Focus: Luminescent Assay to Track β-Galactosidase Activity

Using the Beta-Glo^® Assay System to Demonstrate Transport of β-Galactosidase Across the Blood-Brain Barrier

Here we review an article in which the Beta-Glo^® Assay System was used to track the delivery of a 116kDa protein, β-galactosidase, across the Blood-Brain Barrier.

From the article: Zhang, Y. and Pardridge, W.M. (2005) Delivery of β-Galactosidase to Mouse Brain via the Blood-Brain Barrier Transferrin Receptor. J. Pharmacol. Exp. Ther. 313, 1075–81.

Introduction

Lysosomal storage disorders result from the inability of lysosomes in cells to degrade unwanted cellular or extracellular substances. These unwanted or “waste” molecules are degraded in the lysosome through the action of a variety of acid hydrolases. Some lysosomal disorders result from defects in the processes through which the acid hydrolases are targeted to the lysosome, while others arise from distinct mutations that result in the inactivity of a particular acid hydrolase. For instance, Tay Sachs disease arises when individuals are homozygous for a mutation that eliminates β-hexosaminidase A activity in the lysosome. Tay Sachs disease affects a step in the degradation of gangliosides, thereby adversely affecting central nervous system function through the accumulation of gangliosides in brain tissue. The neurological effects of many lysosomal storage disorders arising from a defective acid hydrolase cannot be treated because the therapeutic enzymes are too big to cross the Blood-Brain Barrier (BBB).

Delivery of Molecules Across the Blood-Brain Barrier using a Trojan Horse

One way of delivering large lipid insoluble molecules, such as proteins, across the BBB involves “tricking” the protein gate keepers (transporters) of the cell membrane. These membrane transporters selectively allow proteins and other molecules to cross the membranes of the endothelial cells that form the BBB. One such transporter is the transferrin receptor. The authors of this study use a peptidomimetic monoclonal antibody (mAb) against the transferrin receptor to deliver β-galactosidase across the BBB. Peptidomimetic antibodies bind to an epitope on the receptor protein that is distinct from the normal ligand-binding site for the receptor. When the antibody binds, it stimulates receptor-mediated transport of the antibody. β-galactosidase is attached to the mAb using the strong streptavidin-biotin interaction, creating a antibody-streptavidin conjugate. Such conjugates have successfully delivered proteins of up to 40kDa across the BBB.

However, the enzymes disrupted in lysosomal storage disorders range in size from 50 to 100kDa, much larger than the 40kDa proteins delivered across the BBB to date. In this work, the authors design a system to deliver the 116kDa protein, β-galactosidase, across the BBB using the transferrin receptor and mAb-streptavidin conjugate.

Creating the Antibody-β-Galactosidase Conjugate

The authors used the rat monoclonal antibody (mAb 8D3) previously generated against mouse transferrin receptor (Tfr). The mAb was purified by affinity chromatography, and the streptavidin (SA) conjugate of the mAb was created using thiolated 8D3 and S-SMPB activated SA as described in Zhang and Pardridge (2005).

To create the biotinylated β-galactosidase, recombinant β-galactosidase was mixed with sulfo-NCH-LC-LC-biotin in the presence of sodium bicarbonate. The enzymatic activity of the biotinylated β-galactosidase was determined either spectrophotometrically or using the Beta-Glo^® Assay System (Cat.# E4720). The procedure yielded a ratio of approximately 1 to 1.5 biotin moieties per molecule of β-galactosidase.

After creating the monobiotinylated β-galactosidase and the 8D3-SA conjugate, the two molecules were mixed at a 1:1 molar ratio and incubated at room temperature to generate the β-galactosidase-8D3 conjugate. Enzyme activity assays indicated no loss of β-galactosidase activity in the final conjugate.

