Uncovering the catalytic direction of chondroitin ac exolyase-from the reducing end towards the non-reducing end

Article abstract of DOI:10.1074/jbc.C115.708396

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes

Full text of DOI:10.1074/jbc.C115.708396

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 9, pp. 4399–4406, February 26, 2016
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Uncovering the Catalytic Direction of Chondroitin AC
Exolyase
FROM THE REDUCING END TOWARDS THE NON-REDUCING END*
Received for publication, December 3, 2015, and in revised form, December 28, 2015 Published, JBC Papers in Press, January 7, 2016, DOI 10.1074/jbc.C115.708396
Feng-Xin Yin (尹风新)‡, Feng-Shan Wang (王凤山)‡§1, and X Ju-Zheng Sheng (生举正)‡§2
From the ‡Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and
Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China and §National
Glycoengineering Research Center, Shandong University, Jinan 250012, China
Glycosaminoglycans (GAGs) are polysaccharides that play will therefore enhance our collective understanding of the mode
vital functional roles in numerous biological processes, and of action of ChnAC.
compounds belonging to this class have been implicated in a
wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC
Glycosaminoglycans (GAGs)3 are long unbranched polysac-
charides consisting of repeating disaccharide units that can be
found in the connective tissues of animals (1, 2). In terms of
their structure, GAGs are composed of repeating disaccharide
units containing a glucosamine (e.g. galactosamine or glucosa-
mine) and uronic acid, such as glucuronic acid (GlcA) or idu-
ronic acid (IdoA) (2). Heparan sulfate (HS), chondroitin sulfate
(CS), and hyaluronic acid (HA) are three of most extensively
studied classes of GAGs (2). The glucosamine and uronic acid
residues in HS and CS are modified by the sulfation process,
whereas HA is not naturally sulfated. These polysaccharides are
thought to be involved in the regulation of various cellular pro-
cesses such as adhesion, differentiation, migration, and prolif-
eration (1, 3–6). GAGs and their low molecular weight
derivates, which can be prepared by various methods of depoly-
merization, have therefore received considerable interested in
terms of their potential clinical applications as anticoagulant,
antitumor, anti-inflammatory, and anti-complement agents
(7–11). Several different degradation methods, including
chemical and enzymatic approaches, can be used to provide
access to structurally distinct GAG chains, which can display a
wide variety of different pharmacological and pharmacokinetic
properties (12–14). Among the GAGs, CS consists of repeating
disaccharide units of GlcA and N-acetyl-galactosamine (Gal-
NAc) with ␤-1,3 and ␤-1,4 linkages; each of which is capable of
carrying sulfonate groups. CS can be classified into several dif-
ferent types depending on the position of the sulfate groups. For
example, CS-A, CS-C, and CS-E contain GalNAc-4-O-sulfate,
GalNAc-6-O-sulfate, and GalNAc-4,6-O-sulfate groups,
respectively. CS-B (dermatan sulfate, DS) differs from CS-A in
that the GlcA unit has been replaced by an IdoA unit. Com-
pared with CS-E, CS-D contains an additional sulfated modifi-
cation on carbon 2 of the GlcA unit.
4.2.2.5) catalyzes the degradation of various GAGs, including
chondroitin sulfate and hyaluronic acid, to give the correspond-
ing disaccharides containing an ⌬4-unsaturated uronic acid at
their non-reducing terminus. ChnAC has been isolated from
various bacteria and utilized as an enzymatic tool for study and
evaluating the sequencing of GAGs. Despite its substrate speci-
ficity and the fact that its crystal structure has been determined
to a high resolution, the direction in which ChnAC catalyzes the
cleavage of oligosaccharides remain unclear. Herein, we have
determined the structural cues of substrate depolymerization
and the cleavage direction of ChnAC using model substrates and
recombinant ChnAC protein. Several structurally defined
oligosaccharides were synthesized using a chemoenzymatic
approach and subsequently cleaved using ChnAC. The degrada-
tion products resulting from this process were determined by
mass spectrometry. The results revealed that ChnAC cleaved
the ␤1,4-glycosidic linkages between glucuronic acid and gluco-
samine units when these bonds were located on the reducing
end of the oligosaccharide. In contrast, the presence of a
GlcNAc-␣-1,4-GlcA unit at the reducing end of the oligosac-
charide prevented ChnAC from cleaving the GalNAc-␤1,4-
GlcA moiety located in the middle or at the non-reducing end of
the chain. These interesting results therefore provide direct
proof that ChnAC cleaves oligosaccharide substrates from their
reducing end toward their non-reducing end. This conclusion
* This work was supported in part by the National Natural Science Founda-
tion of China (Project no. 31300673), the Specialized Research Fund for the
Doctoral Program of Higher Education (Project no. 20130131120046), the
Projects of Science and Technology Dept. of Shandong Province (Project
no. 2015GSF121002 and 2015ZDJS04002), and the Fundamental Research
Funds (Project no. 2014YQ002) of Shandong University. A patent
(CN201510289566) has been published relating to this article. Despite this
publication, the authors have strictly adhered to all the policies provide by
the The Journal of Biological Chemistry regarding the sharing of data and
materials.
