A CatalyCEST MRI Contrast Agent That Detects the Enzyme-Catalyzed Creation of a Covalent Bond
Dina Hingorani, Edward Randtke and Mark Pagel
I am a sucker for NMR-based bioanalytical assays. These experiments appeal to my desire to move NMR beyond its role in structural biology and into other arenas of biology. Additionally, I have watched these CEST methods with a passing interest, but I have never had a chance to sink my teeth into this kind of work.
In this JACs communication Hingorani et al. describe a MRI contrast agent designed to detect catalysis by the enzyme transglutaminase (TGase). The authors call this technique CataylCEST. Here is a rough sketch of how it works. The authors design a paramagnetic tag with a moiety that looks like the substrate to an enzyme. (In this case the substrate is lysine and the enzyme is TGase). Then the enzyme covalently attached the paramagnetic tag to a second substrate (in this case a protein or peptide via glutamine residues). Once the tag is attached, protons (and 13C, 15N, etc) in the neighborhood to it broaden and resonate at dramatically different frequencies due to hyperfine contact shift. Let's assume that some of these protons are in slow exchange with the water. If you saturate these spins using a presat-type experiment, then during the saturation pulse, some of this saturation is transferred to the water by chemical exchange and the integral of the water signal will decrease relative to a control. Figure 1A of the paper (below) describes this experiment:
The specific enzyme the author's query, TGase, catalyzes covalent bond formation between side chains of glutamine (Q) and lysine (K). TGase forms cross-links in the extracellular matrix in tissues and in cancer. The authors do not discuss their motivation for monitoring TGase in the introduction and only touch on it in the conclusion. I can only surmise that their long-term goal is to develop in vivo CEST MRI with exogenous CEST agents that do not suffer from rapid pharmacokinetic washout.
The results of this study -
The author's synthesize TM-DO3A-cadaverine paramagnetic CEST agent and couple this molecule to 5 different substrates using inexpensive microbial TGase. The substrates are: Boc-Gln-OH, CBZ-protected QG peptide, QR peptide, GQR peptide and bovine serum albumin (BSA). They acquire their NMR data on a 600 MHz NMR at 37 C using (essentially) the presat experiment with a saturation time of 4 s and a field of 20 uT (or (20 uT/14.1 T)*600 MHz = 850 Hz field). The saturation offset is arrayed in 1 ppm steps from -30 to 30 ppm, relative to water, which is set at 0 ppm. The data is presented as a saturation profile showing the intensity of the water signal as a function of the saturation offset. Finally, the authors fit Lorentzian line shapes to assess the chemical shift of the species in slow exchange with water. The authors do not report error bars, which I find troubling, because the peaks are quite broad. As we'll see later, the authors will argue that a difference of 1.8 ppm is significant, but a difference of 1 ppm is not. I am not sure what to make of this difference, myself.
The most convincing result is with BSA. The authors make an NMR sample with 25 mM CEST agent, 0.75 mM BSA and 10 mM glutathione (to maintain the reducing environment required by the eznyme) in pH 7 Tris buffer. Then they react with 0.327 uM TGase for 24 h under aerobic conditions. The authors repeat this experiment in triplicate. Before catalysis, the saturation profile shows CEST at +4.6 ppm upfield from water. After cataysis, the saturation profiles shows a CEST at +4.6 ppm and -9.2 ppm.
If the publication ended right here, I'd be sold. This paper confuses me, though, when the authors report their controls. Their results reinforce a long held suspicion about CEST: how do you assign the peaks in your saturation profiles?
Let's discuss their controls. The most logical control is each of the components individually. The CEST agent alone shows a CEST at -5.2 ppm, BSA alone shows a CEST at +5.6 ppm and glutathione alone shows a CEST at +5.4 ppm. By the way, I'll note that the text says that BSA and glutathione have a CEST at +4.6 ppm, but the figure caption says +5.6 and +5.4 ppm, respectively. You see what I mean about error bars! I guess 1 ppm is not significant. So why isn't the CEST profile of the reaction mixture before catalysis equal to a superposition of the reactants? (I think it probably is, if you correct for concentration, but the authors do not do this for the readers and we are left wondering). Next the authors try control peptides. The authors mix 25 mM CEST agent, 25 mM peptide, 10 mM glutathione in pH 7 Tris buffer with and without TGase. For GQR and QR, there is a CEST at -9.0 ppm before catalysis and CESTs at +5.8 ppm and -10.8 ppm after. (In the discussion, the authors assign the signals at -9 ppm and -10.8 ppm to a supramolecular adduct and paramagnetic amides, respectively, implying that they can tell a difference between signals the differ by 1.8 ppm. See what I mean about error bars!) For CBZ-protected QG, there is no CEST signal before catalysis and CESTs at 4.6 ppm, 9.8 ppm and 22.5 ppm after. Finally for Boc-protected Q there is a CEST at +7.2 ppm before and a broad CEST between -10 ppm and -20 ppm after catalysis. Why do none of the controls show the same signal before catalysis as BSA? (Once again, I presume the issue is concentration, but the authors leave it up to their reader to discern). Also, after covalent attachment of the CEST agent why does the CEST differ depending so dramatically for different substrates?
The author's interpretation of their results falls into two categories (they don't make this explicit, I am interpreting). 1) Aggregation/Heterogeneity - There is a noncovalent supramolecular adduct or some type of conformational heterogeneity that dramatically alters the chemical shift of the water exchangeable protons. This effect is responsible for CESTs at -5.2 for the CEST agent alone, at -9 ppm for GQR and QR peptides prior to TGase catalysis, at +4.6 ppm, +9.8 ppm and +22.5 ppm for the CEST agent-linked ZQG peptide and at -10 to -20 ppm for the CEST agent-linked Boc-Gln-OH. 2) Change in chemical exchange rate contants - The authors assert that chemically modifying the substrate alters the rate constant and rates of chemical exchange. This effect is responsible for the CEST at +5.8 ppm for GQR and QR peptides after TGase reaction.
As you can tell, I am skeptical of these explanation. I am not saying the authors are incorrect. They know a lot more about this subject than I do. I only mean to say that I believe more justification is needed. For example, there are a few additional controls the authors could do to validate their assignments. If they are concerned about "noncovalent supramolecular adducts", why not reduce concentration or increase ionic strength to break up these interactions? If they are concerned about hydrophobicity causing conformational heterogeneity or heterogeneous ligand conformations, then perhaps the peptides they are using are not good controls? If they are concerned about rate constants, why not play with temperature or field?
In the end, this paper DOES convince me of its main objective: the authors have a catalyCEST MRI contrast agent that can be used to detect the formation of a covalent bond in BSA mediated by TGase. After studying this paper, though, I am concerned that the CEST varies from substrate to substrate - from -9.2 ppm for BSA to 22.5 for ZQG. Let me end my critique with a question: If you wanted to check for TGase activity using a new protein and you were not sure if it was a substrate or if you wanted to test TGase activity in vivo, then what results do you expect from the CEST assay? The fact that you do not know shows how far we have to go.