzymes regulating the addition and removal of the modification (O-GlcNAc transferase and O-GlcNAcase respectively) are often found associated with transcription factors, which are frequently O-GlcNAcylated [7, 8]. Unfortunately, many transcription factors, including nuclear receptor, forkhead, and GATA family transcription factors, are around 50 kDa [9]. This creates a unique challenge in investigating O-GlcNAcylation of many transcription factors by immunoprecipitation. Our proteolytic method of cleaving antibody heavy chain solves the conundrum of evaluating the O-GlcNAcylation status of proteins of interest that previously have been inscrutable.

Our method utilizes the specificity of IdeZ protease, an IgG endopeptidase, isolated from the bacteria Streptococcus equi, subspecies zooepidermicus [10]. IdeZ protease cleaves specifically at the hinge region of IgG, creating a F(ab’)₂ and Fc fragment [11], which collapses to protein fragments of 25 kDa when further exposed to denaturing agents (Fig. 1A). As evidence of the high specificity of the IgG endopeptidases, a homologous IgG endopeptidase, IdeS, has been successfully transfused into human patients to cleave antibodies generated against transplanted organs [12]. Thus, we utilized this high specificity to cleave the precipitating antibody in our IP samples, leaving cellular proteins intact and allowing assessment of O-GlcNAcylation of a protein running at a similar molecular weight to heavy chain (Fig. 1B). Cell lysate preparation and immunoprecipitation procedures are previously described [13]. Briefly, naive CD4+ T cells were isolated by flow cytometry from spleens of 12–20 week-old wild type C57BL/6 mice (Jackson Laboratories). T cells were then incubated with or without 10 μM Thiamet-G (TMG, a highly selective inhibitor of the enzyme that removes O-GlcNac [14]) for six hours and then activated on anti-CD3 and anti-CD28 coated plates in the presence of cytokines (IL-6 and TGFp) to drive differentiation towards the T helper 17 (Th17) lineage. After 4 days of culture in IMDM, Th17 cells were harvested and cell lysate extracted with 20 mM Tris, pH 7.4, 150 mM NaCl, 40 mM GlcNAc, 2 mM EDTA, 1 mM DTT, 1% Nonidet P-40 lysis buffer with protease inhibitors. One μg of anti-RORYt primary antibody was added to 500–1,000 μg of the cell lysate from Th17 cells treated with (+) and without (−) TMG for the IP samples. One μg of non-specific rabbit IgG antibody was also added to an equivalent amount of a 1:1 ratio of TMG treated and untreated cell lysate to rule out non-specific antibody binding. Two extra control samples containing only lysate buffer spiked with 1 μg of primary antibody to eventually be treated with and without addition of IdeZ protease were also included. Treatment of the primary antibody with and without IdeZ protease serves as assurance that the digestion of the antibody occurs to completion in the digested samples. Samples were rotated overnight at 4°C, then rotated with anti-Fc receptor binding beads for two hours at 4°C, and finally washed vigorously. Per the manufacturer’s instructions (Promega), the optimal buffer for IdeZ protease activity is 50 mM sodium phosphate, pH 6.6, 150 mM NaCl. Thus, after washing IP samples in high salt buffer sufficient to remove non-specific protein binding, the washing buffer should be transitioned to a neutral pH buffer with minimal salt. In our case, we washed with RIPA buffer containing 1M NaCl for three washes and then two washes in PBS. Five units of IdeZ protease (Promega) were then added to all samples, except the undigested primary antibody control, and resuspended in PBS to a total volume of 20 μΕ After an incubation of 30 minutes at 37°C, 5 μL of 5X Laemmli buffer was added and the samples were boiled for 2 minutes to fully denature the IP samples. Samples were then loaded onto SDS-PAGE gels along with 40 μg of cell lysate inputsand developed for Western blot analysis as previously described [13].

Fig. 1.

IdeZ protease cleaves specifically at the hinge region, allowing for collapse of heavy chain from 50 kDa to 25 kDa and visualization of proteins of interest that run at a similar molecular weight. A. IdeZ protease cleaves antibody at the hinge region, resulting in generation of F(ab’)2 and Fc fragments. With disruption of disulfide bridges by denaturing buffer, the bands collapse to 25 kDa. B. Schematic of modified immunoprecipitation procedure with addition of IdeZ protease incubation step of 30 min. An additional control of primary antibody without IdeZ protease addition provides reassurance that the antibody is fully digested in samples of interest. The unfilled rectangle represents the protein of interest (POI), and hexagons represent glycosylation present on both the antibody and protein of interest. The black rectangles represent antibody components (heavy and light chain).