A variety of bacterial CS lyases have been prepared with the
ability to affect the enzymatic degradation of CS polysaccha-
rides (15, 16). Although these CS lyases followed the same gen-
1 To whom correspondence may be addressed: Institute of Biochemical
and Biotechnological Drugs, School of Pharmaceutical Science, Shan-
dong University, Jinan 250012, China. Tel.: ϩ86-0531-88382658; E-mail:
fswang@sdu.edu.cn.
2 To whom correspondence may be addressed: Institute of Biochemical 3 The abbreviations used are: GAG, glycosaminoglycan; ChnAC, chondroitin
and Biotechnological Drugs, School of Pharmaceutical Science, Shan-
dong University, Jinan 250012, China. Tel.: ϩ86-0531-88380288; E-mail:
shengjuzheng@sdu.edu.cn.
AC lyase; CS, chondroitin sulfate; HA, hyaluronan; AsChnAC, Arthrobacter
ChnAC; PAMN-HPLC, polyamine-based anion exchange; ESI-MS, electros-
pray ionization mass spectrometry; HS, heparan sulfate.
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Catalytic Direction of Chondroitin AC Lyase
eral mechanism, in that they catalyzed a ␤-elimination reaction units. This selection was based on the fact that the CS backbone
at the C4 position of GlcA to yield ⌬4,5-unsaturated GlcA at the structure (-GalNAc-␤-1,4-GlcA-) can be cleaved by ChnAC,
non-reducing end of the oligosaccharide products, these whereas the HS backbone structure (-GlcNAc-␣-1,4-GlcA-)
enzymes exhibited different substrate specificities (17–19). remains unchanged (16, 19). The site resistant to being cleaved
Briefly, chondroitin AC lyase (ChnAC, EC 4.2.2.5) cleaves by the ChnAC enzyme was positioned on the reducing or the
HA, CS-A, and CS-C. Chondroitin-sulfate-ABC exolyase non-reducing terminus, and the products resulting from the
(exChnABC, EC 4.2.2.21) and chondroitin-sulfate-ABC treatment of these substrates with ChnAC were identified by
endolyase (enChnABC, EC 4.2.2.20) can both degrade CS-A, electrospray ionization mass spectrometry (ESI-MS). The
CS-C, and DS, although the latter of these enzymes shows results of these experiments therefore provided an opportunity
endolytic activity. Notably, chondroitin B lyase (ChnB, EC to determine the preference of this enzyme for endolytic cleav-
4.2.2.19) is specific for DS. To date, a number of ChnAC age at the reducing or non-reducing ends of the different sub-
enzymes have been obtained from various bacteria, including strates. The ChnAC isolated from Arthrobacter sp.5 displayed
Arthrobacter sp (16, 20), Flavobacterium heparinum (21, 22), cleavage activity toward the oligosaccharide substrate when the
Serratia marcescens (23) and Bacteroides stercoris (24), and GalNAc-␤1,4-GlcA residue was present at the reducing end. In
these enzymes have been subjected to extensive biochemical contrast, this enzyme displayed no activity toward the degrada-
and x-ray crystallographic analyses. Interestingly, the enzy- tion of the oligosaccharide substrate were the GalNAc-␤-1,4-
matic characterization of the ChnAC from F. heparinum GlcA residues at the reducing end had been replaced with
showed that it acted as an endolyase toward a polysaccharide GlcNAc-␣-1,4-GlcA residues. These data demonstrate, for the
substrate, producing a mixture of unsaturated oligosaccharides first time, that ChnAC cleaves polysaccharides from their
of different sizes (25). This behavior was in contrast to that of reducing end toward their non-reducing end. The results of this
the other bacterial ChnAC lyases listed above, which acted as study will therefore advance our understanding of the mode of
exolyases. Crystal structures of the ChnAC isolated from action of ChnAC enzymes.
