Gill GS and Schultz MC (2022), Multienzyme activity profiling for evaluation o...

XB-ART-59458

FASEB Bioadv 2022 Nov 17;411:709-723. doi: 10.1096/fba.2022-00073.

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Multienzyme activity profiling for evaluation of cell-to-cell variability of metabolic state.

Gill GS, Schultz MC.

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In solid organs, cells of the same "type" can vary in their molecular phenotype. The basis of this state variation is being revealed by characterizing cell features including the expression pattern of mRNAs and the internal distribution of proteins. Here, the variability of metabolic state between cells is probed by enzyme activity profiling. We study individual cells of types that can be identified during the post-mitotic phase of oogenesis in Xenopus laevis. Whole-cell homogenates of isolated oocytes are used for kinetic analysis of enzymes, with a focus on the initial reaction rate. For each oocyte type studied, the activity signatures of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase 1 (MDH1) vary more between the homogenates of single oocytes than between repeat samplings of control homogenates. Unexpectedly, the activity signatures of GAPDH and MDH1 strongly co-vary between oocytes of each type and change in strength of correlation during oogenesis. Therefore, variability of the kinetic behavior of these housekeeping enzymes between "identical" cells is physiologically programmed. Based on these findings, we propose that single-cell profiling of enzyme kinetics will improve understanding of how metabolic state heterogeneity is related to heterogeneity revealed by omics methods including proteomics, epigenomics, and metabolomics.

