Computational design and experimental characterisation of a stable human heparanase variant

Abstract

A mutant heparanase that exhibits wild type structure and activity but can be heterologously produced in bacterial protein expression systems.

A Universal and Modular Scaffold for Heparanase Activatable Probes and Drugs

“…Compared to our previous probe, HADP , probe 4F exhibits approximately half of the catalytic efficiency ( HADP k cat / K M = 0.11 μM –1 ·min –1 ), one-third of the turnover number ( HADP k cat = 0.75 min –1 ), and approximately twice the affinity for the enzyme ( HADP K M = 7.0 μM). The values determined here between HPSE and HADP (Supplementary Figure S3) are lower than those in our initial report using HADP ; this difference likely reflects the batch-to-batch variability in the activity of expressed HPSE protein used in our previous and current reports, a phenomenon also described by other laboratories . Nevertheless, all compounds in this report were reacted with the same batch of HPSE, meaning HADP here serves as a positive control against which 2F and 4F can be compared.…”

“…The values determined here between HPSE and HADP (Supplementary Figure S3) are lower than those in our initial report using HADP; 20 this difference likely reflects the batch-to-batch variability in the activity of expressed HPSE protein used in our previous and current reports, a phenomenon also described by other laboratories. 11 Nevertheless, all compounds in this report were reacted with the same batch of HPSE, meaning HADP here serves as a positive control against which 2F and 4F can be compared. Considering the kinetic values determined here, as an exemplary fluorogenic probe, 4F demonstrates a robust response to HPSE.…”

“…3,4,10 Since its discovery and the elucidation of its involvement in various pathological conditions, HPSE has met with growing interest and the concomitant development of new tools for its examination. 11 Historically, heparan sulfate polysaccharides were used to develop radioisotope-based 14,15 or fluorescent 12 assays; however these assays inherently suffer from the heterogeneity of the polysaccharides that can be a result of varying sources and manufacturing, 13,14 prohibiting the advancement of targeting heparanase in diagnosis and disease treatment. The development of small-molecule HPSE probes has long been considered the "gold standard" of HPSE detection 13 by providing simple, sensitive, and rapid methods to detect and quantify HPSE activity.…”

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

“…This has recently allowed for the expression of human heparanase (a glucuronidase) from E. coli with identical kinetics, inhibition, structure, and structural/protein dynamics to the wild-type protein . The use of the PROSS-2 server (the protein repair one-stop shop) to improve the expression levels/stability of known enzymes by incorporating evolutionally conserved and functionally neutral mutations could be a game changer in optimizing proteins which exhibit favorable expression levels and stability to make many of these enzymes attractive to industrial applications .…”

“…This has recently allowed for the expression of human heparanase (a glucuronidase) from E. coli with identical kinetics, inhibition, structure, and structural/protein dynamics to the wild-type protein. 148 The use of the PROSS-2 server (the protein repair one-stop shop) to improve the expression levels/stability of known enzymes by incorporating evolutionally conserved and functionally neutral mutations could be a game changer in optimizing proteins which exhibit favorable expression levels and stability to make many of these enzymes attractive to industrial applications. 149 Finally, for the provision of glycan building blocks for new technologies (such as AGA) or traditional chemical building blocks for synthesis, one of the defining bottlenecks is scalable access to such materials; biocatalytic approaches are showing they can innovate here.…”