Glycosylation engineering of S-layer protein for bioactivity

Glycans are critical components in many biological processes. Cells use them to communicate with each other, bacteria and viruses use them to infect their hosts, and cancer cells use them to chart new territory in the body. Frequently, carbohydrate-mediated phenomena fall in the categories of biostimulation and biotargeting, with the carbohydrates in many cases being coupled to a protein. Thus, glycosylation engineering to obtain customized protein glycosylation will decisively increase our capabilities in influencing and controlling complex biological systems. Bacteria are regarded promising candidates for this endeavor, because they are easily tractable, have favorable process economics to produce glycoproteins, and, most importantly, according to recent data, there are effective ways to make homogeneously glycosylated proteins in bioengineered bacteria. Despite of the power of bacterial cell surface display for protein display in basic and applied research, this strategy has not yet been exploited for customized carbohydrates. In the present project, we propose to undertake a biomimetic approach, in which bacterial protein glycosylation engineering shall be combined with multivalent surface display of customized glycans by using the naturally glycosylated bacterial cell surface layer protein of the Gram-positive bacterium Paenibacillus alvei CCM 2051T as a 2D crystalline, nanometer-scale display matrix. This matrix is generated through self-assembly, with the attached glycan chains being naturally orientated towards the ambient environment. We hypothesize that due to the similarities between the S layer protein glycosylation pathway and other bacterial polysaccharide biosynthesis systems, engineering of customized S layer glycoprotein glycans will be possible. As a way to analyze the basis for S layer glycosylation engineering and to test our hypothesis, our research goals are: A) Investigation of the native tyrosine O glycosylation sites on the S layer protein; B) Unraveling mechanisms for glycan chain length regulation, export and oligosaccharide:protein transfer; C) Engineering S layer protein SpaA glycosylation in selected glycosylation mutants of Pa with diverse heterologous saccharides to demonstrate the convergence of the S-layer glycosylation machinery with foreign bacterial glycosylation systems; D) Extension of the glycosylation potential of the S layer protein SpaA by exchanging the native O glycosylation site(s) by bacterial N glycosylation site(s) that are recognized by the cognate glycosylation machineries. This system shall allow ultimate control over the position, quantity and type of carbohydrate structure present on the bacterium. It provides a unique framework for studying the biostimulation and biotargeting behavior of various carbohydrates of interest in a setting that mirrors the natural dynamic behavior of a cell and may help to enhance the affinity and specificity of interaction between carbohydrates and their ligands through multivalent presentation. The present project follows the current trend of exploiting means for organizing biological functions at the nanometer level aiming at the development of novel concepts for life and non-life sciences. It provides an innovative approach to advance the opportunities emanating from glycobiology for the fields of human health and biomaterial design.

Carbohydrates are pivotal to many biological processes reflected by their biostimulation and biotargeting functions. Thus, glycosylation engineering to obtain customized carbohydrates will decisively increase our capabilities in influencing and controlling complex biological systems. Bacteria are regarded promising candidates for this endeavor, because they are easily tractable and have favorable process economics to produce carbohydrates. Cell surface display of rationally designed epitopes is regarded a powerful strategy. We proposed to undertake a biomimetic approach, in which bacterial glycosylation engineering should be combined with multivalent surface display of rationally designed carbohydrates by using the naturally glycosylated bacterial cell surface (S-) layer protein of the Gram-positive bacterium Paenibacillus alvei (Pa) as a 2D crystalline, self-assembling display matrix. The combination of modules from different glycosylation systems is a prerequisite for the chosen approach. The major achievements of this project were: (i) Establishment of an in vivo (glyco)protein co-display system based on the Pa S-layer and the newly identified protein SlhA. (ii) Eluci- dation of a novel mechanism governing cell surface display on Pa cells, relying on so-called S-layer homology domains present in triplicate on terminal position of the displayed proteins and possessing recognition function for peptidoglycan and a cell wall polysaccharide linker. (iii) Characterization of the oigosaccharyl:protein transferase from the Pa S-layer protein glycosylation system as a key module for glycosylation engineering, revealing that the en- zyme appears to be more stringent than comparable enzymes. Additionally, it seems to rely on the endogenous folding pathway of the S-layer target protein. (iv) Demonstration of a second Pa protein O-glycosylation system targeting flagellin. Glycosylation engineering of the flagella may provide future opportunities for customized epitope display. (v) Metabolic engineering of Pa allowed modification of the Pa S-layer glycan with sialic acid. (vi) The L. buchneri S-layer was characterized as a safe display matrix and tested for the construction of a model vaccine. A novel O-glycosylation motif was identified in the species L. buchneri.The established system shall allow ultimate control over the position, quantity and type of carbohydrate structure present on the bacterium. It provides a unique framework for studying the functions of various carbohydrates of interest in a setting that mirrors the natural dynamic behavior of a cell and may help to enhance the affinity and specificity of interaction between carbohydrates and their ligands through multivalent presentation. The present project follows the current trend of exploiting means for organizing biological functions at the nanometer level aiming at the development of novel concepts for life and non-life sciences. It provides an innovative approach to advance the opportunities emanating from micro- and glycobiology for the fields of human health and biomaterial design.