Anne Line Norberg

Production of Chitooligosaccharides and Their Potential Applications in Medicine

Chitooligosaccharides (CHOS) are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine. CHOS can be produced using chitin or chitosan as a starting material, using enzymatic conversions, chemical methods or combinations thereof. Production of well-defined CHOS-mixtures, or even pure CHOS, is of great interest since these oligosaccharides are thought to have several interesting bioactivities. Understanding the mechanisms underlying these bioactivities is of major importance. However, so far in-depth knowledge on the mode-of-action of CHOS is scarce, one major reason being that most published studies are done with badly characterized heterogeneous mixtures of CHOS. Production of CHOS that are well-defined in terms of length, degree of N-acetylation, and sequence is not straightforward. Here we provide an overview of techniques that may be used to produce and characterize reasonably well-defined CHOS fractions. We also present possible medical applications of CHOS, including tumor growth inhibition and inhibition of TH2-induced inflammation in asthma, as well as use as a bone-strengthener in osteoporosis, a vector for gene delivery, an antibacterial agent, an antifungal agent, an anti-malaria agent, or a hemostatic agent in wound-dressings. By using well-defined CHOS-mixtures it will become possible to obtain a better understanding of the mechanisms underlying these bioactivities.

Comparative studies of chitinases A, B and C from Serratia marcescens

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.

Degradation of chitosans with a family 46 chitosanase from Streptomyces coelicolor A3(2).

We have studied the degradation of well-characterized soluble heteropolymeric chitosans by a novel family 46 chitosanase, ScCsn46A from Streptomyces coelicolor A3(2), to obtain insight into the enzymes mode of action and to determine its potential for production of different chitooligosaccharides. The degradation of both a fully deacetylated chitosan and a 32% acetylated chitosan showed a continuum of oligomeric products and a rapid disappearance of the polymeric fraction, which is diagnostic for a nonprocessive endomode of action. The kinetics of the degradation of the 32% acetylated chitosan demonstrated an initial rapid phase and a slower second phase, in addition to a third and even slower kinetic phase. The first phase reflects the cleavage of the glycosidic linkage between two deacetylated units (D-D), the primary products being fully deacetylated dimers, trimers, and tetramers, as well as longer oligomers with increasing degrees of acetylation. In the subsequent slower kinetic phases, oligomers with a higher degree of acetylated units (A) appear, including oligomers with As at the reducing or nonreducing end, which indicate that there are no absolute preferences for D in subsites -1 and +1. After maximum degradation of the chitosan, the dimers DA and DD were the dominant products. The degradation of chitosans with varying degrees of acetylation to a maximum degree of scission showed that ScCsn46A could degrade all chitosan substrates extensively, although to decreasing degrees of scission with increasing F(A). The potential use of ScCsn46A to prepare fully deacetylated oligomers and more highly acetylated oligomers from chitosan substrates with varying degrees of acetylation is discussed.

Human Chitotriosidase-Catalyzed Hydrolysis of Chitosan

Chitotriosidase (HCHT) is one of two family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the human defense mechanism against fungal parasites, but its precise role and the details of its enzymatic properties have not yet been fully unraveled. We have studied the properties of HCHT by analyzing how the enzyme acts on high-molecular weight chitosans, soluble copolymers of β-1,4-linked N-acetylglucosamine (GlcNAc, A), and glucosamine (GlcN, D). Using methods for in-depth studies of the chitinolytic machinery of bacterial family 18 enzymes, we show that HCHT degrades chitosan primarily via an endoprocessive mechanism, as would be expected on the basis of the structural features of its substrate-binding cleft. The preferences of HCHT subsites for acetylated versus nonacetylated sugars were assessed by sequence analysis of obtained oligomeric products showing a very strong, absolute, and a relative weak preference for an acetylated unit in the -2, -1, and +1 subsites, respectively. The latter information is important for the design of inhibitors that are specific for the human chitinases and also provides insight into what kind of products may be formed in vivo upon administration of chitosan-containing medicines or food products.

Determination of substrate binding energies in individual subsites of a family 18 chitinase

Thermodynamic parameters for binding of N‐acetylglucosamine (GlcNAc) oligomers to a family 18 chitinase, ChiB of Serratia marcescens, have been determined using isothermal titration calorimetry. Binding studies with oligomers of different lengths showed that binding to subsites −2 and +1 is driven by a favorable enthalpy change, while binding to the two other most important subsites, +2 and +3, is driven by entropy with unfavorable enthalpy. These remarkable unfavorable enthalpy changes are most likely due to favorable enzyme‐substrate interactions being offset by unfavorable enthalpic effects of the conformational changes that accompany substrate‐binding.

