Hijacking the Peptidoglycan Recycling Pathway of Escherichia coli to Produce Muropeptides.

Soluble fragments of peptidoglycan called muropeptides are released from the cell wall of bacteria as part of their metabolism or as a result of biological stresses. These compounds trigger immune responses in mammals and plants. In bacteria, they play a major role in the induction of antibiotic resistance. The development of efficient methods to produce muropeptides is, therefore, desirable both to address their mechanism of action and to design new antibacterial and immunostimulant agents. Herein, we engineered the peptidoglycan recycling pathway of Escherichia coli to produce N-acetyl-ß-D-glucosaminyl-(1→4)-1,6-anhydro-N-acetyl-ß-D-muramic acid (GlcNAc-anhMurNAc), a common precursor of Gram-negative and Gram-positive muropeptides. Inactivation of the hexosaminidase nagZ gene allowed the efficient production of this key disaccharide, providing access to Gram-positive muropeptides through subsequent chemical peptide conjugation. E. coli strains deficient in both NagZ hexosaminidase and amidase activities further enabled the in vivo production of Gram-negative muropeptides containing meso-diaminopimelic acid, a rarely available amino acid.

Lipo-Chitooligosaccharides Induce Specialized Fungal Metabolite Profiles That Modulate Bacterial Growth.

Lipo-chitooligosaccharides (LCOs) are historically known for their role as microbial-derived signaling molecules that shape plant symbiosis with beneficial rhizobia or mycorrhizal fungi. Recent studies showing that LCOs are widespread across the fungal kingdom have raised questions about the ecological function of these compounds in organisms that do not form symbiotic relationships with plants. To elucidate the ecological function of these compounds, we investigate the metabolomic response of the ubiquitous human pathogen Aspergillus fumigatus to LCOs. Our metabolomics data revealed that exogenous application of various types of LCOs to A. fumigatus resulted in significant shifts in the fungal metabolic profile, with marked changes in the production of specialized metabolites known to mediate ecological interactions. Using network analyses, we identify specific types of LCOs with the most significant effect on the abundance of known metabolites. Extracts of several LCO-induced metabolic profiles significantly impact the growth rates of diverse bacterial species. These findings suggest that LCOs may play an important role in the competitive dynamics of non-plant-symbiotic fungi and bacteria. This study identifies specific metabolomic profiles induced by these ubiquitously produced chemicals and creates a foundation for future studies into the potential roles of LCOs as modulators of interkingdom competition. IMPORTANCE The activation of silent biosynthetic gene clusters (BGC) for the identification and characterization of novel fungal secondary metabolites is a perpetual motion in natural product discoveries. Here, we demonstrated that one of the best-studied symbiosis signaling compounds, lipo-chitooligosaccharides (LCOs), play a role in activating some of these BGCs, resulting in the production of known, putative, and unknown metabolites with biological activities. This collection of metabolites induced by LCOs differentially modulate bacterial growth, while the LCO standards do not convey the same effect. These findings create a paradigm shift showing that LCOs have a more prominent role outside of host recognition of symbiotic microbes. Importantly, our work demonstrates that fungi use LCOs to produce a variety of metabolites with biological activity, which can be a potential source of bio-stimulants, pesticides, or pharmaceuticals.

Expanding the Biological Role of Lipo-Chitooligosaccharides and Chitooligosaccharides in Laccaria bicolor Growth and Development.

The role of lipo-chitooligosaccharides (LCOs) as signaling molecules that mediate the establishment of symbiotic relationships between fungi and plants is being redefined. New evidence suggests that the production of these molecular signals may be more of a common trait in fungi than what was previously thought. LCOs affect different aspects of growth and development in fungi. For the ectomycorrhizal forming fungi, Laccaria bicolor, the production and effects of LCOs have always been studied with a symbiotic plant partner; however, there is still no scientific evidence describing the effects that these molecules have on this organism. Here, we explored the physiological, molecular, and metabolomic changes in L. bicolor when grown in the presence of exogenous sulfated and non-sulfated LCOs, as well as the chitooligomers, chitotetraose (CO4), and chitooctaose (CO8). Physiological data from 21 days post-induction showed reduced fungal growth in response to CO and LCO treatments compared to solvent controls. The underlying molecular changes were interrogated by proteomics, which revealed substantial alterations to biological processes related to growth and development. Moreover, metabolite data showed that LCOs and COs caused a downregulation of organic acids, sugars, and fatty acids. At the same time, exposure to LCOs resulted in the overproduction of lactic acid in L. bicolor. Altogether, these results suggest that these signals might be fungistatic compounds and contribute to current research efforts investigating the emerging impacts of these molecules on fungal growth and development.

