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Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.

Lamppa JW, Ackerman ME, Lai JI, Scanlon TC, Griswold KE - PLoS ONE (2011)

Bottom Line: To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented.In both cases, the PEGylated enzyme was found to be substantially less immunoreactive.In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, United States of America.

ABSTRACT
Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

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Sites of cysteine substitution.Ribbon diagram of alginate Lyase A1-III (PDB file 1HV6). A trisaccharide                            reaction product is bound in the active cleft and shown as a grey ball                            and stick model. Amino acid residues targeted for cysteine substitution                            are shown in space filling mode, and are color coded as follows:                            S32C = Red, A41C = Orange,                            A53C = Green, A270C = Yellow,                            and A328C = Purple.
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pone-0017042-g001: Sites of cysteine substitution.Ribbon diagram of alginate Lyase A1-III (PDB file 1HV6). A trisaccharide reaction product is bound in the active cleft and shown as a grey ball and stick model. Amino acid residues targeted for cysteine substitution are shown in space filling mode, and are color coded as follows: S32C = Red, A41C = Orange, A53C = Green, A270C = Yellow, and A328C = Purple.

Mentions: To facilitate site-specific PEGylation, mutant A1-III genes encoding single cysteine substitutions were constructed by total gene synthesis [21]. The synthetic genes were codon optimized for expression in E. coli, and each gene encoded a single site-specific cysteine substitution: S32C, A41C, A53C, A270C or A328C, where residue numbering is per Yoon et al. [22] The five sites for mutation were selected based on an analysis of PDB structure 1HV6 [22]. Priority was placed on small amino acids that, when substituted with a cysteine, would result in a solvent exposed thiol group (Fig. 1 and Movie S1). Particular emphasis was given to residues with spatial proximity to the S32-C49 peptide segment, a motif that has previously been reported as constituting an immunodominant region of the enzyme [23]. A C-terminal hexahistidine tag (his-tag) was appended to each mutant to facilitate purification by immobilized metal-ion affinity chromatography (IMAC). A construct encoding the corresponding his-tagged version of the wild type enzyme (WT-his) was generated as a control.


Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.

Lamppa JW, Ackerman ME, Lai JI, Scanlon TC, Griswold KE - PLoS ONE (2011)

Sites of cysteine substitution.Ribbon diagram of alginate Lyase A1-III (PDB file 1HV6). A trisaccharide                            reaction product is bound in the active cleft and shown as a grey ball                            and stick model. Amino acid residues targeted for cysteine substitution                            are shown in space filling mode, and are color coded as follows:                            S32C = Red, A41C = Orange,                            A53C = Green, A270C = Yellow,                            and A328C = Purple.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3038863&req=5

pone-0017042-g001: Sites of cysteine substitution.Ribbon diagram of alginate Lyase A1-III (PDB file 1HV6). A trisaccharide reaction product is bound in the active cleft and shown as a grey ball and stick model. Amino acid residues targeted for cysteine substitution are shown in space filling mode, and are color coded as follows: S32C = Red, A41C = Orange, A53C = Green, A270C = Yellow, and A328C = Purple.
Mentions: To facilitate site-specific PEGylation, mutant A1-III genes encoding single cysteine substitutions were constructed by total gene synthesis [21]. The synthetic genes were codon optimized for expression in E. coli, and each gene encoded a single site-specific cysteine substitution: S32C, A41C, A53C, A270C or A328C, where residue numbering is per Yoon et al. [22] The five sites for mutation were selected based on an analysis of PDB structure 1HV6 [22]. Priority was placed on small amino acids that, when substituted with a cysteine, would result in a solvent exposed thiol group (Fig. 1 and Movie S1). Particular emphasis was given to residues with spatial proximity to the S32-C49 peptide segment, a motif that has previously been reported as constituting an immunodominant region of the enzyme [23]. A C-terminal hexahistidine tag (his-tag) was appended to each mutant to facilitate purification by immobilized metal-ion affinity chromatography (IMAC). A construct encoding the corresponding his-tagged version of the wild type enzyme (WT-his) was generated as a control.

Bottom Line: To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented.In both cases, the PEGylated enzyme was found to be substantially less immunoreactive.In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, United States of America.

ABSTRACT
Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

Show MeSH
Related in: MedlinePlus