From the Division of Biological Engineering and
** Division of Health Sciences and Technology,
Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Received for publication, November 8, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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In the previous paper (Myette, J. R.,
Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G.,
and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157-12166), we described the molecular cloning, recombinant
expression, and preliminary biochemical characterization of the
heparin/heparan sulfate 2-O-sulfatase from
Flavobacterium heparinum. In this paper, we extend our
structure-function investigation of the 2-O-sulfatase.
First, we have constructed a homology-based structural model of the
enzyme active site, using as a framework the available crystallographic
data for three highly related arylsulfatases. In this model, we have
identified important structural parameters within the enzyme active
site relevant to enzyme function, especially as they relate to its
substrate specificity. By docking various disaccharide substrates, we
identified potential structural determinants present within these
substrates that would complement this unique active site architecture.
These determinants included the position and number of sulfates present
on the glucosamine, oligosaccharide chain length, the presence of a
Heparin and heparin sulfate glycosaminoglycans
(HSGAGs)1 are
structurally complex linear polysaccharides (1, 2) composed of
repeating disaccharides of uronic acid ( As members of a large enzyme family, the sulfatases hydrolyze a wide
array of sulfate esters (for a review, see Refs. 3 and 4). Their
respective substrates include sulfated complex carbohydrates such as
the glycosaminoglycans (GAGs), steroids, sphingolipids, xenobiotic
compounds, and amino acids such as tyrosine. Additionally, many of
these enzymes are able to hydrolyze in vitro smaller
synthetic substrates (e.g. 4-nitrophenyl sulfate and
catechol sulfate). It is for this reason that these enzymes are often
generically described as "arylsulfatases" (even when their
preferred in vivo substrate is ill defined). Despite their
disparate substrate specificities, the members of this enzyme family
share both considerable structural homology and a common catalytic
mechanism with one another (5). Each sulfatase possesses a signature
catalytic domain toward its amino terminus, which is readily identified
by the consensus sequence (C/S)XPXRXXXX(S/T)G. The conserved
cysteine (or less commonly serine) within this sulfatase motif is of
particular functional importance, since it is covalently modified to an
L-C The crystal structures of two human lysosomal sulfatases, arylsulfatase
A (cerebroside-3-sulfate 3-sulfohydrolase) (9, 10) and arylsulfatase B
(N-acetylgalactosamine-4-sulfate 4-sulfohydrolase) (11), and a bacterial arylsulfatase from Pseudomonas
aeruginosa (12) have been solved. These three sulfatases share an
identical alkaline phosphatase-like structural fold (according to the
Structural Classification of Proteins
Database)2 composed of a
series of mixed parallel and antiparallel Using the strong structural and functional similarities that have been
observed among the members of the sulfatase family of enzymes, we
extend our characterization of the recombinant 2-O-sulfatase
in this paper to include a structure-function description of the
enzyme's substrate specificity. To do so, we first constructed a
homology-based structural model for the wild type
2-O-sulfatase using the three crystal structures (cited
above) as a molecular scaffold. From this model, we present a picture
of the enzyme active site from which we have identified specific
residues likely to be involved in substrate binding and catalysis.
Predictions concerning substrate discrimination made from this model
are supported by the biochemical and kinetic data presented in the
previous paper. In addition, a specific prediction regarding the
sulfatase's exolytic mode of action is made; this prediction is
subsequently validated by the biochemical data. Finally, we use a
combination of aldehyde-specific chemical labeling and peptide mapping
methods in parallel with site-directed mutagenesis to identify the
predicted active site cysteine, which is covalently modified to a formylglycine.
Reagents--
The unsaturated heparin decasaccharide
Homology Modeling of 2-O-Sulfatase--
The crystal structures
of human arylsulfatase A, human arylsulfatase B, and the P. aeruginosa
arylsulfatase4 were used to
obtain a structural model for the 2-O-sulfatase enzyme. A
multiple sequence alignment of the 2-O-sulfatase with different bacterial and lysosomal sulfatases was performed using ClustalW (14) (Fig. 1). Based on this multiple sequence alignment, three model structures of 2-O-sulfatase were obtained
corresponding to its alignment with the other three sulfatases. The
models were constructed using the Homology module of the Insight II
molecular simulations package (Accelrys, San Diego, CA). The side chain of the critical Cys-82, which is shown to undergo posttranslational modification in the active enzyme, was replaced by the geminal diol
(C Molecular Docking of Disaccharide Substrates into the Active Site
of the Modeled 2-O-Sulfatase--
Heparin-derived disaccharides with a
The orientation of the cleavable sulfate group relative to O Recombinant Expression and Protein Purification of the
Flavobacterial 2-O-Sulfatase--
The molecular cloning, recombinant
expression in Escherichia coli, and subsequent purification
of the 6× histidine-tagged 2-O-sulfatase by nickel affinity
chromatography is as described in the preceding paper. The sulfatase