Delivering β-Galactosidase to the Brain

Adult female BALB/c mice were injected in the jugular vein with either β-galactosidase or the β-galactosidase-8D3 conjugate. Two treatment regimens were followed: a high- dose treatment using 150µg/mouse of biotinylated β-galactosidase conjugated to 300µg/mouse of 8D3-SA or 150µg/mouse unconjugated β-galactosidase, or a low-dose treatment using 15µg/mouse biotinylated β-galactosidase conjugated to 30µg/mouse 8D3-SA or 15µg/mouse unconjugated β-galactosidase. Mice were sacrificed at 1 or 4 hours after injection.

Measuring β-Galactosidase Activity in Tissue Homogenates Using a Luminescent Assay

Because of interference of absorbance readings from tissue pigments, the Beta-Glo^® luminescent activity assay was used to measure β-galactosidase activity from tissue samples using the following protocol.

1. Tissue was extracted with a working dilution of 5X Reporter Lysis Buffer (Cat.# E3971) at a ratio of 2ml buffer to 0.5g of tissue according to manufacturer instructions and then homogenized using a Brinkman Polytron PT3000.
2. The homogenate was centrifuged for 10 minutes at 12,000 × g, and the supernatant was retained.
3. Beta-Glo^® Substrate was reconstituted in Beta-Glo^® Assay Buffer, and the resulting Beta-Glo^® Reagent was mixed at a 1:1 ratio with the supernatant (pH 7.6).
4. The mixture was incubated in the dark at room temperature for 1 hour.
5. Luminescence (relative light units) was measured, and relative light units were converted to milliunits of enzyme activity based on a β-galactosidase standard curve.
6. Protein content of organ extract was determined, and organ enzyme activity was measured as milliunits per mg of protein, milliunits per gram of organ weight, or percent of injected does per gram of organ weight.

Histochemical staining of brain sections did not clearly reveal β-galactosidase activity in brain parenchyma because the parenchymal β-galatosidase activity was less than the minimum that the authors determined necessary for colorimetric detection. To address the question of whether the β-galactosidase completely crossed the BBB into the brain parenchyma, the authors used a capillary depletion technique to separate the vascular endothelium from the brain parenchyma by centrifugation. The luminescent Beta-Glo^® Assay was used to detect β-galactosidase activity in the fractions.

Figure 1. High-dose injection study. Percent of injected dose (ID) per gram of tissue is shown for mouse liver, spleen, kidney, heart and brain (inset) at 60 minutes after an I.V. injection of a high dose (150μg/mouse) of β-galactosidase in either the unconjugated form (dark bars) or as a conjugate with the 8D3 TfRmAb (open bars). Data are mean ± S.E. (n = 3). The ID per gram of organ was computed from the specific activity of the injected enzyme or enzyme-conjugate (milliunits per microgram) and the ID of enzyme (micrograms). The endogenous β-galactosidase activity (Table 1) was subtracted for each organ. Figure used with permission from W.M. Pardridge and the American Society for Pharmacology and Experimental Therapeutics (http:\\www.jpet.aspetjournals.org) from Zhang and Pardridge (2005) J. Amer. Soc. Pharmacol. Exp. Ther. 313, 1075–81.

Results

The Beta-Glo^® Assay demonstrates delivery of β-galactosidase across the BBB into brain parenchyma. Figure 1 shows the percent of injected dose for mouse liver, spleen, kindney, heart and brain 1 hour after the high-dose injections into mice. There is a tenfold increase in uptake of β-galactosidase in mice injected with the β-galactosidase-8D3 compared to mice injected with unconjugated β-galactosidase. The capillary depletion study followed by luminescent detection of β-galactosidase activity demonstrated the presence of the enzyme in the brain parenchyma, which was not detectable using colorimetric methods (Table 1). β-galactosidase activity was higher in the homogenate and postvascular supernatant than in the vascular pellet.

Conclusion

The luminescent Beta-Glo^® Assay was able to detect β-galactosidase activity in brain parenchyma not detectable by colorimetric assay, demonstrating the successful delivery of a large (116kDa) enzyme across the BBB. These data indicate that use of receptor mediated transport may provide a viable mechanism for delivery of larger, lipid-nonsoluble molecules to brain tissue without invasive techniques, expanding the range of molecules and compounds that might serve as therapeutics for neurological disorders.