Size-Controlled Synthesis of ß(1→4)-GlcNAc Oligosaccharides Using an Endo-Glycosynthase.

Chitin and peptidoglycan fragments are well recognized as pathogen associated molecular patterns (PAMPs). Long-chain oligosaccharides of ß(1→4)-linked N-acetyl-D-glucosamine (GlcNAc) units indeed activate plants and mammals innate immune system. However, the mechanisms underlying PAMPs perception by lysine motif (LysM) domain receptors remain largely unknown because of insufficient availability of high-affinity molecular probes. Here, we report a two-enzyme cascade to synthesize long-chain ß(1→4)-linked GlcNAc oligomers. Expression of the D52S mutant of hen egg-white lysozyme (HEWL) in Pichia pastoris at 52 mg L-1 provided a new glycosynthase catalyzing efficient polymerization of α-chitintriosyl fluoride. Selective N-deacetylation at the non-reducing unit of the glycosyl fluoride donor by Sinorhizobium meliloti NodB chitin-N-deacetylase abolished its ability to be polymerized by the glycosynthase but not to be transferred onto an acceptor. Using NodB and D52S HEWL in a one-pot cascade reaction allowed the synthesis on a milligram scale of chitin hexa-, hepta- and octasaccharides with yields up to 65 % and a perfect control over their size.

Optimizing Chitin Depolymerization by Lysozyme to Long-Chain Oligosaccharides.

Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box-Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.

Distinct genetic basis for root responses to lipo-chitooligosaccharide signal molecules from different microbial origins.

Lipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling the nodulation of legumes by rhizobia. More recently, LCOs were also found in symbiotic fungi and, more surprisingly, very widely in the kingdom Fungi, including in saprophytic and pathogenic fungi. The LCO-V(C18:1, fucosylated/methyl fucosylated), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants such as Medicago truncatula can discriminate between Nod-LCOs and Fung-LCOs. To address this question, we performed a genome-wide association study on 173 natural accessions of M. truncatula, using a root branching phenotype and a newly developed local score approach. Both Nod-LCOs and Fung-LCOs stimulated root branching in most accessions, but the root responses to these two types of LCO molecules were not correlated. In addition, the heritability of the root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, of which only one was common. This suggests that Nod-LCOs and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.

Lipo-chitooligosaccharides as regulatory signals of fungal growth and development.

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.

A lysin motif effector subverts chitin-triggered immunity to facilitate arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) fungi greatly improve mineral uptake by host plants in nutrient-depleted soil and can intracellularly colonize root cortex cells in the vast majority of higher plants. However, AM fungi possess common fungal cell wall components such as chitin that can be recognized by plant chitin receptors to trigger immune responses, raising the question as to how AM fungi effectively evade chitin-triggered immune responses during symbiosis. In this study, we characterize a secreted lysin motif (LysM) effector identified from the model AM fungal species Rhizophagus irregularis, called RiSLM. RiSLM is one of the highest expressed effector proteins in intraradical mycelium during the symbiosis. In vitro binding assays show that RiSLM binds chitin-oligosaccharides and can protect fungal cell walls from chitinases. Moreover, RiSLM efficiently interferes with chitin-triggered immune responses, such as defence gene induction and reactive oxygen species production in Medicago truncatula. Although RiSLM also binds to symbiotic (lipo)chitooligosaccharides it does not interfere significantly with symbiotic signalling in Medicago. Host-induced gene silencing of RiSLM greatly reduces fungal colonization levels. Taken together, our results reveal a key role for AM fungal LysM effectors to subvert chitin-triggered immunity in symbiosis, pointing to a common role for LysM effectors in both symbiotic and pathogenic fungi.

