Acidic mammalian chitinase (AMCase) has been implicated in various pathophysiological conditions including asthma, allergic inflammation and food processing. AMCase is most active at pH 2.0, and its activity gradually decreases to up to pH 8.
Carbohydrate Polymers 164 (2017) 145–153 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Improved fluorescent labeling of chitin oligomers: Chitinolytic properties of acidic mammalian chitinase under somatic tissue pH conditions Satoshi Wakita a,1 , Masahiro Kimura a,1 , Naoki Kato a , Akinori Kashimura a , Shunsuke Kobayashi a , Naoto Kanayama a , Misa Ohno a , Shotaro Honda a , Masayoshi Sakaguchi a , Yasusato Sugahara a , Peter O Bauer b,c , Fumitaka Oyama a,∗ a Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA c Bioinova Ltd., Prague 142 20, Czechia b a r t i c l e i n f o Article history: Received 14 June 2016 Received in revised form January 2017 Accepted 29 January 2017 Available online 31 January 2017 Keywords: Acidic mammalian chitinase Chitin Chitin degradation products Chitin oligomers Fluorophore Pre-acidification method a b s t r a c t Acidic mammalian chitinase (AMCase) has been implicated in various pathophysiological conditions including asthma, allergic inflammation and food processing AMCase is most active at pH 2.0, and its activity gradually decreases to up to pH Here we analyzed chitin degradation by AMCase in weak acidic to neutral conditions by fluorophore-assisted carbohydrate electrophoresis established originally for oligosaccharides analysis We found that specific fragments with slower-than-expected mobility as defined by chitin oligosaccharide markers were generated at pH 5.0 ∼ 8.0 as by-products of the reaction We established an improved method for chitin oligosaccharides suppressing this side reaction by preacidification of the fluorophore-labeling reaction mixture Our improved method specifically detects chitin oligosaccharides and warrants quantification of up to 50 nmol of the material Using this strategy, we found that AMCase produced dimer of N-acetyl-d-glucosamine (GlcNAc) at strong acidic to neutral condition Moreover, we found that AMCase generates (GlcNAc)2 as well as (GlcNAc)3 under physiological conditions © 2017 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Chitin is a -1,4-linked polymer, insoluble in most solvents, composed primarily of N-acetyl-d-glucosamine (GlcNAc) residues It is a major component of the exoskeletons of crustaceans and insects, the microfilarial sheaths of parasitic nematodes and fungal cell walls (Khoushab & Yamabhai, 2010; Koch, Stougaard, & Spaink, 2015) Thus, chitin is the second most abundant polysaccharide in nature Chitinases are glycosidases that break down glycosidic bonds in chitin They are important enzymes responsible for chitin metabolism in a wide range of organisms, including bacteria, fungi, nematodes and arthropods (Bueter, Specht, & Levitz, 2013; Hamid et al., 2013; Khoushab & Yamabhai, 2010; Koch et al., 2015; Lee et al., 2011) Although mammals not produce chitin, mice and ∗ Corresponding author E-mail address: f-oyama@cc.kogakuin.ac.jp (F Oyama) These authors contributed equally to this article humans express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) (Bussink, van Eijk, Renkema, Aerts, & Boot, 2006; Lee et al., 2011) Chit1 was the first mammalian chitinase to be purified and its gene was cloned (Boot, Renkema, Strijland, van Zonneveld, & Aerts, 1995; Renkema, Boot, Muijsers, Donker-Koopman, & Aerts, 1995) AMCase was the second mammalian chitinase discovered and was named for its acidic isoelectric point (Boot et al., 2001) AMCase has attracted considerable attention due to its increased expression under certain pathological conditions related to immune response, for example in an induced asthma mouse model and antigen-induced mouse models of allergic lung inflammation (Reese et al., 2007; Zhu et al., 2004) Some polymorphisms