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Drifting toward polymer perfection

The addition of polysialic acid to proteins and cells is emerging as a promising therapeutic strategy. Polysialyltransferases synthesize polymers of widely varying lengths not optimal for therapeutic reagents, but the development of enzyme variants using neutral genetic drift offers a new way to overcome this problem.

Karen J Colley

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olysialic acid (polySia) is a sugar polymer synthesized by bacteria and mammals that has great promise for several therapeutic approaches. In particular, polySia enhances the bioavailability of therapeutic proteins1,2, promotes repair of the damaged nervous system3 and serves as a key component of bacterial meningitis vaccines4. The therapeutic utility of polySia has stimulated investigators to explore the ability of bacterial polysialyltransferases (polySTs) such as Neisseria meningitis B polysialyltransferase (polySTNmB) to add polySia to proteins and cell surfaces2,4,5. A limitation of these enzymes is that they are processive and synthesize polySia chains in a wide range of lengths2,4. Processive polymerases have extended acceptor substrate binding sites and continue to engage their acceptors as a chain is polymerized. Thus, their polymer products vary greatly in length (Fig. 1a). In contrast, distributive enzymes have more restricted binding sites and release their acceptors after each round of monomer addition; their polymer products are more uniform in length (Fig. 1b). Processive enzymes are not ideal for therapeutic applications where product uniformity is critical for investigating and optimizing therapeutic efficacy. Keys et al.6 explore this issue by using a neutral drift approach to identify distributive variants of polySTNmB that synthesize more uniform polySia chains. Their work provides a glimpse at the basis for processive and distributive polymerization processes while identifying polySTNmB variants for potential use in therapeutic interventions. The large size and negative charge of polySia allows it to block cell interactions and enhance cell migration and plasticity, especially during development and in cancer7,8. Some neuroinvasive bacteria escape the host immune response because their polySia capsules structurally match mammalian polySia9. Although bacterial and mammalian polySTs synthesize similar polySia chains8, they are very different with respect to substrate recognition. 410

The mammalian polySTs modify a limited set of glycoprotein acceptors10, whereas bacterial polySTs such as polySTNmB are more promiscuous and modify many acceptors2,5. This characteristic of polySTNmB has proved to be advantageous for investigators exploring the use of polySia as a therapeutic molecule. Therapeutic proteins chemically conjugated to bacterial polySia exhibit increased circulating half-lives1. A major drawback of chemical polysialylation is that it is not highly site specific; polySia chains can be randomly

conjugated to lysine side chains throughout the protein. In contrast, polySTs cap existing glycans with polySia chains, resulting in a lower likelihood of blocking active sites and compromising overall protein function. With this in mind, Lindhout et al.2 showed that polySTNmB efficiently polysialylates α1antitrypsin, leading to a marked increase in the circulating half-life of this therapeutic protein with little loss of activity. El Maarouf et al.5 also demonstrated that polySTNmB can synthesize polySia chains on multiple acceptors on the

a CMP-Sia +

Processive enzyme with extended binding site

Polymer products of varying lengths

b CMP-Sia +

Distributive variant with restricted binding site

Polymer products of more uniform lengths

Figure 1 | Processive and distributive polymerization mechanisms. (a) Polymerases with processive mechanisms may have extended acceptor substrate binding interfaces that can bind acceptors of varying length. These enzymes continue binding their acceptors through several rounds of monomer addition. Long polymeric acceptor substrates may have a greater affinity for the binding interface and thus are bound and elongated preferentially, leading to a mixture of short and very long polymer products. (b) Polymerases with distributive mechanisms may have more restricted acceptor binding interfaces that accommodate only the very end of the growing polymer. These enzymes release the acceptor substrate after each monomer addition. Polymer products are more uniform because each acceptor substrate molecule has an equal chance of being bound and elongated. In their work, Keys et al.6 identified variants of polySTNmB containing a K69Q exchange, which seems to restrict the acceptor substrate interface, shifting this enzyme to a more distributive polymerization mechanism and allowing the synthesis of more uniform polySia chain products that are preferable for therapeutic applications. Cytidine monophosphate–sialic acid (CMP-Sia) is the nucleotide sugar donor for the polyST. nature chemical biology | VOL 10 | JUNE 2014 | www.nature.com/naturechemicalbiology

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surface of cells in culture as well as in vivo when injected into mouse brain. These findings suggest that targeted application of polySTNmB could replace the forced expression of mammalian polySTs in strategies aimed at enhancing nervous system repair3,5. To generate distributive polySTNmB variants optimal for producing polySia-based therapeutics, Keys et al.6 subjected this enzyme to three rounds of mutagenesis and identified variants that demonstrated neutral genetic drift in that they retained their basic activity—the biosynthesis of polySia—but exhibited altered polymer product profiles with low, medium and high dispersity. Remarkably, they found that the desired low-dispersity variants that synthesized products within a narrow length distribution all carried a K69Q mutation. In contrast, mediumand high-dispersity variants that synthesized products with a broader length distribution retained a lysine at position 69, as found in the wild-type enzyme. Saturating mutagenesis of a thermally stable variant with the K69Q mutation showed that reversion of position 69 back to the wild-type residue (Q69K) led to a nondistributive enzyme, whereas a variant with a Q69D exchange had a slightly more distributive product profile than the original K69Q variant. These results identified amino acid 69 as the key residue, or ‘molecular switch’, that determines the elongation mechanism and product distribution of polySTNmB. Using the identified polySTNmB variants, the investigators also tested predictions about the acceptor binding pockets of processive and

