Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans

Abstract

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a Celegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Keywords: C. elegans; E. coli; molybdenum cofactor; sulfite oxidase.

Figure 1.

C. elegans acquires Moco from dietary E. coli. (A) C. elegans Moco biosynthesis pathway (red) and orthologous enzymes in E. coli (black) are displayed. Moco and its biosynthetic intermediates are displayed (purple): GTP is guanosine triphosphate (1), cPMP is cyclic pyranopterin monophosphate (2), MPT is molybdopterin (3), MPT-AMP is MPT-adenine monophosphate (4), Moco is the molybdenum cofactor (5), Moco (SO) is the sulfite oxidase form of the molybdenum cofactor (6), and Moco (XO) is the xanthine oxidase form of the molybdenum cofactor (7). C. elegans Moco sulfurase (MOCS-1) has no clear homolog in E. coli, although xdhC is the likely functional analog (Neumann et al. 2007). (B) Wild-type and moc-1(ok366) C. elegans were synchronized at the L1 stage and cultured on mixtures of wild-type E. coli (synthesizes Moco) and ΔmoaA E. coli (cannot synthesize Moco) for 72 h. The Y-axis shows animal length (µm), where 1000 µm roughly corresponds to fertile adulthood and 250 µm roughly corresponds to the L1 stage. Average and standard deviation are displayed for each condition analyzed. Sample size (n) was 10 individual animals assayed for each condition.

Figure 2.

C. elegans uses Moco from diverse Moco-containing proteins. (A) moc-1(ok366) mutant C. elegans were synchronized at the L1 stage and cultured for 72 h on ΔmoaA E. coli supplemented with Moco bound to Escherichia coli YiiM (EcYiiM), Volvox carteri Moco carrier protein (VcMCP), Neurospora crassa nitrate reductase (NcNR), or bovine xanthine oxidase (BtXO), or equivalent amounts of VcMCP purified from bacteria that cannot synthesize Moco (−Moco). EcYiiM, VcMCP, and NcNR (+Moco) each contained 7.7 nmol of Moco, while BtXO (+Moco) contained 8.8 nmol of Moco. Box plots display the median, upper, and lower quartiles, while whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment. (B) moc-1(ok366) mutant C. elegans were synchronized at the L1 stage and cultured on ΔmoaA E. coli supplemented with variable amounts of Moco bound to EcYiiM (0.0077, 0.077, 0.77, 1.93, 3.85, or 7.7 nmol of Moco, blue), VcMCP, (0.0077, 0.077, 0.77, 1.93, 3.85, 7.7, or 15.8 nmol of Moco, orange) or BtXO (0.018, 0.18, 1.8, 4.38, 8.75, or 17.5 nmol of Moco, brown). For each experiment, animals were allowed to develop for 72 h and animal lengths were determined. Mean and standard deviation are displayed for each data point. Sample size (n) was 10 individuals assayed for each data point. (C,D) Representative images of moc-1(ok366) C. elegans cultured for 72 h on ΔmoaA E. coli supplemented with 7.7nmol of Moco bound to VcMCP (C) or equivalent amounts of apo-VcMCP (−Moco) (D). Blue arrowhead indicates a fertile adult, while yellow arrowheads denote animals arrested early in larval development. Scale bar, 250 µm.

Figure 3.

Protein-bound Moco is directly ingested by C. elegans. (A) Experimental protocol used to generate “diet A” and “diet B” in B. ΔmoaA mutant E. coli were cultured overnight at 37°C in 500 µL of LB supplemented with 39 nmol of VcMCP-bound Moco. Bacterial cells were then concentrated, and the supernatant was removed for use in “diet A.” Bacterial cells were washed, resuspended in LB, and seeded onto NGM to be fed to moc-1(ok366) C. elegans (“diet B”). The supernatant from this culture (spent LB + VcMCP media) was filtered (0.20-µm filter, Corning) to remove remaining bacterial cells and used to resuspend a separate concentrated culture of ΔmoaA mutant E. coli that was grown only in LB. This was then seeded onto NGM to be fed to moc-1(ok366) C. elegans (“diet A”). (B) moc-1(ok366) mutant C. elegans were synchronized at the L1 stage and cultured on wild-type E. coli, ΔmoaA E. coli, “diet A,” or “diet B” (see A for diet A and B descriptions). For each experiment, animals were allowed to develop for 48 h. Box plots display the median, upper, and lower quartiles while whiskers indicate minimum and maximum data points. Sample size (n) was 10 individuals assayed for each experiment.

Figure 4.

Stability of protein-bound Moco. (A) Moco occupancies for purified EcYiiM, VcMCP, NcNR, and BtXO were determined by measurements of the Moco derivative form A and protein concentration. Moco occupancy is the percentage of protein molecules that are bound by a Moco prosthetic group. Moco occupancy was determined for VcMCP purified from Moco-producing (+Moco) and Moco-deficient (−Moco) E. coli. The sample size (n) is displayed for each protein and each data point is individually presented with the mean and standard deviation. (B) Protein gel demonstrating the purity of VcMCP purified from Moco-deficient E. coli (1), VcMCP (2), EcYiiM (3), and NcNR purified from Moco-producing E. coli (4), and BtXO purified from bovine milk (5). The gel displays all protein using the TGX stain-free system (Bio-Rad). (C–F) The amount of stable Moco retained by VcMCP (C), EcYiiM (D), NcNR (E), and BtXO (F) was determined over 96 h at 4°C (black) or 22°C (red). Moco retention of VcMCP was also assessed at 37°C (pink). The Y-axis displays the Moco retention as a percentage of the original Moco occupancies (time 0) presented in A. The sample size (n) is three to six replicates per protein and time point. The mean and standard deviation are displayed.