and haplotypes in the AMCase gene are associated with bronchial asthma in humans (Bierbaum et al., 2005; Okawa et al., 2016; Seibold et al., 2009) and inhibition of its activity has been suggested as a therapeutic strategy against asthma (Sutherland et al., 2011; Yang et al., 2009) Furthermore, AMCase has been shown to be involved in eye (Bucolo, Musumeci, Maltese, Drago, & Musumeci, 2008; Bucolo, http://dx.doi.org/10.1016/j.carbpol.2017.01.095 0144-8617/© 2017 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4 0/) 146 S Wakita et al / Carbohydrate Polymers 164 (2017) 145–153 Musumeci, Musumeci, & Drago, 2011; Musumeci et al., 2009) and stomach diseases (Cozzarini et al., 2009; Nookaew et al., 2013) We have reported that AMCase mRNA is synthesized in the mouse stomach at exceptionally high levels These levels are comparable to pepsinogen, the precursor of the major digestive enzyme in gastric fluid, pepsin, suggesting a digestive role of AMCase (Ohno et al., 2013; Ohno, Tsuda, Sakaguchi, Sugahara, & Oyama, 2012) Moreover, we recently showed that AMCase is a proteasesresistant glycosidase in mouse digestive system, further supporting the hypothesis of AMCase functioning as a digestive enzyme (Ohno et al., 2016) We have also shown that beside stomach, AMCase mRNA is highly expressed in submaxillary gland and lung (Ohno et al., 2012) In addition, recombinant AMCase and its catalytic domain had the highest activity at around pH 2.0, when it produces primarily (GlcNAc)2 , and lower activities at more neutral pH (pH 3.0 ∼ 7.0) (Boot et al., 2001; Kashimura et al., 2015; Kashimura et al., 2013) The AMCase activity under somatic tissue conditions at pH ∼ remains to be elucidated and chitosan oligosaccharides (N-acetylChitin chitooligosaccharides) prepared either chemically or enzymatically, have been shown to have anti-cancer and antiinflammatory properties (Azuma, Osaki, Minami, & Okamoto, 2015; Masuda et al., 2014) and have various biological activities in mammalian cells (Aam, Heggset, Norberg, Sorlie, Varum, & Eijsink, 2010; Khoushab & Yamabhai, 2010) We hypothesized that upregulated AMCase under certain pathological conditions can generate specific degradation products associated with those pathologies Here we analyzed the chitinase activity of AMCase by incubating the enzyme with chitin substrates at pH 2.0 ∼ 8.0 followed by fluorophore-assisted carbohydrate electrophoresis (FACE), a method based on labeling the reducing ends of oligosaccharides with a fluorophore (Jackson, 1990) FACE is very sensitive (pmol amounts) as compared to high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectrometry, and is often used for detection of very low oligosaccharide quantities (Boot et al., 2001; Jackson, 1990) During our research, we found a pH-dependent generation of an unexpected by-product with a slower mobility than (GlcNAc)2 , the main fragment resulting from chitin substrates digestion by AMCase This by-product was observed at pH 5.0 ∼ 8.0 To optimize the digestion reaction, we established an improved method for a specific detection of chitin oligosaccharides Using this procedure, we found that AMCase generates (GlcNAc)2 at broad pH range of 2.0 ∼ 8.0 Materials and methods 2.1 Recombinant AMCase expressed in Escherichia coli and enzymatic activity assays We expressed and purified Protein A-AMCase-V5-His from the periplasmic fraction of the E coli as described previously (Kashimura et al., 2015; Kashimura et al., 2013) The proteincontaining fractions were desalted using PD MidiTrap G-25 (GE Healthcare, Milwaukee, WI, USA) equilibrated with TS buffer [20 mM Tris-HCl (pH 7.6), 150 mM NaCl and protein inhibitor (Complete Mini; Roche Diagnostics, Basel, Switzerland)] Chitinolytic activity was determined using a synthetic chromogenic substrate, 4-nitrophenyl N,N -diacetyl--d-chitobioside (Sigma-Aldrich, St Louis, MO, USA) as described previously (Kashimura et al., 