was expressed as an amino-terminal truncation lacking its putative
signal sequence (commonly referred to in both papers as 2-O
AT-10 Compositional Analyses by Capillary Electrophoresis and
MALDI-MS--
Approximately 10 µg of the AT-10 oligosaccharide were
incubated with 100 pmol of 2-O 2-O-Sulfatase Active Site Labeling and Peptide
Mapping--
Approximately 500 µg of 6× histidine-tagged
2-O
The labeled sulfatase (and unlabeled control) were proteolyzed with
sequence grade-modified trypsin for 20 h at 37 °C in digestion buffer that contained 0.1 M Tris-HCl, pH 8.5, 1 mM EDTA, 1 mM dithiothreitol, and 10%
acetonitrile (v/v) in a 30-µl reaction volume. Trypsin was first
reconstituted as a 2.5 mg/ml stock in 1% acetic acid and added at a
1:5 ratio (w/w) relative to the target protein. Following trypsin
digestion, peptide cysteines were reduced by the addition of 50 mM dithiothreitol (50 °C under argon, 1 h). Reduced
cysteines were subsequently alkylated for 30 min at 37 °C (in the
dark) by the addition of 150 mM iodoacetamide, added from a
2 M stock made up in 0.1 M Tris-HCl, pH 8.5. This reduction-alkylation cycle was repeated one more time. Molecular masses of select peptides were determined by MALDI-MS as described (18)
using 1 µl of Site-directed Mutagenesis of the C82A Active Site
Mutant--
The site-directed mutant C82A was cloned by recombinant
PCR using outside primers 5'-TCT AGA CAT ATG CAA ACC TCA AAA GTA GCA GCT-3' (forward) and (5'-GT CTC GAG GAT CCT TAT TTT TTT AAT GCA TAA AAC
GAA TCC-3' (reverse) in addition to the following mutagenic primer
pair: 5'-C CAG CCG CTC GCT ACA CCT TCA CG-3' (forward) and
5'-CG TGA AGG TGT AGC GAG CGG CTG G-3' (reverse). The
engineered codon change for each DNA strand is underlined. Subcloning
into pET28a, recombinant expression in the E. coli strain
BL21 (DE3), and subsequent purification by nickel chelation
chromatography using the N-terminal 6× histidine purification tag are
as described for 2-O 2-O-Sulfatase Cysteine Modification with
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and
Dithiothreitol--
Sulfatase cysteines were titrated based on molar
absorptivity using Ellman's reagent (DTNB) generally as described
(19). 50 µM 2-O-sulfatase (2-O
Structure-based Homology Modeling of the 2-O-Sulfatase Active
Site--
In comparing the structures of human arylsulfatase A (9,
10), arylsulfatase B (N-acetylgalactosamine-4-sulfatase)
(11), and a bacterial arylsulfatase from Pseudomonas
aeruginosa (12), we observed an obvious structural homology
between each of them, especially as it pertained to a conservation of
critical active site residues and their spatial arrangement. By
extension, most of these amino acids were likewise conserved in the
flavobacterial 2-O-sulfatase as evident by a direct
alignment of their primary sequences (Fig.
1). We used this close structural
relationship to construct three homology-based models for the
flavobacterial 2-O-sulfatase, each one based on one of the
three crystal structures examined. We ultimately chose as our
representative 2-O-sulfatase structure the homology model
constructed using the N-acetylgalactosamine-4-sulfatase (arylsulfatase B) (Fig. 2). This decision
was largely based on arylsulfatase B also being a GAG desulfating
enzyme. In this model, we replaced cysteine 82 with a formylglycine
(FGly-82). We chose to represent FGly-82 in the hydrated state as a
geminal diol (-C
In the structural model, the surface of the active site pocket is
composed of many amino acids that can potentially interact with a
disaccharide substrate (Fig. 3). These
include Lys-107, Lys-175, Lys-238, Gln-237 and -309, Thr-104,
Glu-106, and Asp-159. Lysines and glutamines are commonly occurring
amino acids in heparin binding sites that interact with the sulfate and
carboxylate groups of the GAG chain. Unlike the amino acids proximal to
the FGly-82, these residues are not conserved in the other sulfatases
that we examined (Table I, denoted in
normal type), suggesting a potentially unique role of these amino acids
in dictating oligosaccharide substrate specificity. This disparity is
particularly true when directly comparing the 2-O-sulfatase
and arylsulfatase A; many of the nonconserved amino acids in the
2-O-sulfatase are charged, whereas those in arylsulfatase A
are predominantly hydrophobic. This observation is consistent with the
obvious structural distinction of their respective substrates
(i.e. the highly sulfated HSGAG substrates of the
2-O-sulfatase versus the long hydrophobic alkyl chains of cerebroside 3-sulfate substrate of arylsulfatase A).
Active Site Topology and the Exolytic Action of the
2-O-Sulfatase--
Upon inspection of the 2-O-sulfatase
structure, several amino acids that potentially constitute the active
site were identified (Table I). There are several structurally
conserved basic amino acids in the proximity of FGly-82 including
Arg-86, Lys-134, His-136, and Lys-308. The topology of the active site
as observed in our structural model indicated that the critical FGly-82
and the basic amino acid cluster are located at the bottom of a deep
pocket (Fig. 2B). Such a restrictive access to the active
site would appear to impose a clear structural constraint on the
substrate as it relates to the position of the 2-O-sulfate
group within the oligosaccharide chain (i.e. externally
versus internally positioned) upon which the enzyme acts. We
would predict from this topology that only a sulfate group present at
the nonreducing end of the oligosaccharide will be favorably positioned
for catalysis; the juxtaposition of an internal sulfate into the active
site would require a substantial bending of the oligosaccharide chain.