distributive enzymes. They observe that a more processive variant (lysine in position 69) shows increasing affinity for acceptor substrates as chain length increases, consistent with a more extended binding pocket. The more distributive variant (glutamine at position 69) showed no such preference, consistent with a smaller binding pocket that recognizes only the very end of the acceptor polymer. Finally, five additional mutations were identified in Lys69 variants that, when introduced individually into the wild-type enzyme, either increased or decreased polymer dispersity. Notably, the majority of these were changes in basic residues in a region predicted to bind acceptor substrates. These observations support the notion that polySTNmB has an extended, basic acceptor binding pocket and that changes in the composition of this region can influence polySia product length and dispersity. The implications of this work for basic and applied glycoscience are considerable. The new array of polySTNmB variants can be used to dissect its processive and distributive polymerase mechanisms and to generate therapeutically optimal polySia-coated proteins and cells. In principle, neutral genetic drift can be used to create variants of other enzymes that synthesize the capsular polysaccharides of pathogenic bacteria to understand how capsular polysaccharide structure, length and modification affect bacterial virulence in vivo and in turn inform investigators how best to approach the creation of new vaccines in vitro4,9.

Likewise, the polymerization mechanisms of mammalian polySTs can be probed using this approach, and variant enzymes can be used to gain a greater understanding of how polySia chain length affects both protein and cell function in normal processes, in disease and during repair7,8,10. ■ Karen J. Colley is at the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, USA. e-mail: [email protected] Published online 13 April 2014 doi:10.1038/nchembio.1506 References

1. Gregoriadis, G., Jain, S., Papaioannou, I. & Laing, P. Int. J. Pharm. 300, 125–130 (2005). 2. Lindhout, T. et al. Proc. Natl. Acad. Sci. USA 108, 7397–7402 (2011). 3. El Maarouf, A. & Rutishauser, U. Adv. Exp. Med. Biol. 663, 137–147 (2010). 4. McCarthy, P.C. et al. Glycoconj. J. 30, 857–870 (2013). 5. El Maarouf, A. et al. J. Biol. Chem. 287, 32770–32779 (2012). 6. Keys, T.G. et al. Nat. Chem. Biol. doi:10.1038/nchembio.1501 (13 April 2014). 7. Rutishauser, U. Nat. Rev. Neurosci. 9, 26–35 (2008). 8. Falconer, R.A., Errington, R.J., Shnyder, S.D., Smith, P.J. & Patterson, L.H. Curr. Cancer Drug Targets 12, 925–939 (2012). 9. Cress, B.F. et al. FEMS Microbiol. Rev. doi:10.1111/ 1574-6976.12056 (19 December 2013). 10. Mühlenhoff, M., Rollenhagen, M. & Werneburg, S., Gerardy-Schahn, R. & Hildebrandt, H. Neurochem. Res. 38, 1134–1143 (2013).

Competing financial interests

The author declares no competing financial interests.

ANTIMICROBIAL MECHANISMS

A sponge against fungal infections

The finding that the antifungal activity of amphotericin B is primarily due to its ability to extract ergosterol from fungal membranes suggests a new rationale for drug design, which should lead to advanced treatments, particularly for invasive fungal infections.

Karl Lohner

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ungi infect billions of people every year, yet their contribution to the global burden of disease is largely unrecognized1, and the repertoire of antifungal agents is rather limited. Treatment of life-threatening invasive fungal infections is still based on drugs discovered several decades ago, with amphotericin B (AmB) being the gold standard. Anderson et al.2 now provide convincing evidence for a new mechanism of action of AmB. Accordingly, fungi are killed primarily by extracting ergosterol from the plasma membrane via large

extramembranous AmB aggregates. This new mechanistic understanding might open new avenues for the design of antifungal agents that lead to more specific and hence safer therapeutics as well as to a wider armory against fungal infections. Worldwide, a rise in the incidence of invasive fungal infections has been reported. Although true mortality rates are unknown because of a lack of good epidemiological data, this development is to a large extent a result of modern medical interventions and immunosuppressive diseases1.

nature chemical biology | VOL 10 | JUNE 2014 | www.nature.com/naturechemicalbiology

Demographic changes, in particular an increasing elderly population, further demand management of infectious complications common in patients undergoing chemotherapy for cancer, dialysis for renal failure and surgery, especially organ transplantation. Unfortunately, clinically available drugs have had only modest success in reducing the high mortality rates of invasive fungal infections such as candidiasis and cryptococcosis. Roughly 30% of patients with bloodstream infections with 411

Glycobiology: drifting toward polymer perfection.

The addition of polysialic acid to proteins and cells is emerging as a promising therapeutic strategy. Polysialyltransferases synthesize polymers of w...
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