2015; Kashimura et al., 2013) AMCase unit definition was also reported previously (Kashimura et al., 2013) 2.2 Degradation of colloidal chitin and (GlcNAc)6 by E coli-expressed mouse AMCase Colloidal chitin was prepared from shrimp shell chitin (SigmaAldrich), as described previously (Kashimura et al., 2013), and used as a substrate to determine the chitinase activity of AMCase All enzymatic reactions using colloidal chitin (at a final concentration of mg/mL) or N-acetyl-chitohexaose (GlcNAc)6 (0.2 mol/mL) (Seikagaku Corporation, Tokyo, Japan) were carried out in a volume of 50 L containing 0.8 mU or 0.1 mU E coli-expressed mouse AMCase in McIlvaine’s buffer (mixture of 0.1 M citric acid and 0.2 M Na2 HPO4 ; pH 2.0 to pH 8.0) The reaction mixtures were incubated for h at 37 ◦ C 2.3 Fluorophore labeling by the method of Jackson Generated chitin fragments or chitin mono- and oligomers of (GlcNAc)1∼6 (Seikagaku Corporation) as molecular weight markers were labeled covalently at their reducing end groups with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS, Invitrogen, Carlsbad, CA, USA), and separated by 40% polyacrylamide gel electrophoresis (PAGE), as described by Jackson (Jackson, 1990) Briefly, the enzymatic reaction samples were lyophilized and L of 0.2 M ANTS in acetic acid/water (3:17, v/v) and L of 1.0 M NaCNBH3 in dimethyl sulfoxide (DMSO) were added The mixture was incubated at 37 ◦ C for 16 h The reaction was neutralized by 10 L of M NaOH, followed by addition of 10 L Laemmli sample buffer (Laemmli, 1970) without SDS, 2-mercaptoethanol and bromophenol blue The samples were separated by PAGE and quantified using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare), according to the manufacturer’s instructions Exposure condition was fixed as follows: exposure type, precision; sensitivity, high resolution; exposure time, s 2.4 Pre-acidification method for labeling chitin oligomers Enzymatic reactions were lyophilized and L of 0.2 M ANTS in acetic acid/water (3:17, v/v), L of 1.0 M NaCNBH3 in DMSO and L of 17.5 M acetic acid were added for reaction acidification followed by incubation at 37 ◦ C for 16 h The reaction was neutralized by 15 L of M NaOH, followed by loading buffer addition The samples were analyzed by PAGE as described above 2.5 Separation of degradation products from (GlcNAc)6 by AMCase using HPLC Enzymatic reactions using (GlcNAc)6 (0.6 mol/mL) were performed in a volume of 300 L containing 4.2 mU of E coli-expressed mouse AMCase in McIlvaine’s buffer (pH 2.0 or pH 7.0), 30 mM GlyHCl (pH 2.0) or 30 mM Tris-HCl (pH 7.0) The reaction mixtures were incubated at 37 ◦ C for h Generated GlcNAc oligomers were separated by gel permeation chromatography (GPC) essentially as described previously (Kazami et al., 2015) Results 3.1 Detection of pH dependent fluorophore-labeled products at pH 5.0 ∼ 8.0 Previously, we have shown that the E coli-produced AMCase has the highest chitinolytic activity at around pH 2.0 which is decreasing in less acidic environment (pH 3.0 ∼ 7.0) against the synthetic chromogenic substrate, 4-nitrophenyl N,N -diacetyl--dchitobioside [4NP-(GlcNAc)2 ] (Kashimura et al., 2015; Kashimura et al., 2013) To determine whether AMCase can generate distinct S Wakita et al / Carbohydrate Polymers 164 (2017) 145–153 products depending on the pH condition, we incubated the enzyme with colloidal chitin or (GlcNAc)6 substrates at pH 2.0 ∼ 8.0 in McIlvaine’s buffer, followed by fluorophore-assisted carbohydrate electrophoresis (FACE) as originally described by Jackson (Jackson, 1990) AMCase degraded colloidal chitin primarily to (GlcNAc)2 fragments and to a lesser extent to (GlcNAc)3 as well as strong distinct GlcNAc monomer under acidic conditions, which were consistent with our previous observation (Fig 1A) (Kashimura et al., 2013) The hydrolysis was evident to up to pH 6.0 with decreasing activity upon increasing pH (Fig 1A) Thus, the