PRINCIPAL INVESTIGATOR: Stephen G. Withers
GRANT TITLE: Engineered sialyltransferases for sialidase-resistant therapeutic glycoproteinsa)
Abstract Most therapeutic proteins in use are glycoproteins, and their circulatory half-lives are
typically dependent on their constituent glycans, especially of terminal sialic acids. Desialylation ofsialylated biopharmaceuticals occurs in vivo, exposing carbohydrates that are recognized by lectinmediated endocytosis, promoting clearance and resulting in shortened circulatory half-life for the drug.
We proposed that glycoproteins with longer circulatory half-lives could be made by replacing theterminal sialic acids with 3-fluorosialic acids as the 3-fluoro substituent dramatically slows rates of both
enzymatic and spontaneous hydrolysis of sialosides due to inductive destabilisation of reaction transition
states.
Introduction of these 3-fluorosialic acids must be done enzymatically. However the fluoro substituent
also slows enzymatic sialyl transfer, making the preparation of these fluorosialylated glycoproteinsimpractically slow. We proposed to speed up enzymatic fluorosialic acid transfer by directed evolution
using our high-throughput FACS entrapment assay (Figure). This assay depends upon the ability of lacpermease (LacY) to transport fluorescent lactose derivatives in and out of E. coli, but cannot transport
sialylated lactose derivatives out. Thus if E coli hosting anactive sialyltransferase plus the enzyme that makes
CMPsialic acid (SiaB) is incubated with BODIPYlactose
plus sialic acid then BODIPYsialyl-lactose accumulates inthe cells. After washing to remove unreacted
BODIPYlactose fluorescent cells can be sorted by FACS
and those bearing the most active sialyltransferases isolated.
This approach should also work with 3-fluorosialic acid.
We synthesised both epimers of 3-fluorosialic acid as well as BODIPYlactose. The GT80
sialyltransferase from Pasteurella multocida (PmST1) was chosen for evolution and a random mutant
library of PmST1 was generated by error-prone PCR and an appropriate mutation rate (2.3 per gene)confirmed by sequencing. Initial FACS screening was promising as we quickly enriched a populationthat could synthesise a sialylated BODIPYlactose. Unfortunately the sugar being transferred was not 3fluorosialic acid but sialic acid present at low levels in LB growth medium. Switching to minimal media
we saw no transfer in the presence or absence of 3-fluorosialic acid (but it worked with sialic acid).
Increasing the expression level of PmST1 did not help, but if we lysed cells and added CMP3Fsialic acidwe could see (some) transfer, suggesting that 3-fluorosialic acid was not getting into the cells. This could
occur if 3-fluorosialic acid is not a substrate for NanT. Alternatively, since NanT synthesis requires
induction, it may be that 3-fluorosialic acid is not an effective inducer. We therefore transformed E coli
with a plasmid containing the gene for NanT, but still did not get uptake of 3-fluorosialic acid. Given
these difficulties we explored whether other modified sialic acids could resist enzymatic andspontaneous hydrolysis. A good candidate was found and fortunately it can be transferred by thesialyltransferase without the need to engineer. Modified sialyl glycoproteins can be made in this way,
and these are now being studied.