Such chain distortion would be sterically unfavorable. Based on these constraints, therefore, we predict the sulfatase to hydrolyze 2-O-sulfates in an exclusively exolytic fashion. This
exclusivity for the nonreducing end does not necessarily preclude,
however, the enzyme acting on longer chain oligosaccharides
(i.e. those exceeding a disaccharide in length), provided
that they in fact possess sulfates at the terminal 2-OH-position. The
model does suggest a likely kinetic preference for disaccharide
substrates, since they would most readily diffuse into and out of this
narrow active site (see enzyme-substrate structural modeling below).
We addressed this important question using as a substrate the
purified heparin-derived AT-10 decasaccharide
Enzyme-Substrate Structural Complex: Interactions between
2-O-Sulfatase and Disaccharides--
Since the active site can readily
accommodate disaccharide substrates, we modeled several unsaturated
glycosaminoglycan disaccharides as described under "Experimental
Procedures." Our choice of The Requirement for an Unsaturated
To better understand this likely structural constraint, we superimposed
substrate disaccharides containing a nonreducing end iduronic acid in
either the 1C4 or 2S0
conformation (data not shown). The superimposition was such that the
S-O-C-2-C-1 atoms of all of the uronic acids coincided, thereby
fixing the orientation of the 2-O-sulfate group. In this model, the carboxylate groups of the iduronic acid containing disaccharide substrates were, in fact, pointing away from the active
site pocket and were not positioned to interact as favorably with
Lys-175 or Lys-238 as compared with the original disaccharide substrate
possessing a planar C-5 carboxylate.
We assessed this prediction experimentally by testing the ability of
the 2-O-sulfatase to hydrolyze size-fractionated
hexasaccharides derived from the nitrous acid treatment of heparin.
Unlike enzymatic cleavage, these chemically derived heparin saccharides
do not possess a Enzyme-Sulfate Interactions in the 2-O-Sulfatase Active
Site--
In the previous paper (21), we present a biochemical data
demonstrating a clear kinetic preference of the enzyme for highly sulfated disaccharide substrates, namely those possessing a glucosamine sulfated at the 6-OH- and 2-N-positions. Our structural model of the
sulfatase-trisulfated disaccharide complex also points out key
interactions involving these additional sulfates present on the
adjoining glucosamine. In particular, the 6-O-sulfate group interacts with the basic side chain of Lys-107 within the enzyme active
site (Fig. 3). This putative charge interaction would probably play an
important role in stabilizing the orientation of the substrate in the
active site. In contrast, the N-sulfate group of the
disaccharide glucosamine is proximal to a contiguous stretch of
leucines (positions 390-392). In such an arrangement, it is the methyl
group of an N-acetylated glucosamine rather than a sulfate
at this position that is more likely to make favorable hydrophobic
contacts with these residues. This prediction was borne out in one of
our models docking the
We also modeled enzyme-substrate complexes containing two unsaturated
chondroitin sulfate disaccharides
( Metal Ion Coordination--
In discussing this model, we must
briefly consider the potential role of divalent metal ions. We decided
not to include any such metal ions in our theoretical model of the
2-O-sulfatase, since we could find no divalent metal
requirement for enzymatic activity (see preceding paper (21)). A
divalent metal ion is present, however, in all three sulfatase crystal
structures that we examined. In each case, the metal ion coordinates
with the oxygen atoms of the sulfate group of the respective substrate. Additionally, a cluster of four highly conserved acidic amino acids has
been observed to coordinate this divalent metal ion. In the case of
human arylsulfatase B, for example, the oxygen atoms of Asp-53, Asp-54,
Asp-300, and Asn-301 are coordinated with a Ca2+ ion. Three
of the four corresponding amino acids in the flavobacterial sulfatase
model that we have identified as potentially coordinating with a metal
ion are Asp-42, Gln-43, and Asp-295 (Table I). The fourth amino acid in
the 2-O-sulfatase corresponding spatially to Asn-301 of
arylsulfatase B is His-296. The positive charge of this position,
however, does not favor the proximal location of a divalent metal
cation. It is perhaps this unfavorable charge interaction that
interferes with proper metal ion coordination.