recombinant AMCase can degrade high molecular weight chitin substrate at broad pH range However, there was an atypical product generated at pH 5.0 ∼ 8.0 that could represent a mobility shift of the (GlcNAc)2 fragment (Fig 1A and B) AMCase also degraded a lower molecular weight substrate, (GlcNAc)6 , and generated primarily (GlcNAc)2 and to a lesser extent (GlcNAc)3 fragments (Fig 1B) In addition, we detected the by-product, whose presence was evident at pH values above (Fig 1B) These results suggest a pH-dependent generation of an atypical by-product from chitin substrates degradation by recombinant AMCase or from FACE reaction 3.2 By-products generation in the fluoresceination reaction and pre-acidification procedure for detection of the GlcNAc oligomers Next, we explored whether the FACE reaction could lead to the slower mobility of the GlcNAc oligomers in the absence of AMCase at pH 2.0 or 7.0 using McIlvaine’s buffer Bands with expected mobility were obtained when incubated at pH 2.0 (Fig 2A, left and right) In contrast, incubation at pH 7.0 resulted in two bands from each GlcNAc oligomer using Jackson method (Fig 2A, center and right) (Jackson, 1990) Slower migrating by-products of the GlcNAc oligomers appeared when the fluorophore labeling reaction was performed at pH 7.0 (Fig 2A) Moreover, the fluoresceination efficiency was lower at pH 7.0 when compared to that at pH 2.0 (Fig 2A) Thus, caution should be exercised when performing fluorescence labeling of the GlcNAc oligomers directly obtained from the enzymatic reaction at pH 5.0 ∼ 8.0 using McIlvaine’s buffer by the method of Jackson (Jackson, 1990) Next, we attempted to optimize the reaction conditions to suppress the by-product formation at neutral pH When we acidified the reaction with acetic acid before the labeling with fluorophore (pre-acidification), we obtained single bands for all tested oligomers at both conditions (Fig 2B) Thus, the by-products were formed in a pH-dependent manner during the fluorescent labeling These results indicate that our pre-acidification method by concentrated acetic acid just before the labeling reaction effectively prevents the formation of unwanted fragments 3.3 Effect of buffers on the fluorescent labeling We next examined the effects of several buffer systems commonly used in the biochemical evaluation of chitinolytic activities on the fluorescent labeling of (GlcNAc)1∼6 We tested McIlvaine’s (Fig 3A), phosphate or Tris-HCl (Fig 3B), as well as 2-(N-morpholino) ethanesulfonic acid (MES), piperazineN,N -bis (2-ethanesulfonic acid) (PIPES), 4-(2-Hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino) propanesulfonic acid (MOPS) (Fig 3C) buffers with final concentrations adjusted to 150 mM at pH 7.0 Our pre-acidification method for the FACE reaction appeared beneficial irrespective of the used buffer systems (Fig 3) confirming universal enhancement of the reaction at pH 7.0 147 3.4 Application of the pre-acidification method for determination of the GlcNAc oligomers using standard curve To evaluate the ability for GlcNAc dimers quantification, we generated standard curves at pH 7.0 and found a linearity between (GlcNAc)2 and fluorescence intensity to up to 50 nmol (Fig 4) The linearity was preserved with the pre-acidification step (Fig 4) The detection sensitivity at pH 7.0 was not inhibited by increasing levels of acetic acid when the (GlcNAc)2 was labeled using the pre-acidification method Thus, our method enables GlcNAc oligomers quantification in chitinolytic reactions performed at pH 7.0 To analyze a sample that contains less than nmol of (GlcNAc)2 per 50 L, the volume of the sample solution could be modified from 50 L to 150 L Otherwise, addition of concentrated acetic acid to the sample or longer exposure in a luminescent image analyzer is recommended 3.5 Re-evaluation of chitin substrates degradation by AMCase using pre-acidification Using our pre-acidification