Genetically Engineered Alginate Lyase-PEG Conjugates Exhibit Enhanced Catalytic Function and Reduced Immunoreactivity

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.Cystic Fibrosis Foundation (Research Development Program)National Center for Research Resources (U.S.) (P20RR018787-06

Identification of a Pivotal Residue for Determining the Block Structure-Forming Properties of Alginate C5 Epimerases

Alginate is a linear copolymer composed of 1→4 linked β-d-mannuronic acid (M) and its epimer α-l-guluronic acid (G). The polysaccharide is first produced as homopolymeric mannuronan and subsequently, at the polymer level, C-5 epimerases convert M residues to G residues. The bacterium Azotobacter vinelandii encodes a family of seven secreted and calcium ion-dependent mannuronan C-5 epimerases (AlgE1–AlgE7). These epimerases consist of two types of structural modules: the A-modules, which contain the catalytic site, and the R-modules, which influence activity through substrate and calcium binding. In this study, we rationally designed new hybrid mannuronan C-5 epimerases constituting the A-module from AlgE6 and the R-module from AlgE4. This led to a better understanding of the molecular mechanism determining differences in MG- and GG-block-forming properties of the enzymes. A long loop with either tyrosine or phenylalanine extruding from the β-helix of the enzyme proved essential in defining the final alginate block structure, probably by affecting substrate binding. Normal mode analysis of the A-module from AlgE6 supports the results

¹H,¹³C and¹⁵N resonances of the AlgE62 subunit from <em>Azotobacter vinelandii </em>mannuronan C5-epimerase

The 17.7 kDa R2 module from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been isotopically labeled ((13)C,(15)N) and recombinantly expressed. Here we report the (1)H, (13)C, (15)N resonance assignment of AlgE6R2

Acid gel formation in (pseudo) alginates with and without G blocks produced by epimerising mannuronan with C5 epimerases

The main scope of this paper is the characterization, in terms of viscoelastic and mechanical properties, of acid gels formed from solutions of mannuronan ALG (0%G/0%GG) and its enzymatically epimerised products. The epimerised products were obtained using recombinantly produced mannuronan C5 epimerases named AlgE1 and AlgE4, which catalyse the conversion of mannuronic residues into guluronic (G) and guluronic-mannuronic (GM) blocks, respectively. The products used in this study resulted from either the action of AlgE1 on mannuronan for 5 and 24 h (named ALG(44%G/32%GG) and ALG (68%G/59%GG), respectively) or AlgE4 on mannuronan (named ALG (47%G/0%GG)). d-gluconic acid-δ-lactone (GDL) was used as H+-donor to produce acidic gels. ALG (0%G/0%GG) yields strong, stable solid-like structures. As predicted by circular dichroism measurements performed at different pH, gelation of ALG (47%G/0%GG) occurs at lower values of pH (∼1) than those obtainable using GDL. Hydrochloric acid was therefore added to ALG (47%G/0%GG) solutions yielding rapid sol-gel transitions and gels with a remarkable resistance to thermal treatment. The introduction of guluronic residues along the chain (ALG (44%G/32%GG)) causes a reduction in the storage modulus at the equilibrium with respect to that of ALG (0%G/0%GG) and the occurrence of negligible syneresis at the highest polymer concentrations. The increase in the average length of the G blocks (ALG (68%G/59%GG)) is accompanied by a further increase in the storage modulus without the occurrence of any significant syneresis. © 2006 Elsevier Ltd. All rights reserved.The main scope of this paper is the characterization, in terms of viscoelastic and mechanical properties, of acid gels formed from solutions of mannuronan ALG (0%G/0%GG) and its enzymatically epimerised products. The epimerised products were obtained using recombinantly produced mannuronan C5 epimerases named AlgE1 and AlgE4, which catalyse the conversion of mannuronic residues into guluronic (G) and guluronic–mannuronic (GM) blocks, respectively. The products used in this study resulted from either the action of AlgE1 on mannuronan for 5 and 24 h (named ALG(44%G/32%GG) and ALG (68%G/59%GG), respectively) or AlgE4 on mannuronan (named ALG (47%G/0%GG)). Dgluconic acid-d-lactone (GDL) was used as HC-donor to produce acidic gels. ALG (0%G/0%GG) yields strong, stable solid-like structures. As predicted by circular dichroism measurements performed at different pH, gelation of ALG (47%G/0%GG) occurs at lower values of pH (w1) than those obtainable using GDL. Hydrochloric acid was therefore added to ALG (47%G/0%GG) solutions yielding rapid sol–gel transitions and gels with a remarkable resistance to thermal treatment. The introduction of guluronic residues along the chain (ALG (44%G/32%GG)) causes a reduction in the storage modulus at the equilibrium with respect to that of ALG (0%G/0%GG) and the occurrence of negligible syneresis at the highest polymer concentrations. The increase in the average length of the G blocks (ALG (68%G/59%GG)) is accompanied by a further increase in the storage modulus without the occurrence of any significant syneresis

Biomimetic sulphated alginate hydrogels suppress IL-1β-induced inflammatory responses in human chondrocytes

Loss of articular cartilage from ageing, injury or degenerative disease is commonly associated with inflammation, causing pain and accelerating degradation of the cartilage matrix. Sulphated glycosaminoglycans (GAGs) are involved in the regulation of immune responses in vivo, and analogous polysaccharides are currently being evaluated for tissue engineering matrices to form a biomimetic environment promoting tissue growth while suppressing inflammatory and catabolic activities. Here, we characterise physical properties of sulphated alginate (S-Alg) gels for use in cartilage engineering scaffolds, and study their anti-inflammatory effects on encapsulated chondrocytes stimulated with IL-1β. Sulphation resulted in decreased storage modulus and increased swelling of alginate gels, whereas mixing highly sulphated alginate with unmodified alginate resulted in improved mechanical properties compared to gels from pure S-Alg. S-Alg gels showed extensive anti-inflammatory and anti-catabolic effects on encapsulated chondrocytes induced by IL-1β. Cytokine-stimulated gene expression of pro-inflammatory markers IL-6, IL-8, COX-2 and aggrecanase ADAMTS-5 were significantly lower in the sulphated gels compared to unmodified alginate gels. Moreover, sulphation of the microenvironment suppressed the protein expression of COX-2 and NF-κB as well as the activation of NF-κB and p38-MAPK. The sulphated alginate matrices were found to interact with IL-1β, and proposed to inhibit inflammatory induction by sequestering cytokines from their receptors. This study shows promising potential for sulphated alginates in biomimetic tissue engineering scaffolds, by reducing cytokine-mediated inflammation and providing a protective microenvironment for encapsulated cells