Arthrobacter sp. bound to various oligosaccharide substrates
Experimental Procedures
revealed that the lytic mechanism of this enzyme was depen-
Materials—1-O-(para-nitrophenyl)-glucuronide (GlcA-pNP),
uridine 5Ј-diphosphate (UDP)-N-acetylglucosamine (GlcNAc),
UDP-N-acetylgalactosamine (GalNAc), and UDP-GlcA were
purchased from Sigma Aldrich.
dent of the (␣/␣)5 toroidal fold. These data also showed that the
Tyr-242 residue acted as a base to abstract a proton from the C5
position of GlcA, while the Asn-183 and His-233 residues neu-
tralized the charge on the acidic group of GlcA (16). ChnAC
and several other CS lyases have been widely used in conjunc-
tion with modern separation and analytical methods for disac-
charide analysis, polysaccharide sequencing, and biological
evaluations (19). However, very little is known about the direc-
tion of the ChnAC-catalyzed degradation of polysaccharides.
This lack of understanding regarding the direction of the deg-
radationismainlyduetothelackofstructurallysuitableoligosac-
charides. An advanced chemoenzymatic method was recently
developed for the efficient synthesis of GAG oligosaccharides
with high purity (26–28). Furthermore, the bacterial enzymes
involved in the synthesis of the GAG backbone, including
N-acetyl glucosaminyl transferase from Escherichia coli K5
strain (KfiA), heparosan synthase 2 from Pasteurella multocida
(PmHS2), and E. coli K4 chondroitin polymerase (KfoC), show
broad donor substrate specificities and acceptor promiscuity
(29).4 These developments have allowed us to prepare structur-
ally defined chimeric oligosaccharide substrates to probe the
directionality of this process in greater detail.
Enzymes—Arthrobacter species chondroitin AC lyase genes,
AsChnAC (GenBankTM accession number KT985049), were
cloned into a pET28a(ϩ) expression plasmid2, followed by a
transformation step into E. coli BL21 (DE3). The resulting
recombinant strain was used as a source of recombinant
ChnAC. The N-His-tagged AsChnAC were purified over a Ni
Sepharose 6 Fast Flow column (GE Healthcare Bio-Sciences
AB, Uppsala, Sweden) following the protocol provided by the
manufacturer. KfiA and PmHS2 were expressed in E. coli and
purified using an appropriate affinity chromatography system
as described previously (27, 32–34). KfoC (GenBankTM acces-
sion number AB079602) was recombinantly expressed in E. coli
BL21 (DE3) cells as a soluble N-His6-tagged fusion protein and
purified using an appropriate affinity chromatography system.4
Preparation of Model Oligosaccharide Substrates—Four
structurally defined oligosaccharides were prepared using
enzymatic synthesis following previously published procedures
from the literature (32, 33, 35). All four of the oligosaccharides
prepared in the current study were synthesized from the com-
mercially available monosaccharide of GlcA-pNP. Notably, the
␣-(1,4) linkages between the GlcNAc and GlcA residues and the
␤-(1,4) linkages between the GalNAc and GlcA residues were
built with PmHS2 and KfoC, respectively. Briefly, the CS-trisac-
charide (GlcA-␤1,3-GalNAc-␤1,4-GlcA-pNP) was synthesized
using KfoC, and the subsequent elongation of this trisaccharide
to the HP-CS-hexasaccharide was completed by KfiA and
PmHS2. In contrast, the HP-trisaccharide (GlcA-␤1,4-Glc-
NAc-␣1,4-GlcA-pNP) was initially built by KfiA and pmHS2,
The primary goal of this study was to uncover the direction of
the ChnAC-catalyzed polysaccharide degradation using a
library of structurally defined oligosaccharides. A similar strat-
egy to this was successfully applied to the bacterial heparosan
lyase and mammalian heparanase (30, 31). However, the oligo-
saccharides involved in these previous studies were synthesized
based on the natural structure of the GAG polysaccharides. In
this study, we decided to synthesize chimeric oligosaccharides
concurrently containing repeating HS and CS disaccharide
4 J. J. Xue, L. Jin, X. K. Zhang, F. S. Wang, P. X. Ling, and J. Z. Sheng, manuscript
in preparation.