method, we re-evaluated the pHdependent degradation of chitin substrates by AMCase Incubation of the enzyme (0.8 mU) with colloidal chitin for h in McIlvaine’s buffer at pH 2.0 ∼ 8.0 resulted in production of primarily (GlcNAc)2 with a low amount of (GlcNAc)3 as well as strong distinct GlcNAc monomer (Fig 5A) Incubation of AMCase with (GlcNAc)6 for h in McIlvaine’s buffer at pH 2.0 ∼ 8.0 also resulted in production of primarily (GlcNAc)2 (Fig 5B) The pH-dependent increase of (GlcNAc)3 levels was more pronounced as compared to colloidal chitin (Fig 5B) 3.6 Chitinolytic properties of AMCase in weak acidic and neutral conditions The results described above indicate that AMCase can degrade colloidal chitin and (GlcNAc)6 with relatively high activity even in weak acidic and neutral environment To further clarify this finding, we incubated a reduced AMCase amount (0.1 mU) with (GlcNAc)6 or 4NP-(GlcNAc)2 , a chromogenic substrate used for evaluating chitinolytic properties of the recombinant enzyme, at pH ranging from 2.0 to 8.0 at 37 ◦ C for h FACE analysis using pre-acidification step indicated that AMCase produced primarily (GlcNAc)2 and (GlcNAc)3 at pH 2.0 ∼ 5.0 with a second peak (GlcNAc)2 at pH 6.0 and seemed to be active up to pH 8.0 (Fig 6A) The colorimetric analysis using 4NP(GlcNAc)2 at 405 nm indicated that its cleavage reached maximum at pH 2.0 and it decreased at more neutral pH (pH 3.0 ∼ 7.0) (Fig 6B) Although both analysis showed similar results, FACE analysis could detect more pronounced second peak at pH 6.0 (Fig 6C) Our results indicate that AMCase can degrade chitin substrates to (GlcNAc)2 at pH 2.0 ∼ 8.0 These data show that the FACE analysis with pre-acidification is much more sensitive than the assay using chromogenic substrate, 4NP-(GlcNAc)2 Our results suggest that AMCase can degrade chitin substrates not only in stomach but also in other tissues including lungs and submaxillary glands 3.7 Detection of GlcNAc oligomers using HPLC or FACE Next, using HPLC or FACE, we evaluated the degradation of (GlcNAc)6 by AMCase at strong acidic and neutral conditions First, we degraded (GlcNAc)6 in McIlvaine’s buffer using AMCase and analyzed the HPLC-separated the products having absorbance at 210 nm We obtained buffer-derived peaks, which are eluted at approximate positions of (GlcNAc)4∼6 , whereas our improved FACE method detected proper (GlcNAc)2 and (GlcNAc)3 signal (Supple- 148 S Wakita et al / Carbohydrate Polymers 164 (2017) 145–153 Fig Detection of pH-dependent fluorophore-labeled bands at pH 5.0 ∼ 8.0 Chitinolytic activity of mouse AMCase was investigated by incubating the enzyme (0.8 mU) with colloidal chitin or (GlcNAc)6 at pH 2.0 ∼ 8.0 in McIlvaine’s buffer, followed by FACE as described in Materials and methods Chitin oligomers are shown in the left margin as standards Recombinant AMCase degraded colloidal chitin (A) or (GlcNAc)6 (B) and generated primarily (GlcNAc)2 fragments, and in lower amounts the GlcNAc monomer and (GlcNAc)3 The quantification is shown right Fluorescence intensity estimated from the results in Fig 1A and B Thin arrows, (GlcNAc)2 fragments; thick arrows, slow mobile bands Fig Comparison of the fluorescent labeling between Jackson’s and our pre-acidification methods A Efficiencies of the Jackson’s method at pH 2.0 (left) or 7.0 (center) using McIlvaine’s buffer The quantification is shown right B The efficiency of the fluorescent labeling chitin oligosaccharide with pre-acidification at pH 2.0 (left) or 7.0 (center) The quantification is shown right S Wakita et al / Carbohydrate Polymers 164 (2017) 145–153 149 Fig Effect of different buffers on the labeling of the ANTS conjugate We tested for labeling of (GlcNAc)1∼6 using McIlvaine’s buffer (A), phosphate or Tris-HCl buffer (B) and Good buffers (MES, PIPES, HEPES and MOPS) (C) The final concentration of each buffer was adjusted to 150 mM at pH 7.0 M, molecular weight markers of N-acetyl