Inhibition of angiogenesis by chitooligosaccharides with specific degrees of acetylation and polymerization.

Chitooligosaccharides (CHOS) inhibit angiogenesis and may be used in the treatment of cancer tumors. We have studied the effect of the fraction of acetylation (FA) and the degree of polymerization (DP) on CHOS anti-angiogenic activity. We tested enzymatically produced CHOS-mixtures with FA0.15, FA0.3 and FA0.6, and DP≤12 in initial experiments with chorioallantoic membranes. All of the samples reduced the formation of new blood vessels, CHOS with FA0.3 giving the best effect. Single-DP fractions from the FA0.3 sample purified by size-exclusion chromatography (DP3-DP12) were then tested for inhibition of migration of human endothelial cells, which is an important element of the angiogenesis process. All of the fractions inhibited migration, meaning that, within the DP area tested in this study, FA is more important than DP for the effect. Generally, the results reveal that DP3-DP12 CHOS have considerable potential as anti-angiogenic compounds.

Substrate positioning in chitinase A, a processive chito-biohydrolase from Serratia marcescens

The contributions of the –3 subsite and a putative +3 subsite to substrate positioning in ChiA from Serratia marcescens have been investigated by comparing how ChiA and its –3 subsite mutant W167A interact with soluble substrates. The data show that Trp – GlcNAc stacking in the –3 subsite rigidifies the protein backbone supporting the formation of the intermolecular interaction network that is necessary for the recognition and positioning of the N‐acetyl groups before the –1 subsite. The +3 subsite exhibits considerable substrate affinity that may promote endo‐activity in ChiA and/or assist in expelling dimeric products from the +1 and +2 subsites during processive hydrolysis.

Analysis of noncovalent chitinase-chito-oligosaccharide complexes by infrared-matrix assisted laser desorption ionization and nanoelectrospray ionization mass spectrometry.

Transferring noncovalently bound complexes from the condensed phase into the gas phase represents a challenging task due to weak intermolecular bonds that have to be maintained during the phase transition. Currently, electrospray ionization (ESI) is the standard mass spectrometric (MS) technique to analyze noncovalent complexes. Although infrared matrix-assisted laser desorption ionization (IR-MALDI)-MS also provides particular soft desorption/ionization conditions, this method has so far hardly been applied for the analysis of noncovalent complexes. In this study, we employed IR-MALDI orthogonal time-of-flight (o-TOF)-MS in combination with the liquid matrix glycerol to characterize the specific complex formation of chito-oligosaccharide (CHOS) ligands with two variants of Chitinase A (ChiA) from Serratia marcescens, the inactive E315Q mutant and the active W167A mutant, respectively. The IR-MALDI-o-TOF-MS results were compared to those obtained using nano-ESI-quadrupole (q)-TOF-MS and ultraviolet (UV)-MALDI-o-TOF-MS. Using IR-MALDI-o-TOF-MS, specific noncovalent complexes between ChiA and CHOS were detected with distributions between enzymes with bound oligosaccharides vs free enzymes that were essentially identical to those obtained by nano-ESI-q-TOF-MS. Chitinase-CHOS complexes were not detected when UV-MALDI was employed for desorption/ionization. The results show that IR-MALDI-MS can be a valuable tool for fast and simple screening of noncovalent enzyme-ligand interactions.

Analysis of productive binding modes in the human chitotriosidase

Human chitotriosidase (HCHT) is a family 18 chitinase that is an innate part of the immune system. We have mapped preferred productive binding modes of chito‐oligosaccharide substrates to HCHT and the data show that HCHT has strong binding affinity in the +3 subsite. Moreover, HCHT shows anomer‐specific binding affinities in subsites +2 and +3. These features could endorse HCHT with higher endo‐activity and a higher transglycosylation potential.

Processivity and substrate-binding in family 18 chitinases

Enzymatic depolymerisation of polysaccharides is a key technology in the biorefining of biomass. The enzymatic conversion of the abundant insoluble polysaccharides cellulose and chitin is of particular interest and complexity, because of the bi-phasic nature of the process, the seemingly complicated tasks faced by the enzymes, and the importance of these conversions for the future biorefinery. Here we review recent work on family 18 chitinases that sheds light on important aspects of the catalytic action of these depolymerising enzymes, including the structural basis of processivity and its direction, the energies involved in substrate-binding and displacement.