5 F. X. Yin, F. S. Wang, and J. Z. Sheng, unpublished observations.
4400 JOURNAL OF BIOLOGICAL CHEMISTRY
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Catalytic Direction of Chondroitin AC Lyase
and its subsequent elongation to the desired size was achieved their N-acetylhexosamine and GlcA residues. The HP-CS-hex-
using a KfoC-catalyzed glycosyl transferase reaction. For a typ-
ical elongation reaction, 20 mg of acceptor was incubated with
1.5 equivalents of UDP-sugar and 2 mg of enzyme in 40 ml of
buffer containing 25 mM Tris-HCl (pH 7.2) and 10 mM MnCl2 at
37 °C for 12 h. To confirm that the acceptor had been com-
pletely converted to the desired elongation product, the reac-
tions were monitored by polyamine-based anion exchange
(PAMN)-HPLC. For HPLC, the column (4.6 ϫ 250 mm, Shi-
mogyoku, Kyoto, Japan) was eluted with a linear gradient of
KH2PO4 from 0 to 0.6 M over 40 min at a flow rate of 0.5
ml/min. The para-nitrophenyl (pNP) group was detected at
310 nm. The product was then purified over a Bio-Gel P-2
column, with the desired fractions being detected based on
their UV absorbance at 310 nm. In this way, we synthesized
milligram quantities of these model oligosaccharide sub-
strates and confirmed their structures by ESI-MS and NMR
analyses.
asaccharide had two repeating -GalNAc-␤-1,4-GlcA- units at
its reducing end. In contrast, the repeating units in the CS-HP-
hexasaccharide were configured with the opposite arrange-
ment to those of the HP-CS-hexasaccharide, in that it has one
␣-1,4glycosidicbondatitsreducingend.Allfouroftheoligosac-
charides in this study were synthesized from the commercially
available monosaccharide GlcA-pNP. Notably, the progress of
eachreactionwasreadilymonitoredbyexploitingtheUVabsor-
bance of the pNP tag at 310 nm. The elongation of GlcA-pNP to
give the HP-trisaccharide was accomplished using two bacterial
glycosyltransferases: KfiA and pmHS2. The synthesis of the CS-
HP-hexasaccharide was initiated from the HP-trisaccharide,
and the subsequent elongation steps were accomplished using
KfoC. Although the HP-trisaccharide behaved as a suitable
acceptor for the ␤-1,4-N-acetylgalactosaminyltransferase
activity of KfoC,4 the synthesis of the first ␤-1,4 glycosidic bond
remained a significant synthetic challenge because of the low
reaction rate. A carefully designed ratio of acceptor to donor
was therefore employed to deliver the desired product with
high purity and yield from this step. Similar procedures and
approaches were also used to synthesize the CS-trisaccharide
and the HP-CS-hexasaccharide. All of these oligosaccharides
were purified using conventional techniques and their purities
and structures were confirmed by (PAMN)-HPLC and ESI-MS
analyses, respectively (Figs. 2, A, B, D, E; 3, A, B; 4, A, B).
Cleavage of Synthetic Oligosaccharides—To uncover the
catalytic direction of ChnAC, we evaluated the elimination
products of this enzyme toward several structurally defined
oligosaccharides, including CS-trisaccharide, HP-trisaccharide,
HP-CS-hexasaccharide, and CS-HP-hexasaccharide. A typical
elimination reaction was conducted in a total volume of 1.5 ml
(0.4 mM HP-CS-hexasaccharide, pH 7.0) containing 1 ␮g of
purified ChnAC for 0, 1, 10 min, or 16 h at 37 °C, followed by the
subsequent heating of the mixture at 100 °C for 5 min and sep-
aration by centrifugation. The reaction mixture was monitored
by (PAMN)-HPLC, followed ESI-MS analysis.