chitooligosaccharides; J, Jackson’s method; P, pre-acidification method Next, we changed the buffer system using Gly-HCl (pH 2.0) and Tris-HCl (pH 7.0), respectively We first confirmed that these buffer systems gave no significant background except for a minor peak at pH 2.0 at around expected (GlcNAc)2 signal (Supplementary Fig S2A and B) Using these buffer systems, we analyzed the degraded products by HPLC and our improved FACE methods The results presented in Figs and can be replicated by HPLC at pH 2.0 and pH 7.0, indicating that AMCase produces (GlcNAc)2 at strong acidic and neutral conditions (Fig 7A and B) For FACE analysis, we used a 10× diluted sample because our improved method is very sensitive (pmol amounts) when compared with the conventional HPLC (Fig 7C and D) Based on these results, we conclude that AMCase generates (GlcNAc)2 under physiological conditions and that our improved FACE method is very sensitive and effective for the detection and quantification of chitin oligomers Discussion Fig Quantification of (GlcNAc)2 using standard curve A To evaluate the ability for GlcNAc dimers quantification, we labeled (GlcNAc)2 fragments at pH 7.0 B Quantification of GlcNAc oligomer was tested at up to 50 nmol of (GlcNAc)2 There was a linearity between (GlcNAc)2 and fluorescence intensity in the whole range at pH 7.0 The linearity was preserved when using pre-acidification mentary Fig S1A and B) This is probably caused by the citrate and phosphate in McIlvaine’s buffer having absorbance at 210 nm AMCase has been shown to be predominantly produced in mouse stomach (Boot et al., 2005; Ohno et al., 2013; Ohno et al., 2012) and to have maximal enzymatic activity at pH 2.0 (Boot et al., 2001; Kashimura et al., 2013) Previous studies were carried out using natural chitin substrates at acidic conditions concluding that AMCase produces primarily (GlcNAc)2 at pH 2.0 (Boot et al., 2001; Kashimura et al., 2015; Kashimura et al., 2013) It has also been shown that AMCase can function as a digestive enzyme in the mouse gastrointestinal tract (Ohno et al., 2016; Ohno et al., 2013; Ohno et al., 2012) 150 S Wakita et al / Carbohydrate Polymers 164 (2017) 145–153 Fig Re-examination of the pH degradation of chitin substrates by AMCase using pre-acidification method We performed same experiments in Fig 1A and B except for labeling by ANTS using our pre-acidification method Fig Chitinolytic properties of AMCase under somatic tissue pH conditions A The enzymatic reactions using (GlcNAc)6 were carried out in a volume of 50 L containing 0.1 mU E coli-expressed mouse AMCase in McIlvaine’s buffer B The pH dependence of chitinolytic activity of AMCase using 4NP-(GlcNAc)2 as a substrate The values represent the percentage of the maximum activity at pH 2.0 C Comparison of the chitinolytic activity of recombinant AMCase analyzed by our improved FACE method (Fig 6A) and the colorimetric analysis using 4NP-(GlcNAc)2 at 405 nm (Fig 6B) The values were represented as percentage of the maximum activity at pH 2.0 Our previous gene expression analysis revealed that AMCase mRNA is also expressed at high levels in salivary gland and lung tissues (Ohno et al., 2012) Chitin oligomers (Nacetyl-chitooligosaccharides) have various biological activities in mammalian cells (Aam et al., 2010; Khoushab & Yamabhai, 2010) Anti-cancer and anti-inflammatory properties have been reported for both chitin and chitosan oligosaccharides (Azuma et al., 2015; Masuda et al., 2014) Mouse AMCase has been shown to be active at up to pH 8.0, although its activity substantially decreases with higher pH (Boot et al., 2001; Kashimura et al., 2013) We hypothesized that under pathological conditions, AMCase is upregulated and can generate specific degradation products different from those in stomach condition and that they can modify the bioactivity of the chitin degradation products Therefore, we have been interested in S