Enzyme-Substrate Model: Mechanism for Catalysis--
Nearly
identical mechanisms for the hydrolysis of the sulfate ester bond
involving the conserved active site amino acids have been proposed for
human arylsulfatases A and B and the bacterial sulfatase from P. aeruginosa. Thus, the mechanism proposed for these other
sulfatases should logically apply to the 2-O-sulfatase. The
resting state of the active sulfatase in each of the crystal structures
is proposed to contain the geminal diol, which is stabilized by
interactions with basic residues. His-136 and Arg-86 of the flavobacterial 2-O-sulfatase enzyme are positioned
appropriately in the active site to do so (Fig. 2B). A
critical step in catalysis involves the correct positioning the
2-O-sulfate group such that the sulfur atom is accessible to
the O-
An SN2 mechanism has been proposed to follow the above
steps and eventually lead to the cleavage of the sulfate ester bond (10, 12). In this mechanism, the exocyclic oxygen atom on the leaving
substrate may be protonated by water or potentially by neighboring
amino acids. In the 2-O-sulfatase active site model, Lys-308
is juxtaposed to protonate the leaving group (Fig. 2). The resulting
sulfate group on the geminal diol is subsequently eliminated by
abstraction of a proton from O- 2-O-Sulfatase Peptide Mapping and Chemical Modification of Active
Site Formylglycine--
Finally, in describing the structure-function
relationship of the 2-O-sulfatase active site, we come to
the central catalytic player itself, the formylglycine at position 82. The recombinant expression of catalytically active
2-O-sulfatase in E. coli functionally argues for
this covalent modification of the active site in vivo. We
further established the catalytic function of Cys-82 by site-directed mutagenesis. The mutant (C82A) was recombinantly expressed and purified
as a histidine-tagged protein in the same manner employed for the wild
type enzyme. Comparable expression levels of soluble protein were
achieved. The C82A mutant, however, was completely inactive (data not
shown). Both the wild type and mutant possessed the same secondary
structure as exhibited by their virtually superimposible CD spectra
(data not shown), arguing against any adverse global conformational
changes induced by the molecular replacement of the cysteine by alanine.
We also set out to demonstrate the physical presence of the FGly at
position 82 by the tandem use of protein chemistry and mass
spectrometry. 10 nmol of wild-type sulfatase (2-O
To confirm and extend these findings, we also completed chemical
modification experiments on the wild type enzyme using the cysteine-specific reagent DTNB (Ellman's reagent). Apart from the
catalytic Cys-82 that is converted to a formylglycine, none of the
remaining seven cysteines appear to be highly conserved among other
members of the sulfatase family (Fig. 1). We were unable to effectively
inhibit enzyme activity with the addition of DTNB or dithiothreitol
(data not shown). This general lack of inhibition by these two
cysteine-reactive agents suggests at least two probabilities. First,
the 2-O-sulfatase does not require intramolecular disulfide
linkages to critically stabilize a catalytically active conformation.
This presumption is not surprising, given the fact the native
2-O-sulfatase is a bacterially derived protein. Second, free
sulfhydryls are not directly participating in catalysis. It is
possible, however, that a few of these cysteines are buried and are
therefore not accessible to sulfhydryl exchange. At least five of the
eight cysteines, however, do react with DTNB under nondenaturing
conditions (data not shown). This latter fact may suggest an alternate
role for these solvent-accessible cysteines (along with specific
histidines), perhaps as metal-coordinating thiolates. This possibility
is a reasonable one when one makes the comparison between the
2-O-sulfatase and alkaline phosphatase. Both of these
enzymes are esterases with similar catalytic mechanisms, including the
presumptive formation of a covalent intermediate. The two hydrolytic
enzymes also possess structurally related domains, in particular a
highly superimposible active site that includes a divalent metal
binding pocket. In the case of alkaline phosphatase, it is zinc rather
than calcium (or Mg2+) that is coordinated within this pocket.
In the previous paper, we described the molecular cloning of a HSGAG
2-O-sulfatase from F. heparinum and its
recombinant expression in E. coli as a soluble, highly
active enzyme in milligram quantities. We also presented a
characterization of the enzyme's biochemical properties as it pertains
to optimal in vitro reaction conditions and substrate
kinetics. In this paper, we build upon this biochemical characterization of the enzyme by providing an invaluable structural framework to address the molecular basis of enzyme function. To the
best of our knowledge, this paper represents one of the first structural descriptions of an HSGAG desulfating enzyme. As stated before, our homology model of the 2-O-sulfatase has already
provided valuable insight into likely structural determinants for
substrate specificity and catalysis. Many of these insights are
validated by our biochemical and kinetic evaluation of this
specificity. Importantly, we now have in place a meaningful structural
framework in which to address additional questions pertaining to
oligosaccharide-sulfatase interactions.
4,5-unsaturated double bond, and the exolytic versus
endolytic potential of the enzyme. The predictions made from our model
provided a structural basis of substrate specificity originally
interpreted from the biochemical and kinetic data. Our modeling
approach was further complemented experimentally using peptide mapping
in tandem with mass spectrometry and site-directed mutagenesis to
physically demonstrate the presence of a covalently modified cysteine
(formylglycine) within the active site. This combinatorial approach of
structure modeling and biochemical studies provides insight into the
molecular basis of enzyme function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-L-iduronic or
-D-glucuronic) linked 1
4 to
-D-glucosamine. This structural complexity derives principally from the variable chemical modifications made to the polysaccharide chain. Such modifications include acetylation or sulfation at the N-position of the glucosamine, epimerization of
glucuronic acid to iduronic acid, and additional O-sulfation at the 2-O-position of the uronic acid in addition to the 3-O, 6-O-position of the adjoining glucosamine. It is a highly variable sulfation pattern, in particular, which ascribes to each GAG chain a
unique structural signature. In turn, this signature dictates specific
GAG-protein interactions underlying critical biological processes
related to cell and tissue function. Given this critical structure-function relationship of GAG sulfation, enzymes that can
hydrolyze these sulfates in a structurally specific manner are
important tools for the determination of GAG fine structure to better
ascertain these structure-function relationships. In the previous paper
(21), we described the cloning, recombinant expression, and biochemical
characterization of one such sulfatase, the 2-O-sulfatase
from Flavobacterium heparinum.