The results of x-ray crystallography studies pertaining to
GAG eliminase and its enzyme-substrate complexes have
allowed for the molecular mechanisms of polysaccharide deg-
radation to be elucidated, as well as providing information con-
cerning the potential direction of the reaction (16, 22, 36, 37,
39). However, the lack of conclusive evidence to support the
direction of this process means that the direction currently
remains unclear, which is primarily due to a lack of appropriate
homologous model substrates. Herein, we have prepared struc-
turally defined oligosaccharides to determine the catalytic
direction of the ChnAC directly. We decided to synthe-
size chimeric oligosaccharides containing repeating HS
(-GlcNAc-␣-1,4-GlcA-) and CS (-GalNAc-␤-1,4-GlcA-) disac-
charide units, and positioned the ChnAC-resistant site on the
reducing or non-reducing end of the oligosaccharide, respec-
tively. First, the degradation products resulting from the treat-
ment of the HP- and CS-trisaccharides with purified AsChnAC
were purified and analyzed by (PAMN)-HPLC and ESI-MS,
respectively (Fig. 2). As expected, two new compounds (Gal-
NAc-GlcA and ⌬Di-pNP) were obtained following the depoly-
merization of the CS-trisaccharide (Fig. 2C). In contrast, no
peaks were detected at 232 nm following the treatment of the
HP-trisaccharide sample with ChnAC, which suggested that
the HP-trisaccharide was a poor substrate for ChnAC (Fig. 2F).
Taken together, these results once again confirmed that
ChnAC can selectively recognize and cleave the ␤-1,4 linkages
between the N-acetylhexosamine and GlcA residues, but con-
not act on the ␣-1,4 linkages (40, 41). These results also sug-
gested that the pNP tag did not affect the substrate specificity of
the ChnAC enzyme and that trisaccharides bearing a pNP tag
ESI-MS Analysis—ESI-MS analyses were performed on a
Thermo LCQ-Deca system (Thermo Fisher Scientific Inc.).
The samples were dissolved in 50% methanol. Experiments
were performed in the negative ionization mode with a spray
voltage of 3.5 kV and a capillary temperature of 275 °C.
Results and Discussion
Our efforts for determining the direction of the chondroitin
AC lyase-catalyzed degradation of the chondroitin sulfate (CS)
chains required the expression of recombinant chondroitin AC
lyase and the preparation of structurally defined oligosaccha-
ride substrates. A novel eliminase gene from an Arthrobacter
species capable of degrading CS and hyaluronan (HA) was
cloned and successfully expressed in E. coli.5 Studies pertaining
to the substrate specificity of this enzyme clearly demonstrated
that AsChnAC was a chondroitin AC exolyase (EC 4.2.2.5).
Briefly, purified recombinant AsChnAC affected the degrada-
tion of polysaccharides containing 1,4-␤-D-hexosaminyl and
1,3-␤-D-glucuronosyl linkages to give the corresponding unsat-
urated disaccharides containing 4-deoxy-␤-D-gluc-4-enurono-
syl groups.
Two trisaccharides (HP- and CS-trisaccharides) and two
hexasaccharides (CS-HP- and HP-CS-hexasaccharides) were
synthesized as model substrates according to a similar proce-
dure to that described in our previous work (32, 33, 35) (Fig. 1).
The structures of these homogenous oligosaccharides differed
from each other in terms of the arrangement of their repeating
units (i.e. -GlcNAc-␣-1,4-GlcA- or -GalNAc-␤-1,4-GlcA-) and
the fact that they contain different glycosidic bonds between were not too big to be degraded by ChnAC.