Unprecedented Affinity Labeling of Carbohydrate-Binding Proteins with s-Triazinyl Glycosides.

Carbohydrate-protein interactions trigger a wide range of biological signaling pathways, the mainstays of physiological and pathological processes. However, there are an incredible number of carbohydrate-binding proteins (CBPs) that remain to be identified and characterized. This study reports for the first time the covalent labeling of CBPs by triazinyl glycosides, a new and promising class of affinity-based glycoprobes. Mono- and bis-clickable triazinyl glycosides were efficiently synthesized from unprotected oligosaccharides (chitinpentaose and 2'-fucosyl-lactose) in a single step. These molecules allow the specific covalent labeling of chitin-oligosaccharide-binding proteins (wheat germ agglutinin WGA and Bc ChiA1 D202A, an inactivated chitinase) and fucosyl-binding lectin (UEA-I), respectively.

Mycorrhizal lipochitinoligosaccharides (LCOs) depolarize root hairs of Medicago truncatula.

Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca2+ sensitive reporter dyes, to study the relations between cytosolic Ca2+ signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca2+ level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca2+ signals and nuclear Ca2+ spiking.

Determining the Site of Action of Strigolactones during Nodulation.

Strigolactones (SLs) influence the ability of legumes to associate with nitrogen-fixing bacteria. In this study, we determine the precise stage at which SLs influence nodulation. We show that SLs promote infection thread formation, as a null SL-deficient pea (Pisum sativum) mutant forms significantly fewer infection threads than wild-type plants, and this reduction can be overcome by the application of the synthetic SL GR24. We found no evidence that SLs influence physical events in the plant before or after infection thread formation, since SL-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or Nod lipochitooligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that SL deficiency may influence the bacterial partner. We found that this influence of SL deficiency was not due to altered flavonoid exudation or the ability of root exudates to stimulate bacterial growth. The influence of SL deficiency on infection thread formation was accompanied by reduced expression of some early nodulation genes. Importantly, SL synthesis is down-regulated by mutations in genes of the Nod LCO signaling pathway, and this requires the downstream transcription factor NSP2 but not NIN This, together with the fact that the expression of certain SL biosynthesis genes can be elevated in response to rhizobia/Nod LCOs, suggests that Nod LCOs may induce SL biosynthesis. SLs appear to influence nodulation independently of ethylene action, as SL-deficient and ethylene-insensitive double mutant plants display essentially additive phenotypes, and we found no evidence that SLs influence ethylene synthesis or vice versa.

High yield production of Rhizobium NodB chitin deacetylase and its use for in vitro synthesis of lipo-chitinoligosaccharide precursors.

Lipo-chitinoligosaccharides (LCOs) are key molecules for the establishment of plant-microorganisms symbiosis. Interactions of leguminous crops with nitrogen-fixing rhizobial bacteria involve Nod factors, while Myc-LCOs improve the association of most plants with arbuscular mycorrhizal fungi. Both Nod factors and Myc-LCOs are composed of a chitinoligosaccharide fatty acylated at the non-reducing end accompanied with various substituting groups. One straightforward way to access LCOs is starting from chitin hydrolysate, an abundant polysaccharide found in crustacean shells, followed by regioselective enzymatic cleavage of an acetyl group from the non-reducing end of chitin tetra- or pentaose, and subsequent chemical introduction of N-acyl group. In the present work, we describe the in vitro synthesis of LCO precursors on preparative scale. To this end, Sinorhizobium meliloti chitin deacetylase NodB was produced in high yield in E. coli as a thioredoxin fusion protein. The recombinant enzyme was expressed in soluble and catalytically active form and used as an efficient biocatalyst for N-deacetylation of chitin tetra- and pentaose.

Nod Factor Effects on Root Hair-Specific Transcriptome of Medicago truncatula: Focus on Plasma Membrane Transport Systems and Reactive Oxygen Species Networks.

Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.

Enzymatic Activity of Xyloglucan Xylosyltransferase 5.

Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a ß-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.

The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.

Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice.

Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.

Redox-stimuli responsive micelles from DOX-encapsulating polycaprolactone-g-chitosan oligosaccharide.

Chitosan-based amphiphilic graft copolymers are commonly obtained by modification of chitosan backbones with synthetic polymers hampering both bioactivity and biodegradability. In this work, we report the preparation of a series of chitosan oligosaccharide-grafted copolymers (PCL-g-COs) from coupling reactions between azide-pendent polycaprolactones (PCL-N3) and reducing-end alkynyl-modified chitosan oligosaccharides (COs-alkynyl). The resulting PCL-g-COs self-organized in water into nanoscale micelles (Rh<20 nm) having a COs shell and a PCL core. Locking of the core-micelles structure employing a disulfide-containing bis-alkyne cross-linker resulted in the formation of nano-vehicles which can be degraded in response to physiological (redox) stimuli. This feature was advantageously exploited to preferentially release an anticancer drug, doxororubicin, in response to the intracellular glutathione level.

Aniline-catalyzed reductive amination as a powerful method for the preparation of reducing end-"clickable" chitooligosaccharides.

Functionalized oligosaccharides are useful intermediates to prepare products for biological research or for the development of advanced functional materials. Here, we report the unprecedented use of aniline as an efficient organocatalyst reaction with "clickable" (azide or alkyne) amine for the transimination-mediated reductive amination of a chitooligosaccharide. Moreover, we demonstrate that alkyne-bearing aniline constitutes an excellent tool for the easy derivatization of chitosan oligosaccharides. Evidence for such improvement has been illustrated by the straightforward design of a FRET substrate to probe chitinase activity and of amphiphilic polycaprolactone-grafted-chitosan. This efficient methodology paves the way to the preparation of novel chitosan oligosaccharide-based advanced materials.

The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2.

Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate pre-symbiotic growth of the fungus, which releases lipochito-oligosaccharides (Myc-LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up-regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc-LCOs. Fungal colonization was much reduced by over-expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc-LCOs, that prevents over-colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA-mediated negative regulation of NSP2.

Toward understanding of carbohydrate binding and substrate specificity of a glycosyl hydrolase 18 family (GH-18) chitinase from Trichoderma harzianum.

Surface plasmon resonance (SPR) has been used to assay the roles of amino acid residues in the substrate binding cleft of Trichoderma harzianum chitinase Chit42, which belongs to the glycoside hydrolase family 18 (GH-18). Nine different Chit42 variants having amino acid mutations along the binding site cleft at subsites -4 to +2 were created and characterized with regard to their affinity toward chitinous and non-chitinous oligosaccharides. The catalytically inactive Chit42 mutant E172Q was used as the template for making the additional mutations. The E172Q mutant bound chitinoligosaccharides (tetra-, penta- and hexamer) with an increasing affinity from 12 to 0.2 microM whereas no binding of chitinbiose, -triose or 3'-sialyl-N-acetyllactosamine (Neu5Acalpha-3Galbeta-4GlcNAc) could be measured, indicative of significantly lower affinity for these shorter oligosaccharides. The strongest binding affinity was displayed toward allosamidin, a transition state analog (K(d) = 3 nM), and this was shown to be dependent on the E172 residue, the acid/base catalyst of Chit42. Hydrogen bonding by the glutamic acid E317 between subsites -2 and -3 and particularly the stacking interactions by tryptophanes at subsites -3 and +2 provided to be important, as mutations to these amino acids had a substantial negative effect to the overall binding affinity. Moreover, the substrate binding specificity of Chit42 could be altered toward binding of GlcNbeta-4(GlcNAc)(4) by providing a counter charge through substitution of residue T133 at subsite -3 against aspartic acid. In addition, the introduction of glutamine and particularly an asparagine residue at position 133 seemed to broaden the substrate preference of Chit42 toward Galbeta-4(GlcNAc)(4).