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	FIGURE 1. Experimental approach for analysis of enzyme activity in single oocytes. (A) Reactions catalyzed by cytosolic GAPDH and MDH1. Enzyme concentrations in the oocyte nucleus and cytoplasm are from Kirli et al. (B) GAPDH (35,698 kDa) and MDH1 (36,425 kDa) are abundant in the oocyte. Isolated stage VI oocytes were dissected to remove the nucleus and therefore obtain samples of whole nuclei (Nuc) and whole cytoplasms (Cyto) for Western blotting analysis. As expected, both enzymes are present in the nucleus and cytoplasm. 17 , 18 Sample amount 1 = content of 1 nucleus or cytoplasm. *cross‐reacting bands likely are processing products of vitellogenin, which oocytes take up by endocytosis. The GAPDH lanes are from a single blot. (C) Representative progress curves for GAPDH (left) and MDH1 (right) activity in homogenate of a single‐stage VI oocyte. The pullouts show NADH synthesis (GAPDH) and consumption (MDH1) during the first 5 min of the reactions; these data were obtained in triplicate and the slopes of the linear trendlines averaged to obtain the initial reaction rate for the pool of an enzyme in a whole cell. This estimate is referred to as cell v 0. The A340 readings for MDH1 progress curves are brought into the negative range by subtraction of the blanks. (D) Homogenates of six stage VI oocytes. Samples of these turbid homogenates were diluted for enzyme assays.
	FIGURE 2. Variability of GAPDH and MDH1 cell v 0 (whole‐cell initial rate) in individual stage VI oocytes. (A) Two sets of 18 oocytes were analyzed, each from a different animal. Each oocyte homogenate was assayed separately for GAPDH and MDH1 activity to obtain cell v 0 as the change in absorbance at 340 nm with time (ΔA340/min). The cell v 0 data for GAPDH and MDH1 are shown in the left and middle panels, respectively. Technical reproducibility (right panel) was assessed by assaying enzyme activity in 18 aliquots of a control homogenate prepared from multiple oocytes. RSD, relative standard deviation (shaded in gray). (B) Relationship between variability of GAPDH and MDH1 cell v 0 in individual stage VI oocytes of animal 2 of this study. (C) Relationship between variability of GAPDH and MDH1 cell v 0 in individual stage VI oocytes of animal 3 of this study. In panels B and C, dot colors for individual cells were randomly assigned by Excel.
	FIGURE 3. Enzyme expression level in individual cytoplasms and relationship of cell v 0 to cell volume. (A) Compartment markers NASP (Nuclear Autoantigenic Sperm Protein, nucleus) and EEF2.1 (Eukaryotic Translation Elongation Factor 2.1, cytoplasm) have the expected expression level in the cytoplasm. Six oocytes (late stage V) were dissected to obtain near‐native nuclei and cytoplasms. The low‐speed supernatants of individual cytoplasms (cyto S7500) were then analyzed by LC‐MS to obtain their proteomes. Protein abundance was estimated by spectral counting (% of total ∑# PSMs) and ranked according to these estimates (%rank). (B) Relative expression of GAPDH and MDH1 in isolated cytoplasms (GAPDH, red dots; MDH1 blue dots). Fluctuation of GAPDH abundance is matched by fluctuation of MDH1 abundance. TKTL2, a pentose phosphate pathway enzyme, does not co‐vary with either GAPDH or MDH1. For example, TKTL2 expression is lower than GAPDH and MDH1 in cytoplasm 1, but higher than both in cytoplasm 3. The expression level of translation factor EEF2.1 is remarkably similar in the cytoplasms analyzed. (C) Relationship between the abundance of GAPDH and MDH1 (% of total ∑# PSMs) in isolated cytoplasms. (D) Relationship between cell v 0 and cell volume for 18 stage VI oocytes (animal 3). Each dot is an individual cell. Equations for the lines of best fit in 3C and D are presented in Table S2A,B.
	FIGURE 4. Variability of GAPDH and MDH1 cell v 0 in individual oocytes of three types. (A) cell v 0 was determined for GAPDH (red) and MDH1 (blue) in individual stage II, IV, and VI oocytes from two animals (top two panels; each box and whisker plot shows the data for 18 cells). v 0 for GAPDH and MDH1 was also determined for control ensemble homogenates of the same oocyte types (technical replicates in bottom panel). Variability as relative standard deviation (RSD shaded in gray) is higher for each set of 18 individual oocytes (top panels) than for variability of activity associated with repeat sampling of the corresponding (stage‐matched) technical replicate homogenates. For each type of oocyte, the variability of enzyme activity between individual cells was higher than the variability between replicate samples from the corresponding control homogenate. For example, the RSD for GAPDH in the 18 stage II cells of animal 1 was 27.7% (top panel, left‐most plot); the RSD for the corresponding technical replicate control was 1.57% (bottom panel, left‐most plot). (B) Plots of the relative standard deviation (RSD) associated with assay of GAPDH and MDH1 activity in individual cells (cellRSD, left two panels) and in repeat samples of technical replicate homogenates (trRSD, right panel). This is a representation of the RSD data in A.
	FIGURE 5. Covariation of GAPDH and MDH1 cell v 0 for oocytes at three stages of development. The data for individual cells from two animals are shown in the left and middle columns. For the technical replicate controls (plots at right), eighteen aliquots of an ensemble homogenate of the indicated oocyte type were subsampled for separate assays of MDH1 and GAPDH activity. The highest variability of cell v 0 was for GAPDH in stage II cells (R 2 = 0.9312). For the corresponding control homogenate R 2 = 0.0003. Equations for the lines of best fit are provided in Table S2C.
	FIGURE 6. Relationship of cell volume to GAPDH and MDH1 cell v 0 for oocytes at three stages of development. The data for GAPDH (red) and MDH1 (blue) are shown in the left and right panels, respectively. When considered as a temporal series, the data are consistent with developmental regulation of the strength of the relationship between cell volume and cell v 0. The pattern is similar for GAPDH and MDH1: a robust increase of correlation strength from stage II to stage IV, followed by a stronger decline from stage IV to VI (compare top, middle, and bottom plots for each enzyme). Equations for the lines of best fit are presented in Table S2D. Note that the R 2 values shown here for stage VI oocytes of animal 1 approach those determined for stage VI oocytes of animal 3 (Figure 3D).
	FIGURE 7. Profiling of MDH1 cellKm and cellVmax from titrations of OAA into homogenates of two stage VI oocytes. (A) Representative set of progress curves at increasing concentrations of OAA (cell 8). The expected dependence of NADH consumption on OAA concentration is readily apparent. (B) Michaelis‐Menten plot of data from OAA titration curves for the same amount of homogenate of cells 8 and 16. Km and Vmax were estimated using nonlinear regression for data fitting.

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