-formylglycine (L-2-amino-3-oxopropionic
acid) (4, 6, 7). The ubiquitous importance of this chemical
modification was first functionally identified by its relationship to
the etiology of multiple sulfatase deficiency, a genetically
recessive disorder in which there is a complete loss of sulfatase
activity due to a lack of this critical aldehyde (FGly) within the
active site of all expressed sulfatases (8).
-strands flanked by long
and short
-helices on either side (9-12). In addition to their
common structural fold, these sulfatase structures also possess a high
degree of homology within their respective active sites, especially in
the region localized around the modified cysteine (FGly). Taken
together, these crystal structures present a clear and consistent
description of conserved active site residues and their potential
catalytic role in sulfate ester hydrolysis. At the same time, this
strong structural homology is somewhat surprising, considering that at
least two of these sulfatases act on notably different substrates
(e.g. sulfated sphingolipid versus sulfated GAG).
The structural basis for substrate specificity, therefore, remains to
be determined. The question of this relationship of enzyme active site
structure to substrate specificity would appear to be especially
relevant to GAGs where multiple sulfate esters are present.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
U2S
HNS,6SI2SHNS,6SI2SHNS,6SIHNAc,6SGHNS,3S,6S3
(AT-10) was generated by a partial heparinase digestion and purified as
described (13). Modified trypsin (sequencing grade) was purchased from
Roche Molecular Biochemicals. Texas Red hydrazine was purchased from
Molecular Probes, Inc. (Eugene, OR). Ellman's reagent was purchased
from Pierce. Enzymes for molecular cloning and PCR were obtained from
New England Biolabs (Beverly, MA). All other reagents were purchased
from Sigma unless otherwise noted.
(OH)2). The potentials for the model structures were
assigned using the AMBER force field (15). The deletions in the modeled structures were closed using 200 steps of steepest descent minimization without including charges by keeping most of the structure rigid and
allowing the regions close to the deletion move freely. The final
refined structure was subjected to 400 steps of steepest descent
minimization without including charges and 400 steps of conjugate
gradient minimization including charges.
U at the nonreducing end were modeled as follows. The coordinates of
the trisulfated
U containing disaccharide
(
U2SHNS,6S) were obtained from the co-crystal structure of a heparinase-derived hexasaccharide with fibroblast growth factor 2 (Protein Data Bank code 1BFC). This trisulfated disaccharide structure was used as a reference to generate
the structural models for other disaccharides including
U2SHNS,
U2SHNAc,
and
U2SHNAc,6S. The coordinates of
trisulfated disaccharides (I2SHNS,6S)
containing iduronic acids in the 1C4 and
2S0 conformations were also obtained from 1BFC.
Similarly, chondroitin sulfate-derived disaccharides
U2SGalNAc,4S and
U2SGalNAc,6S were modeled using a reference
structure of a chondroitin-4 sulfate disaccharide
UGalNAc,4S whose coordinates were obtained from its
co-crystal structure with the chondroitinase B enzyme (Protein Data
Bank code 1DBO). The potentials for these disaccharides were assigned
using the AMBER force field modified to include carbohydrates (15) with
sulfate and sulfamate groups (16).
1 of the
geminal diol in the active site of human arylsulfatase A and the
bacterial arylsulfatase was identical as observed in their respective
crystal structures. This orientation was such that one of the faces of
the tetrahedral formed by the three oxygen atoms of
SO
1, facilitating the nucleophilic attack of the sulfur atom and the transfer of the SO
1.
This highly specific orientation of the sulfate group helped in
positioning the disaccharide substrates relative to the active site of
the 2-O-sulfatase. After fixing the orientation of the
2-O-sulfate group, the glycosidic torsion angles and
exocyclic torsion angles were adjusted manually to remove unfavorable
steric contacts with the amino acids in the active site. The enzyme
substrate complexes were minimized using 200 steps of steepest descent
followed by 400 steps of Newton-Raphson minimization including charges.
Most of the enzyme was kept rigid, and only the loop regions
constituting the active site were allowed to move freely. On the other
hand, a force constant of 7000 kcal/mol was applied to constrain the ring torsion angles to fix the ring conformation of the disaccharides during the energy minimization. The manual positioning of the substrates was done using the Viewer module, building of the
disaccharide structures from the reference structures was done using
the Builder module, and the energy minimization was done using
the Discover module of Insight II.
1-24).
N1-24 in a
40-µl reaction volume at 30 °C. 15-µl aliquots were removed at 4 and 17 h and heat-inactivated at 95 °C. The oligosaccharide reaction products (along with 15 µl of a minus sulfatase control) were subjected to an exhaustive heparinase I and III digestion prior to
capillary electrophoresis-based compositional analysis. Desulfation of
the decasaccharide was assayed in parallel by MALDI-MS using
established methods (17).
