Abstract
Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.
Highlights
Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a lysosomal storage disease (LSD), and is one of the most frequently occurring members of the family of Batten diseases [1,2]
Creating a tripeptidyl-peptidase 1 (TPP1) variant with improved pharmacokinetic properties could provide significant clinical benefits compared to the recombinant WT TPP1 that is currently used in the treatment of LINCL
A TPP1 variant with an increased physiological half-life could potentially provide the basis for a more effective therapy, decreasing the amount of protein required per treatment, and/or an increasing the interval between doses, leading to an improved quality of life for LINCL patients
Summary
Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a lysosomal storage disease (LSD), and is one of the most frequently occurring members of the family of Batten diseases [1,2]. The crystal structure of the human proenzyme reveals a suboptimal catalytic triad geometry with the pro-piece linker partially blocking the substrate binding site. This arrangement is believed to prevent premature activation of the enzyme [7,8,9]. When exposed to pH ≤ 4.0, TPP1 undergoes endoproteolytic cleavage at one of three cleavage sites located along the linker region resulting in an enzymatically active mature protein [7,10,11] This is likely due to a rearrangement of the catalytic triad at low pH. TPP1 has two enzymatic functions: a primary tripeptidyl exopeptidase activity where TPP1 catalyzes the sequential release of tripeptides from the N-terminus of a broad range of substrates [15,16] and a weak endoproteolytic activity [12,17]
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