Glycosylation engineering of S-layer proteins for bioactivity

Background.

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 (Wu et al., 2008), 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 (Steiner et al., 2008; Zarschler et al., 2009, 2010a).

Aims and methods.

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 (Zarschler et al., 2010b). 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 (Faridmoayer et al., 2008); 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. Applied methods include i) MS and NMR approaches for the characterization of the products of the enzymatic reactions by; (i) performance of kinetic studies of selected key modules of S-layer protein glycosylation; (iii) structural analysis of those enzymes by CD and FT-IR spectroscopy, (iv) crystallization of carbohydrate active enzymes and co-crystallization with substrates (Steiner et al., 2010), and set-up of functional systems for combining native and heterologous glycosylation modules.

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.

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Zarschler, K., Janesch, B., Zayni, S., Schäffer, C., Messner, P. (2009). Construction of a gene knockout system for application in Paenibacillus alvei CCM 2051T, exemplified by the S-layer glycan biosynthesis initiation enzyme WsfP. Appl. Environ. Microbiol. 75, 3077-3085
Zarschler K., Janesch, B, Pabst, M., Altmann, F., Messner, P., Schäffer, C. (2010a). Protein tyrosine O-glycosylation – A rather unexplored prokaryotic glycosylation system. Glycobiology, 20, 787-798.
Zarschler K., Janesch, B., Kainz, B., Ristl, R., Messner, P., Schäffer, C. (2010b). Cell surface display of chimeric glycoproteins via the S layer of Paenibacillus alvei. Carbohydr. Res., 345, 1422-1431.
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