While developing this method, we evaluated a transcription factor of interest to us, retinoic acid receptor-related orphan receptor gamma t variant (RORYt). RORYt is the master transcription factor that dictates the differentiation of CD4+ T cells towards a T helper 17 (Th17) lineage [15]. Th17 cells are pro-inflammatory CD4+ T cells, which aid in the eradication of fungal and bacterial infections [16]. However, these inflammatory cells are also implicated in autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis [17, 18], as well as the pathogenesis of chronic inflammatory diseases such type 2 diabetes and obesity [19, 20]. We observed that CD4+ T cells treated with Thiamet-G (TMG), a highly selective inhibitor of the enzyme that removes the O-GlcNAc modification (O-GlcNAcase) [14], secreted more of the pro-inflammatory cytokine, interleukin-17 (IL-17), made by Th17 cells [13]. We then determined by chromatin immunoprecipitation that TMG treatment also led to more RORYt remaining bound to the il17 gene promoter consistent with increased IL-17 secretion. Since O- GlcNAc regulates many transcription factors, we hypothesized RORYt is O- GlcNAcylated, and direct O-GlcNAcylation of RORYt is a mechanism for increased RORYt retention at the il17 gene promoter. Unfortunately, RORYt’s molecular weight is 57 kDa and thus obscured by heavy chain. Using our novel proteolytic method, we were successfully able to visualize RORYt (anti-RORYt, Abcam), without interference of the heavy chain band and determine that it is not O-GlcNAcylated in CD4+ T cells differentiated towards the Th17 lineage ex vivo (Figure 2). Thus, we determined direct O-GlcNAcylation of RORYt is an unlikely mechanism for its retention at the il17 gene promoter.

Fig. 2.

Heavy chain is completely cleaved, revealing that RORγt is not O− GlcNAcylated. A. OGlcNAc levels are appropriately increased in the input sample treated with TMG (+) compared to un-treated sample (− ). However, no O-GlcNAc signal is detected at 57 kDa where RORγt runs. Importantly, while heavy chain is seen in the undigested primary antibody sample (1◦), it is absent in the primary antibody sample treated with IdeZ protease and there is a concomitant increase in protein running in the 25 kDa light chain region due to heavy chain’s cleavage by the IdeZ protease. A nonspecific isotype matched antibody (IgG) was included to rule out non-specific immunoprecipitation of RORγt. B. RORγt is successfully and specifically immunoprecip-itated and present at its predicted molecular weight of 57 kDa. Heavy chain remains present in the undigested primary antibody sample and runs slightly below RORγt but again is absent in the digested primary antibody only sample.

Using IdeZ protease to digest heavy chain represents a significant improvement to current techniques to mask heavy chain bands on Western blot. It is the only method that allows for evaluation of the presence of a protein of interest, protein-protein interactions, as well as modification by O-GlcNAc. In addition to cellular lysates, this technique could be used on tissue samples with high endogenous antibody binding being evaluated by Western blot (spleen, lymph nodes, organs in autoimmune disease models, etc.). Potentially, the presence of the IdeZ protease could obscure proteins of interest, since a faint band often appears on the Western blot (Figure 2). Formulations of IdeZ with a His-tag (New England Biolabs) are available, which would allow for its removal by running samples over a nickel column if desired. The high specificity of IdeZ protease is ideal for alleviating concerns of off-target digestion of proteins in the cellular lysate, but also limits the types of antibodies it can cleave. All human, humanized, rabbit, sheep, monkey, and Fc-fusion chimeric IgG antibodies can be digested by IdeZ protease. However, other immunoglobulin subtypes (i.e. IgM, IgA), mouse IgG1, rat, porcine, bovine, and goat IgG are not able to be cleaved. IdeZ has reduced activity against mouse IgG2a and IgG3, which may require longer incubation times to achieve complete digestion. Thus, careful selection of an appropriate immunoprecipitating antibody is essential for successful evaluation of a protein of interest using this method.

In this technical note, we have demonstrated a proteolytic method allowing for the evaluation of O-GlcNAcylation of proteins of interest that are typically obscured by heavy chain during immunoprecipitation. To our knowledge, this is the only modified immunoprecipitation procedure that allows for evaluation of the O-GlcNAcylation status of these proteins as well as the presence of a protein of interest and any interacting proteins. Potentially, this method will allow more rapid identification of transcription factors and other proteins regulated by O-GlcNAcylation, advancing our understanding of this unique modification’s effect on cellular processes.

Highlights.

• Adding IdeZ protease to immunoprecipitated proteins cleaves antibody heavy chain

• Formerly obscured ~50 kDa proteins and presence of O-GlcNAcylation can be observed

• Conveniently, only the addition of a single, short incubation step is required