N1-24 (wild type enzyme and C82A
site-directed mutant) were lyophilized by Speed-Vac centrifugation and
vigorously resuspended in 90 µl of denaturation buffer containing 6 M guanidinium hydrochloride, 0.1 M Tris-HCl, pH
7.5. Active site aldehydes were fluorescently labeled by adding 25 µl
of Texas Red hydrazine made up as a 10 mM stock in dimethyl formamide. Labeling was carried out for 3 h at room temperature with gentle mixing on a rotating platform. The hydrazone linkage was
stabilized by the addition of 10 µl of a fresh 5 M sodium cyanoborohydride stock made up in 1 N NaOH. Reduction was
carried out for 1 h at room temperature. Unreacted fluorophore was
removed by repeated acetone precipitation (added 5:1 (v/v)).
Acetone was prechilled at
20 °C. Samples were chilled at
85 °C for 20 min prior to spinning in a microcentrifuge for 10 min, maximum speed, at 4 °C. Pellets were dried by Speed-Vac centrifugation.
-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.3% trifluoroacetic acid as a matrix.
1-24 (previous paper
(21)).
N1-24) was reacted with 500 µM DTNB in a
1.4-ml reaction volume containing 0.1 M sodium phosphate,
pH 8.0, and 5 mM EDTA. Absorbance was measured at 412 nm
following a 15-min incubation at room temperature with a minus enzyme
control used as a blank. For measurements made under denaturing
conditions, SDS was added to a final concentration of 2%. Molar
extinction coefficients used were as follows: nondenaturing (minus
SDS), 14,150 M
1; denaturing (2% SDS), 12,500 M
1.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(OH)2), consistent with the
proposed resting state (before catalysis) of the enzyme (5, 9).
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Fig. 1.
Multiple sequence alignment of the sulfatases
using ClustalW. The flavobacterial enzyme is a member of a large
sulfatase family. The putative active site is boxed, with
critically modified cysteine noted by an asterisk. Invariant
residues are shaded in dark gray, those with
partial identity in light gray, and conservative
substitutions in charcoal. Multiple sequence alignment
was generated by ClustalW using only select sequences identified from a
BLASTP search of the protein data base. Enzymes are abbreviated as
follows. FH2S, F. heparinum
2-O-sulfatase; PARS, P. aeruginosa
arylsulfatase; MDSA, Prevotella sp. MdsA gene;
HGal6S, human N-acetylgalactosamine-6-sulfate
sulfatase (chondroitin 6-sulfatase); HARSA, human
cerebroside-3-sulfate sulfatase (arylsulfatase A);
HARSB, human N-acetylgalactosamine-4 sulfate
sulfatase (arylsulfatase B); HI2S, human iduronate-2-sulfate
sulfatase; cons, consensus sequence. The
GenBankTM protein accession numbers for sulfatases listed
in Fig. 1 are as follows: CAA88421, P. aeruginosa
arylsulfatase; AAF72520, Prevotella sp.
MdsA mucin desulfating gene; AAC51350, Homo sapiens
N-acetylgalactosamine-6-sulfate sulfatase; AAB03341, H. sapiens cerebroside-3-sulfate sulfatase (arylsulfatase A);
AAA51784, H. sapiens
N-acetylgalactosamine-4-sulfate sulfatase (arylsulfatase B);
AAA63197, H. sapiens iduronate-2-sulfate sulfatase.
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Fig. 2.
Structural model of the
2-O-sulfatase and topology of active site.
A, ribbon diagram of the proposed
2-O-sulfatase structure constructed using homology modeling
of the crystal structure of human arylsulfatase B. The strands are colored red, and the
-helices are colored
blue. The geminal diol form of the modified cysteine is
rendered as CPK (carbon colored green and oxygen colored
red). The direction of substrate diffusing into the active
site is indicated by an arrow. B, CPK rendering
of the top view of the structure shown in A. The modified
cysteine is colored purple, and the surrounding basic amino
acids (Arg, His, and Lys) are colored dark purple, acidic
amino acids (Asp and Glu) are colored red, and Gln and Asn
are colored light blue. Note that the active site geminal
diol is located in the bottom of a deep cleft.
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Fig. 3.
Active site amino acids and their interaction
with U2SHNS,6S.
A, stereo view of the 2-O-sulfatase active site
highlighting important amino acids (shown here by a stick
representation). Basic residues are colored blue, acidic
amino acids are colored red, Gln is colored light
blue, Thr is colored orange, Leu is colored
brown, and FGly-82 is colored purple. The docked
disaccharide (colored in green) is also shown using a stick
representation. The sulfur atom of 2-O-sulfate group is
colored yellow, and oxygen atoms of the
2-O-sulfate group and the planar carboxyl group are colored
red. B, schematic representation of the amino
acids shown in A using the same color
scheme. Potential metal ion coordination is also shown, with
the divalent cation (M2+) depicted as a gray
circle.
Structure-based comparison of sulfatase active site residues
traces using the
combinatorial extension algorithm (20). Regions of
deletion in the structural alignment are noted with a minus sign.