Wakita et al / Carbohydrate Polymers 164 (2017) 145–153 151 Fig Detection of GlcNAc oligomers using HPLC and FACE methods (GlcNAc)6 was degraded by AMCase at pH 2.0 or pH 7.0 as described in Materials and Methods The samples were analyzed by HPLC (A) or FACE (B) Numbers with arrows indicate the number of GlcNAc units in the corresponding peaks For FACE, we used a 10 x diluted sample C Analysis of HPLC-separated degradation products by HPLC D Quantification of data generated by HPLC and FACE the AMCase-mediated degradation pattern of chitin substrates at neutral conditions The widely-used Jackson method for oligosaccharides detection (Jackson, 1990) is used for reducing ends labeling in the analyzed molecules followed by high resolution PAGE We usually conduct chitin degradation reaction using McIlvaine’s buffer, which is a citrate/phosphate buffer system allowing us to set the pH over a broad range However, we found two drawbacks of this method for chitin oligosaccharides labeling directly used in high concentration of non-volatile buffer By FACE analysis, we noticed that AMCase produced not only the predominant GlcNAc dimer but also certain number of fragments with slower mobility at pH > 5.0 We found that these additional fragments (here called “by-products”) were generated at pH 5.0 ∼ 8.0 not during the digestion by AMCase but during the oligomers labeling (Fig 1) Importantly, we observed reduced labeling at higher pH values when compared to pH 2.0 (Fig 2A) To overcome these problems, we developed an improved method for fluorescent labeling enabling us to efficiently label the reducing ends of the GlcNAc oligomers generated during the enzymatic reactions performed at pH 2.0 ∼ 8.0 We achieved this by acidification of the sample by concentrated acetic acid just before the labeling step with the fluorophore Importantly, during labeling, pH has to be adjusted to pH < 4.0 The original Jackson method, however, is suitable for pH < 4.0, adjusted by addition of concentrated acetic acid to the dried sample (Jackson, 1990) Although we not know the chemical structure of the by-products, our preacidification procedure was able to suppress the side reaction and form single bands corresponding to the GlcNAc oligomer markers (Fig 2B) This method is simple and can be applied to various buffers commonly used for biochemical analyses (Fig 3) In addition, the fluorescence efficiency increased allowing better quantification of the GlcNAc oligomers Our pre-acidification method presented in this report [diagrammatic scheme of overall our improved method and the formation of ANTS-(GlcNAc)2 are described in Supplementary Figs S3 and S4] is essentially identical with the methods reported by Jackson (Jackson, 1990) except for addition of concentrated acetic acid before fluorescent labeling reaction In the original Jackson method (Jackson, 1990), the saccharide samples to be labeled were initially dissolved in water or else dissolved in 0.1 M ammonium acetate, then dried under vacuum and re-dissolved in water Because ammonium acetate is volatile, the dried sample would be buffer-free before re-dissolving in water Then, for the labeling reaction the diluted acetic acid is sufficient to maintain the acidic environment needed to catalyze the opening of the reducing end sugar But, in our experiments, all samples were dissolved in high concentration of non-volatile buffer before labeling This is why we found it necessary to add more acetic acid, in order to ensure an acidic solution during the labeling reaction Our improved method provides a simple solution to the observation of unknown side products in the fluorescent labeling of chitin oligosaccharides at the reducing end, for use in FACE analysis of chitin oligosaccharides It could become widely adopted by other laboratories Finally, we re-examined the pH-dependent degradation of the chitin substrates using our pre-acidification method for