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Catalytic Direction of Chondroitin AC Lyase
FIGURE 1. Scheme for the synthesis of homogenous oligosaccharides and the schematic representation of AsChnAc cleavage of these substrates. The
synthesis was initiated from GlcUA-pNP using a chemoenzymatic approach. The starting monosaccharide was elongated to the desired size using glycosyl-
transferases according to the methods described in “Experimental Procedures.” Four oligosaccharides were synthesized in this way, and their purities were
assessed by (PAMN)-HPLC (Figs. 2, A, D; 3A; and 4A). The structures of these oligosaccharides were evaluated by ESI-MS analysis (Figs. 2, B and E; 3B; and 4B). KfiA
is an N-acetyl glucosaminyl transferase from E. coli strain K5. pmHS2 was obtained from P. multocida heparosan synthase. KfoC is a chondroitin polymerase
from E. coli K4. A, scheme for the synthesis of the CS-trisaccharide and HP-CS-hexasaccharide, and the cleavage of these oligosaccharide substrates by
AsChnAC. The degradation products resulting from the treatment of CS-trisaccharide with AsChnAC were determined by (PAMN)-HPLC and ESI-MS analyses,
which revealed the presence of two new compounds (GalNAc-GlcA and ⌬Di-pNP). When the ␤-1,4 linkages between the N-acetylhexosamine and GlcA
residues (i.e. residues Cys and Asp or residues Glu and Phe) were present at the reducing end of the HP-CS-hexasaccharide, AsChnAC displayed a consecutive
cleavage pattern. The products of this degradation process were determined to contain GlcNAc-GlcA-GalNAc, ⌬Di-GalNAc, and ⌬Di-pNP by ESI-MS analysis.
The pentasaccharide GlcNAc-GlaA-GalNAc-GlaA-GalNAc was identified following the partial digestion of the HP-CS-hexasaccharide. B, scheme for the synthe-
sis of the HP-trisaccharide and CS-HP-hexasaccharide, and the cleavage of these oligosaccharide substrates by AsChnAC. When there was one ␣-1,4 linkage
betweentheN-acetylhexosamineandGlcAresiduesatthenon-reducingendofthesubstrate, asintheHP-trisaccharideandtheCS-HP-hexasaccharide, ChnAC
did not exhibit any cleavage activity. By setting the ChnAC-uncleavable site on the reducing or non-reducing terminus, we have shown that ChnAC cleaves
oligosaccharide chains from the reducing end toward the non-reducing end.
We then sought to determine whether the ChnAC-catalyzed nity to determine whether this enzyme exhibited a preference
cleavage occurred in a specific direction using two homologous for endolytic cleavage at the reducing or non-reducing end of
oligosaccharides (i.e. the CS-HP- and HP-CS-hexasaccharides). these substrates. The reactions were incubated for 0, 1, and 10
It is noteworthy that these two substrates contained different min at 37 °C, followed by a subsequent period of heating at
linkages between the N-acetylhexosamine and GlcA residues at 100 °C for 5 min. The resulting mixtures of partially and com-
the ends of their chains. For example, the CS-HP- and HP-CS- pletely ChnAC-digested HP-CS-hexasaccharide were analyzed
hexasaccharides had ␣-1,4 and ␤-1,4 linkages at their reducing by ESI-MS (Fig. 3). The complete digestion of the HP-CS-hexa-
ends, respectively. The degradation products resulting from the saccharide substrate yielded three products. ESI-MS analysis
treatment of these two oligosaccharides with ChnAC were revealed that one of these products had a molecular mass of
identified by ESI-MS analysis, thereby providing an opportu- 599.02 Da, which was very close to the molecular mass calcu-
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Catalytic Direction of Chondroitin AC Lyase
FIGURE 2. HPLC and MS analysis of CS-trisaccharide and HP- trisaccharide treated with AsChnAC. The CS-trisaccharide and HP-trisaccharide were
prepared using a chemoenzymatic process, and their purities were analyzed by (PAMN)-HPLC (panels A and D). The structures of these two trisaccharides were
also analyzed by ESI-MS according to the methods described in “Experimental Procedures.” B, ESI-MS spectrum of the CS-trisaccharide. E, ESI-MS spectrum of
the HP-trisaccharide. C, ESI-MS spectrum of the CS-trisaccharide was after it had been treated with AsChnAC for 16 h at 37 °C. This treatment process resulted
in the formation of two newly compounds with m/z values of 396.05 and 295.92, which were attributed to the [M-H]ϪϪ ions of GalNAc-GlcA and ⌬Di-pNP,
respectively. F, (PAMN)-HPLC analysis of the HP- trisaccharide was after it had been treated with AsChnAC for 16 h at 37 °C. The result of this analysis revealed
that there were no peaks with measurable absorption at 232 nm, suggesting that the HP-trisaccharide was a poor substrate for ChnAC.
