Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Abstract

Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a "biomolecular construction kit" for the design of new biomaterials, by utilizing the self-assembly system of the crystalline cell surface (S)-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O-glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans. By using a combined carbohydrate/protein engineering approach, two types of S-layer neoglycoproteins are produced in Escherichia coli. Based on the identification of a suitable periplasmic targeting system for the SgsE self-assembly protein as a cellular prerequisite for Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts protein glycosylation, and on engineering of one of the natural protein O-glycosylation sites into a target for N-glycosylation, the heptasaccharide from the AcrA protein of Campylobacter jejuni and the O7 polysaccharide of E. coli are co-or post-translationally transferred to the S-layer protein by the action of the oligosaccharyltransferase PglB. The degree of glycosylation of the Slayer neoglycoproteins after purification from the periplasmic fraction reaches completeness. Electron microscopy reveals that recombinant glycosylation is fully compatible with the S-layer protein self-assembly system. Tailor-made ("functional") nanopatterned, self-assembling neoglycoproteins may open up new strategies for influencing and controlling complex biological systems with potential applications in the areas of biomimetics, drug targeting, vaccine design, or diagnostics.

Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051 T , Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP

“…Due to the lack of suitable tools for genetic manipulation of bacterial S-layer glycosylation pathways, progress in the elucidation of the glycan biosynthesis mechanism, which is a prerequisite for the desired production of functional S-layer neoglycoproteins, was limited to in vitro testing of individual enzymes from these pathways (34) and to heterologous carbohydrate-engineering approaches in the past (32).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Mycobacterium tuberculosis cell wall TOF Chemical synthesis from monosaccharide building blocks Carbohydrates from bacteria Construction of self-assembly S-layer glycoproteins as nanopatterned biomaterials (Steiner, et al, 2008b) Nematode etc. carbohydrates Schistosoma glycans TOF Complex type from eggs.…”

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…The mono-or multilayered Slayers confer an evolutionary advantage to the cells but are not essential for their survival (539). Their self-assembling capacities make them interesting candidates for study in the field of biomimetics and glycoengineering of therapeutic targets (e.g., vaccine design, drug targeting, and diagnostics) (540). The research field dedicated to these intriguing molecules is called "nanoglycobiology" (541).…”