labeling the GlcNAc oligomers (Figs and 6) We noticed that, at pH ∼ 8, the products generated from chitin substrates are more abundant than those from the synthetic chromogenic substrate of 4NP-(GlcNAc)2 (Fig 6) (Kashimura et al., 2013) These data indicate that the degradation ability of AMCase against colloidal chitin is superior to the synthetic substrates at pH ∼ and that AMCase can work under somatic tissue pH conditions Family 18 chitinases are proposed to utilize the substrate-assisted catalytic mechanism while their catalytic domains having TIM-barrel fold (Terwisscha van Scheltinga et al., 1995) The DXXDXDXE motif included in this domain is thought to have a central role in substrate binding and catalysis in acidic condition (Chou et al., 2006) with His187 of AMCase being responsible for the acidic optimum (Bussink, Vreede, Aerts, & Boot, 2008) This folding mechanism could explain the mouse AMCasemediated (GlcNAc)2 production at pH 2.0 The mechanistic details of the AMCase activity at pH 7.0 remains to be determined Taken 152 S Wakita et al / Carbohydrate Polymers 164 (2017) 145–153 together, AMCase can work under strong acidic to neutral condition and degrade chitin substrates to produce predominantly (GlcNAc)2 We did not obtain any indication of longer GlcNAc oligomers production under physiological conditions One can speculate that AMCase-mediated longer GlcNAc oligomers production may occur under pathological conditions On the other hand, long oligomers produced by inefficient chitin degradation, might be stimuli or enhancers of certain pathologies Conclusions Mouse AMCase has optimal pH at pH 2.0 for the maximum activity and is active at up to pH We performed an extensive analysis of chitin degradation by AMCase not only in strong acidic, but also weak acidic to neutral conditions When we analyzed chitin degradation products in weak acidic to neutral conditions by FACE established originally for oligosaccharides analysis, we found that the by-products in the fluoresceinated reaction were formed by labeling at pH > and established a pre-acidification method for chitin oligosaccharide analysis to suppress these products formations Using this procedure, we found that AMCase generates (GlcNAc)2 and (GlcNAc)3 at conditions mimicking somatic tissue pH conditions as well as at pH 2.0 Acknowledgements We are grateful to Daisuke Yamanaka, Naohito Ohno, Tadatomo Kawai, Shinji Nagumo, Yasutada Imamura and Yoshiaki Furukawa for their suggestions and encouragement, to Kazuaki Okawa, Daisuke Mizutani, Eri Tabata for valuable suggestions This work was supported by the Project Research Grant from the Research Institute of Science and Technology, Kogakuin University, and Grants-in-Aid for Scientific Research (15J10960 and 16K07699) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Grant from the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan and in part by a grant of the Strategic Research Foundation Grant-aided Project for Private Universities (S1411005) from the Ministry of Education, Culture, Sport, Science and Technology, Japan P.O.B received support from ALS Association and Mayo Clinic Center for Regenerative Medicine Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.01 095 References Aam, B B., Heggset, E B., Norberg, A L., Sorlie, M., Varum, K M., & Eijsink, V G (2010) Production of chitooligosaccharides and their potential applications in medicine Marine Drugs, 8(5), 1482–1517 Azuma, K., Osaki, T., Minami, S., & Okamoto, Y (2015) Anticancer and anti-inflammatory properties of chitin and chitosan oligosaccharides Journal of Functional Biomaterials, 6(1), 33–49 Bierbaum, S., Nickel, R., Koch, A., Lau, S., Deichmann, K A., Wahn, U., et al (2005) Polymorphisms and haplotypes of acid mammalian chitinase are associated with bronchial asthma American Journal of Respiratory and Critical Care Medicine, 172(12), 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