U2SHNS,6SI2SHNS,6SI2SHNS,6SIHNAc,6SGHNS,3S,6S. This oligosaccharide possesses a
4,5-unsaturated uronic acid at the
nonreducing end and both externally and internally positioned 2-O-sulfates. The substrate was first exhaustively treated
with the 2-O-sulfatase. The 2-O-desulfated
decasaccharide was then subjected to heparinase treatment. Capillary
electrophoresis-based compositional analyses indicated the
disappearance of the disaccharide
U2SHNS,6S
by only one-third; two-thirds of this trisulfated disaccharide remained
after sequential treatment with the 2-O-sulfatase and heparin lyases (Fig. 4). Loss of a single
sulfate was independently determined by mass spectrometry. Sequencing
analysis of the singularly desulfated product allowed us to assign the
original position of the 2-O-sulfate exclusively to the
uronic acid present at the nonreducing end. This result was confirmed
using other oligosaccharide substrates, including tetra- and
hexasaccharides containing both nonreducing end 2-O-sulfates
and internal 2-O-sulfates (data not shown). Taken together,
these results strongly suggest a sulfatase whose hydrolytic activity
proceeds in an exolytic fashion. This conclusion is supported by our
model, which, given the narrow topology of the enzyme active site,
predicts a strong preference for the cleavage of sulfates positioned at
the nonreducing end.
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Fig. 4.
The exolytic activity of the
2-O-sulfatase. The ability of the sulfatase to
hydrolyze internally positioned 2-O-sulfates within the
AT-10 decasaccharide and subsequent compositional analyses of the
heparinase-treated product. A, AT-10 decasaccharide sequence
with property-encoded nomenclature (22) and outline of experimental
design. B, capillary electrophoretagram for both the control
and sulfatase-pretreated samples along with their saccharide
compositional assignments. Heparinase cleavage products following
sulfatase pretreatment are shown as a dashed line
(with gray fill). Minus sulfatase control is
shown as a white line (no fill). The
tetrasulfated tetrasaccharide (+4-7) is also noted. Disappearance of
the trisulfated disaccharide (D) by one-third and the
corresponding appearance of the 2-O-desulfated product
( UHNS,6S) are depicted by vertical
arrows. The minor tetrasaccharide contaminant is noted by an
asterisk.
4,5-unsaturated substrates was logical
for two reasons: 1)
-eliminative cleavage of a HS polysaccharide by
the flavobacterial lyases that naturally occurs in
vivo results in the formation of disaccharides and other small
oligosaccharides, all possessing a
4,5-unsaturated bond at the
nonreducing end uronic acid and 2) the obligatory substrate-product relationship between the 2-O- sulfatase and the
4,5-glycuronidase that exists both in vitro (as described
in the previous paper (21)) and in vivo. In addition, we
directly tested the requirement of a
4,5 uronic acid directly in
experiments described below. A representative structural complex
involving the trisulfated disaccharide
U2SHNS,6S (Fig. 3) was used to describe the
molecular interactions between the enzyme and the substrate. This
choice was made based on the biochemical data (shown in the
accompanying manuscript (21)) that indicated the trisulfated
disaccharide as the kinetically preferred substrate. A description of
these interactions and their proposed functional roles is shown in
Table II. The functional roles of the
conserved active site amino acids (listed in boldface type in Table I)
were proposed based on their interactions with the
2-O-sulfate group and/or the geminal diol of the
formylglycine at position 82. Identical roles have been proposed for
the corresponding amino acids in the three known sulfatase crystal
structures (Table I).
Functional assignment of 2-O-sulfatase active site amino acids
4,5 Nonreducing
Terminus--
A closer inspection of the modeled enzyme-substrate
complex revealed some interesting possibilities pertaining to the role of the nonconserved amino acids in substrate recognition and binding. The planar carboxylate group attached to the C-5 atom of the
4,5 uronic acid is oriented in such a manner as to potentially interact with Lys-175 and Lys-238. These amino acids could play an important role, therefore, in favorably orienting the 2-O-sulfate
within the active site. We were further interested in this arrangement given the additional constraint imposed upon the planar carboxyl group
of the uronic acid by the presence of the C-4-C-5 double bond. This
constraint may further influence substrate orientation within the
active site. As such, we would predict a substrate discrimination
exhibited by the 2-O-sulfatase that is based on the presence
of the
4,5 double bond at the oligosaccharide nonreducing terminus.
In the absence of this double bond, the favorable orientation of the
C-5 carboxylate afforded by charge interactions with Lys-175 and
Lys-238, respectively, would not occur. The change in the orientation
of the C-5 carboxylate due to the lack of the double bond would, in
turn, disrupt the favorable positioning of the 2-O-sulfate
group for catalysis.
4,5-unsaturated bond at their respective
nonreducing ends. A majority of the resultant tetrasaccharides,
however, do contain an I2S at this end. Using MALDI-MS, we
were unable to detect any enzyme-dependent desulfation of
treated hexasaccharides under conditions that resulted in quantitative
elimination of 2-O-sulfate from
4,5-containing
oligosaccharides. This result strongly suggests a structural
requirement for the
4,5 bond in addition to the exolytic preference
of the enzyme. As we have already pointed out, our model provides a
rationale for this requirement (i.e. the obligatory physical
connection between this bond and the planar carboxylate at the uronic
acid C-5 position). The constraint imposed by a planar C-4-C-5 double
bond on the orientation of this carboxylate in turn confers upon the
same substrate an orientation of the 2-O-sulfate within the
enzyme active site that is favorable for catalysis.
U2SHNAc,6S substrate
in the active site (data not shown).
