Nicolas Laurent

Glycoarrays—tools for determining protein–carbohydrate interactions and glycoenzyme specificity

Carbohydrate arrays (glycoarrays) have recently emerged as a high-throughput tool for studying carbohydrate-binding proteins and carbohydrate-processing enzymes. A number of sophisticated array platforms that allow for qualitative and quantitative analysis of carbohydrate binding and modification on the array surface have been developed, including analysis by fluorescence spectroscopy, mass spectrometry and surface plasmon resonance spectroscopy. These platforms, together with examples of biologically-relevant applications are reviewed in this Feature Article.

Enzyme catalysis on solid surfaces

Enzyme-catalysed reactions in which substrates are bound (immobilised) to solid surfaces are becoming increasingly important in biotechnology. There is a general drive for miniaturisation and automation in chemistry and biology, and immobilisation of the reaction intermediates and substrates, for example on microarrays or nanoparticles, helps to address technical challenges in this area. In bionanotechnology, enzyme catalysis can provide highly selective and biocompatible tools for the modification of surfaces on the nano-scale. Here, we review the range of enzyme-catalysed reactions that have been successfully performed on the solid phase and discuss their application in biotechnology.

A versatile gold surface approach for fabrication and interrogation of glycoarrays

Glycoarrays on gold: A designer gold surface incorporating a self-assembled monolayer with weak protein absorption properties has been optimised for rapid display and interrogation of both native and derivatised glycans in array formats. This rapid, facile approach has diverse applications in glycomics, through exploitation of fluorescence, SPR and MALDI-ToF MS detection methods.

SPOT synthesis of peptide arrays on self-assembled monolayers and their evaluation as enzyme substrates.

amino acid to the other, and therefore the amount and purity of peptides can also vary. Monitoring reactions on the support relies on the use of a coloured dye, and quality control of the peptide requires its cleavage from the support after completion of synthesis for subsequent solution-phase analysis by HPLC or MALDI-ToFMS. [19] Consequently, assay results obtained from an array prepared by SPOT synthesis sometimes need to be confirmed by using purified samples of peptides in a solution-phase assay. [15] By combining the robustness and versatility of SAM-coated gold platforms and the easy-to-handle SPOT peptide synthesis methodology, an efficient method for preparing peptide arrays on gold surfaces is described herein. The method allows onchip monitoring of the synthesis and subsequent evaluation of enzymatic modifications by MALDI-ToFmass spectrometry (Figure 1).

Biochemical correlation of activity of the α-dystroglycan-modifying glycosyltransferase POMGnT1 with mutations in muscle-eye-brain disease.

Congenital muscular dystrophies have a broad spectrum of genotypes and phenotypes and there is a need for a better biochemical understanding of this group of diseases in order to aid diagnosis and treatment. Several mutations resulting in these diseases cause reduced O-mannosyl glycosylation of glycoproteins, including α-dystroglycan. The enzyme POMGnT1 (protein-O-mannose N-acetylglucosaminyltransferase 1; EC 2.4.1.-) catalyses the transfer of N-acetylglucosamine to O-linked mannose of α-dystroglycan. In the present paper we describe the biochemical characterization of 14 clinical mutants of the glycosyltransferase POMGnT1, which have been linked to muscle-eye-brain disease or similar conditions. Truncated mutant variants of the human enzyme (recombinant POMGnT1) were expressed in Escherichia coli and screened for catalytic activity. We find that three mutants show some activity towards mannosylated peptide substrates mimicking α-dystroglycan; the residues affected by these mutants are predicted by homology modelling to be on the periphery of the POMGnT1 surface. Only in part does the location of a previously described mutated residue on the periphery of the protein structure correlate with a less severe disease mutant.

MALDI-ToF MS Analysis of Glycosyltransferase Activities on Gold Surface Arrays

Glycan-processing enzymes such as glycosyltransferases and glycosidases are responsible for the makeup of the glycome. The definition of their substrate specificities is, therefore, a central task in glycomics. In addition, these enzymes are themselves useful synthetic tools for the generation of complex carbohydrate structures as an alternative to tedious chemical synthesis. There has been great interest in using microarrays for studying these glycoenzymes because it allows the specificity of the enzyme to be probed against a panel of immobilized potential substrates, and also expands the repertoire of sugar arrays available for further carbohydrate-protein interaction studies. In particular, self-assembled monolayers (SAMs) of alkanethiols on gold surfaces have proven to be a valuable platform for such studies due to their robustness and their biocompatible, well-defined structure. Furthermore, a direct observation of the change in mass of immobilized substrates due to enzymatic processing is possible through label-free MALDI-ToF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) technique. In this chapter, we describe the preparation of SAMs-coated gold surface arrays presenting carbohydrate or (glyco)peptide substrates, either pre-formed or directly synthesized on-chip, and MALDI-ToF MS analysis of glycosyltransferase activities on these immobilized substrates.

Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

The biosynthesis of mucin-type O-linked glycans in animals is initiated by members of the large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), which play important roles in embryogenesis, organogenesis, adult tissue homeostasis and carcinogenesis. Until now, the mammalian forms of these enzymes have been the best characterized. However, two N-acetylgalactosaminyltransferases (xGalNAc-T6 and xGalNAc-T16) from the African clawed frog (Xenopus laevis), which are most homologous to those encoded by the human GALNT6 and GALNT16 (GALNTL1) genes, were shown to have contrasting roles in TGF-β/BMP signaling in embryogenesis. In this study we have examined these two enzymes further and show differences in their in vivo function during X. laevis embyrogenesis as evidenced by in situ hybridization and overexpression experiments. In terms of enzymatic activity, both enzymes were found to be active towards the EA2 peptide, but display differential activity towards a peptide based on the sequence of ActR-IIB, a receptor relevant to TGF-β/BMP signaling. In summary, these data demonstrate that these two enzymes from different branches of the N-acetylgalactosaminyltransferase do not only display differential substrate specificities, but also specific and distinct expression pattern and biological activities in vivo.