After synthesis and initial carbohydrate transfer from the dolichol phosphate carrier, endogenous and recombinant a-L-iduronidase undergo the trim backprocessing typical for glycoproteins leaving high-mannose chains. For certain chains on the protein, a special two-step glycosylation reaction attaches the N-acetylglucosamine residue via the phosphate, and then cleaves off the sugar uncovering the phosphate group. Other chains undergo further glycosylation to complex or hybrid structures. In the Golgi, the exposed mannose 6-phosphate residues bind to the small cation-dependent M6PR, which leads to the trafficking of the protein through vesicular transport to deliver the enzyme to primary lysosomes. The enzyme then resides in the lysosome and is relatively resistant to the low pH and proteolytic environment, with a half-life of about 4 to 5 d for recombinant enzyme in Hurler fibroblasts . For overexpressed recombinant a-L-iduronidase, about 50% of the enzyme is secreted into the medium compared with only trace secretion in normal cells.
Once outside the cells, the recombinant a-L-iduronidase with mannose 6-phosphate can be rapidly and efficiently taken up via the larger cation-independent M6PR present on the cell surface of CHO cells or other cell types. For uptake into Hurler fibroblasts, the bis-phosphorylated chains interact with high affinity with the overall half-maximal uptake occurring at ~1 nM. Once bound to the surface of cells, the M6PR endocytoses the enzyme and rapidly recycles to the surface. The process is efficient and rapid and can lead to manyfold normal levels of enzyme in Hurler fibroblasts within an hour of exposure to saturating concentration of enzyme. This uptake constant can be measured by assessing the accumulation of recombinant a-L-iduronidase in extracts of the Hurler cells, after exposing plates of Hurler cells to different concentrations of the enzyme. A plot of enzyme activity in the medium vs. enzyme activity in the extract shows receptor saturation as expected (Figure 12.3). By performing a Michaelis-Menten-type kinetic analysis of the uptake data using a double-reciprocal plot, and calculating the KM from the slope and Vmax (1/Y intercept), a Kuptake value can be determined (Figure 12.3). This value is an accurate estimation of the uptake affinity for the enzyme although it is not technically a true KD since no equilibrium state is reached. This uptake assessment is used as a test of enzyme quality and potency during process development and release testing of the Aldurazyme product for commercial use.
Was this article helpful?