lated for GlcNAc-GlcA-GalNAc (599.52 Da). Using a similar
ChnAC, which belongs to the CS lyase family, can be used to
method, we also identified the disaccharide ⌬Di-GalNAc (cal- achieve the eliminative degradation of HA, CS-A, and CS-C
culated molecular mass of 379.32 Da) and the pNP-tagged polysaccharides with the release of the corresponding ⌬4-un-
monosaccharide ⌬Di-pNP (calculated molecular mass of saturated disaccharides. Over a dozen of ChnAC enzymes have
297.22 Da). In contrast, the treatment of the CS-HP-hexasac- been obtained from various bacteria and their biochemical and
charide substrate under the same conditions did not result in zymological characteristics studied extensively. ArthroAC, the
any degradation, even when it was incubated with the enzyme ChnAC lyase from A. aurescens, has been studied extensively,
for 16 h at 37 °C (Fig. 4, C and D). This result suggested that the and the results of these studies provided a satisfactory explana-
presence of the GlcNAc-␣-1,4-GlcA unit at the reducing end of tion for its exolytic mode of action. Lunin et al. recently
the substrate prevented it from being cleavage by ChnAC. reported a high-resolution x-ray crystal structure of ArthroAC
These data therefore indicated that the saccharide chain was as a complex with various oligosaccharides (16). Numerous
being degraded from the reducing end toward the non-reduc- critical interactions between the enzyme and substrate were
ing end. Interestingly, a pentasaccharide with a molecular mass identified, including the (␣/␣)5 toroidal fold responsible for the
of 978.86 Da, which was close to the calculated molecular mass lytic mechanism. This work also identified the Tyr-242 residue,
of GlcNAc-GlaA-GalNAc-GlaA-GalNAc (979.84 Da) (Fig. 3C), which acted as a general base for the abstraction of the proton
was identified after the partial digestion of the HP-CS-hexa- from the C5 position of the GlcA unit, as well as the Asn-183
saccharide. This result therefore provided further evidence in and His-233 residues, which neutralized the charge on the glu-
support of our conclusion regarding the direction of the curonic acid group. A novel ChnAC lyase from an Arthrobacter
ChnAC cleavage process.
species (AsChnAC) was cloned and overexpressed in E. coli by
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Catalytic Direction of Chondroitin AC Lyase
FIGURE 3. HPLC and MS analysis of HP-CS-hexasaccharide treated with AsChnAC. The HP-CS-hexasaccharide was prepared using a chemoenzymatic
process, and the purity and structural characteristics of the resulting material were confirmed by (PAMN)-HPLC (panel A) and ESI-MS (panel B) analyses,
respectively. TheHP-CS-hexasaccharidewasincubatedfor0, 1, or10minat37 °C, followedbyasubsequentperiodofheatingat100 °Cfor5min. Thestructures
of the partially and completely ChnAC-digested HP-CS-hexasaccharide samples were analyzed by ESI-MS. C, ESI-MS spectrum of the fully digested products
resulting from the ChnAC-catalyzed degradation of the HP-CS-hexasaccharide. This reaction was incubated for 10 min at 37 °C, followed by subsequent
heating at 100 °C for 5 min. The ESI-MS spectrum of the fully digested material revealed the presence of three new peaks with m/z values of 599.02, 377.84, and
295.89, which were attributed the [M-H]ϪϪ values of GlcNAc-GlcA-GalNAc, ⌬Di-GalNAc, and ⌬Di-pNP, respectively. D, ESI-MS spectrum of the products
resulting from the partial digestion of the HP-CS-hexasaccharide. The reaction was incubated for 1 min at 37 °C, followed by subsequent heating at 100 °C for
5 min. ESI-MS analysis of this mixture revealed a pentasaccharide with a molecular mass of 978.80 Da.