U2SGalNAc,4S and
U2SGalNAc,6S). In comparison with our
original model using the heparin disaccharide substrate, we found
interactions with the 2-O-sulfate and carboxyl group of the
U monosaccharide that were identical to that of
U2SHNS,6S. There were fewer interactions involving the 4-sulfate and 6-sulfate groups, however. This particular model, therefore, is consistent with our kinetic data describing the
ability of the so-called "heparin/heparan sulfate"
2-O-sulfatase to hydrolyze 2-O-sulfated
chondroitin disaccharides. Given a lack of additional favorable
contacts between the enzyme and substrate (e.g. with either
the 4-O- or 6-O-sulfates), our model also
predicts a lower catalytic efficiency for the chondroitin disaccharides relative to the structurally corresponding heparin disaccharides.
1 of the geminal diol. We have already described how
interactions of specific active site amino acids with the planar
carboxyl group of the uronic acid (Lys-175 and -238), with the
6-O-sulfate of the glucosamine (e.g. Lys-107 and
possibly Thr-104) and with the 2-O-sulfate itself (Lys-134
and -308) are likely to serve in this capacity (Table II). At the same
time, interaction of the 2-O-sulfate group with charged
amino acids would also enhance any electron density withdrawal from the
oxygen atoms, thereby increasing the electrophilicity of the sulfur
center. It has also been suggested that the nucleophilicity of the
O-
1 atom is enhanced by a possible proton donation to a neighboring
aspartic acid residue. In our structural model of the
2-O-sulfatase, this residue would correspond to Asp-295.
2, regenerating the formylglycine.
His-136 is positioned to abstract this proton. Our functional
assignment of these lysine and histidine residues is based entirely on
homologous positions found in the other sulfatases examined and their
proposal to catalytically function in the fashion described (5).
N1-24) and the C82A mutant were reacted with Texas Red
hydrazide (620.74 Da) as described under "Experimental Procedures."
The two sulfatase fractions were subsequently trypsinized under mildly
denaturing conditions followed by reductive methylation of the
unmodified cysteines. The molecular masses of the resultant peptides
were determined by MALDI-MS (Fig. 5). In
this experiment, we identified a single ionized species uniquely
present in the labeled sulfatase experiment (Fig. 5B) but
absent in the active site mutant (Fig. 5C) or in the
unlabeled control (Fig. 5A). The empirical mass of this
species corresponded most closely to the peptide sequence FTRAYCAQPLCTPSR, resulting from a partial trypsin
cleavage. This peptide contains the sulfatase consensus sequence
CXPXR, which includes the critical active site
cysteine (underlined) at position 82. The mass of this peptide is
consistent with first the conversion of this cysteine to a
formylglycine (FGly-82) followed by the covalent hydrazone linkage of
the aldehyde-reactive fluorophore at this position. It also takes into
account the carbamidomethylation of the second (unmodified) cysteine
present in this peptide. These data, taken together with the loss of
function observed for the C82A mutant, establish the important
structure-function relationship for this active site modification.
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Fig. 5.
Identification of
2-O-sulfatase active site modification (FGly) by
chemical labeling and mass spectrometry. Wild type sulfatase
(2-O N1-24) and C82A mutant were reacted
with Texas Red hydrazide and subjected to trypsin proteolysis as
described under "Experimental Procedures." The molecular masses of
the resultant peptides were subsequently characterized by MALDI-MS.
A, unlabeled wild type sulfatase control. B,
covalently labeled wild type sulfatase. C, C82A mutant
refractory to chemical labeling. Unique molecular mass signature in
B is noted by an asterisk.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health (NIH) Grants GM 57073 and CA90940.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Merck/MIT Fellowship.
¶ Recipient of NIH/MIT Toxicology Training Grant 5T32GM08334.
Recipient of NIH Biotechnology Training Grant 5T32GM08334 and
a Whitaker Foundation predoctoral fellowship.
To whom correspondence should be addressed: Division of
Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-258-9494; Fax: 617- 258-9409.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M211425200
2 The Structural Classification of Proteins Database is available on the World Wide Web at scop.mrc-lmb.cam.ac.uk/scop/.
3
Heparin/heparan sulfate monosaccharides are
abbreviated as follows: I, -L-iduronic acid; H,
glucosamine. Subscripts 2S, 3S, and 6S indicate 2-O-,
3-O-, and 6-O-sulfations, respectively. NAc and
NS, N-acetylation and N-sulfation of glucosamine.
4 Protein Data Bank accession numbers are as follows: cerebroside-3-sulfate sulfatase (1E2S); N-acetylgalactosamine-4-sulfatase (1FSU); P. aeruginosa arylsulfatase (1HDH).
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ABBREVIATIONS |
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The abbreviations used are:
HSGAG, heparin/heparan sulfate glycosaminoglycan;
GAG, glycosaminoglycan;
2-O N1-24, recombinant
2-O-sulfatase lacking NH2-terminal signal
sequence composed of first 24 amino acids;
MALDI-MS, matrix-assisted laser desorption ionization spectrometry;
FGly, formylglycine (L-2-amino-3-oxo-propionic acid);
DTNB, 5,5'-dithiobis(2-nitrobenzoic acid);
AT-10, antithrombin-binding
oligosaccharide;
U, uronic acid with a
4,5 double bond.
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