our group,5 and the purified enzyme was employed to deter- saccharide intermediates generated at different stages of the
mine the direction of the ChnAC-catalyzed degradation of the digestion of the enChnABC-treated CS-C. The three-dimen-
polysaccharide in this paper. This enzyme showed the same sional structure of this enzyme (PDB code 1HN0) showed that
substrate specificity and had a high sequence similarity to that the catalytic domain of this chondroitin ABC lyase had an
of the ArthroAC lyase, thereby demonstrating that this enzyme (␣/␣)5 topology, which led to the proposal of a plausible mech-
acted as a typical chondroitin AC exolytic lyase. AsChnAC also anism for this enzyme based on the replacement of an arginine
showed 65% amino acid similarity to ArthroAC, which were residue remote to the linear sequence (39). However, with the
relatively high compared with those of the other lyases belong- exception of the C-terminal domain, the chondroitin ABC lyase
ing to the GAG family. The essential amino acid residues (Tyr- showed poor sequence similarity compared with other lyases
242, Asn-183, and His-233) required for the lytic activity of belonging to the CS family, while the substrate-binding site also
ArthroAC (16), were determined to be Tyr-275, Asn-216, and showed very little sequence conservation (16, 39).
His-266, respectively, in the AsChnAC enzyme. These results
In this study, we have designed and synthesized two homog-
therefore indicate that these two enzymes (AsChnAC and enous hexasaccharides that differed only in terms of the stereo-
ArthroAC) share the same lytic mechanism. Taken together configuration of the first glycosidic bond between the N-acetyl-
with the results of this study, which show that this enzyme hexosamine and GlcA residues at their reducing ends. These
cleaves polysaccharides from their reducing end toward their hexasaccharides were subsequently degraded in the presence of
non-reducing end, these data provide important information purified ChnAC and the resulting cleavage products were ana-
pertaining to the exolytic mode of action of ChnAC lyases. lyzed by (PAMN)-HPLC and ESI-MS. The results revealed that
Interestingly, it has recently been reported that the enChnABC ChnAC cleaves polysaccharides from their reducing end
from proteus vulgaris NCTC 4636 cleaved polysaccharides or toward their non-reducing end, and will therefore provide val-
oligosaccharides from their non-reducing end to their reducing uable information to enhance our collective understanding of
end (38). This conclusion was based on an analysis of the oligo- the mode of action of ChnAC and related enzymes.
4404 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 9•FEBRUARY 26, 2016

Catalytic Direction of Chondroitin AC Lyase
FIGURE 4. HPLC and MS analyses of the CS-HP-hexasaccharide following its treatment with AsChnAC. The CS-HP-hexasaccharide was prepared using a
chemoenzymatic method, and the purity and structural characteristics of the resulting material were confirmed by (PAMN)-HPLC (panel A) and ESI-MS (panel
B) analyses, respectively. The CS-HP-hexasaccharide was incubated for 16 h at 37 °C. Analysis by (PAMN)-HPLC revealed that there were no peaks with
measurable absorption at 232 nm, suggesting that the CS-HP-hexasaccharide was a poor substrate for ChnAC (panel C). D, ESI-MS spectrum of the products
resulting from the complete digestion of the CS-HP-hexasaccharide. The ESI-MS spectrum contained a peak corresponding to the CS-HP-hexasaccharide with
anm/zvalueof1275.05forthe[M-H]ϪϪ ion. Notably, nonewpeakswereidentified, suggestingthattheCS-HP-hexasaccharidewasapoorsubstrateforChnAC.
The presence of the GlcNAc-␣-1,4-GlcA unit at the reducing terminus therefore appeared to prevent the ChnAC-mediated cleavage of the GalNAc-␤-1,4-GlcA
unit located in the middle or at the non-reducing end of the CS-HP-hexasaccharide.
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4406 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 9•FEBRUARY 26, 2016

Uncovering the Catalytic Direction of Chondroitin AC Exolyase: FROM THE
REDUCING END TOWARDS THE NON-REDUCING END
Feng-Xin Yin, Feng-Shan Wang and Ju-Zheng Sheng
J. Biol. Chem. 2016, 291:4399-4406.
doi: 10.1074/jbc.C115.708396 originally published online January 7, 2016
Access the most updated version of this article at doi: 10.1074/jbc.C115.708396
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