From the
The gene encoding a
All of these glycosidases are important reagents
for oligosaccharide analysis.
Native oligosaccharide substrate was
incubated at different concentrations (0.2-1.0 m
M) with
2.5 milliunits/ml of wild-type or recombinant
The K
The peculiar aglycon specificity exhibited by
A better
understanding of the cellular location of the Streptococcal
Second, only a single gene was obtained from expression cloning. If
more than one
This evidence, together with the
substrate specificity data
(3) , implies with great certainty
that both forms of the
The
contribution to substrate configuration made by amino acid residues
outside the active site in glycohydrolytic enzymes is a largely
unexplored phenomenon. The availability of truncated enzymes that have
altered substrate specificities as described here provides a unique
opportunity to study the mechanism by which glycosidases bind and
hydrolyze complex oligosaccharides.
Boldface residues indicate perfect
matching with the S. pneumoniae
A
desialylated, degalactosylated biantennary glycan was incubated with
recombinant enzymes from clone pGXStr8 and -17, the corresponding
factor Xa-cleaved enzyme, StrH8 and -17 or wild-type
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) L36923.
We thank Professor Raymond Dwek for support. We also
thank Ned Siegel and Christine Smith, Monsanto Co. St. Louis, for
providing amino-terminal amino acid analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- N-acetylglucosaminidase from
Streptococcus pneumoniae has been obtained by screening an
expression library for
- N-acetylglucosaminidase activity.
Clones of different nucleotide sizes each having arylglycoside activity
were obtained, and DNA sequencing revealed a gene of 3933 base pairs
possessing typical bacterial transcription initiation and termination
sequences and terminating in an ochre stop codon. Computer analysis of
the translated protein of 1311 amino acids (144,210 Da) identified a
tandem repeat within which lies a sequence homologous with six other
hexosaminidase gene products from a wide variety of species ranging
from bacteria to humans. Also found were an amino-terminal putative
secretion signal peptide and a carboxyl-terminal cell sorting/anchorage
motif typically found in over 20 other Gram-positive surface proteins.
The expression of an almost complete DNA clone in Escherichia coli produced a functional and authentic
- N-acetylglucosaminidase with aglycon specificity
identical to the wild-type enzyme. However, enzymes produced from
truncated DNA clones show more restricted aglycon specificity and are
unable to hydrolyze terminal
1-2GlcNAc residues from
N-glycans containing a bisecting N-acetylglucosamine.
The availability of these clones allows structural analyses to be made
of catalytic and oligosaccharide recognition protein domains that
enhance functional activity.
- N-Acetylglucosaminidase, found in both the
culture medium and in association with undisrupted cells, is one of six
extracellular Streptococcus pneumoniae glycosidases purified
to date
(1, 2, 3) . This repertoire of enzymes,
in addition to the
- N-acetylglucosaminidase, includes a
-galactosidase, three endoglycosidases, and a neuraminidase that
are thought to aid the organism in the breakdown of oligosaccharides in
its surrounding environment for use as a carbon source. In particular,
the neuraminidase has been implicated in the process of pathogenesis.
By cleaving the terminal sialic acids on cell-surface glycolipids, it
is thought that the neuraminidase can expose the carbohydrate ligand by
which the S. pneumoniae attaches to the host
(4, 5) . S. pneumoniae also synthesizes and
secretes a hyaluronidase that is capable of degrading a component of
the extracellular matrix and may aid the organism to invade underlying
host tissue
(6) . Since GlcNAc
1-linked residues are common
components of several cell-surface molecules in host tissues, it is
possible that
- N-acetylglucosaminidase also has a
pathogenic role.
- N-Acetylglucosaminidase
has been demonstrated to have broad specificity using GlcNAc-Gal,
hydrolyzing both
1-3 and
1-6 linkages that are
commonly found in mucins. By contrast, the hydrolysis of
N-linked sugars at low enzyme concentrations is restricted to
GlcNAc
1-2Man linkages except when the Man-
1-6 arm
is substituted with a GlcNAc at both C-2 and C-6 positions, or the Man
-linked to the chitobiose core is substituted at the C-4 position
by a bisecting GlcNAc
(7) . This limited activity can be a
useful tool in the sequencing of oligosaccharides, giving specific
structural information about the cleaved bond and the surrounding sugar
residues. It also raises the issue of defining the intrinsic properties
of the enzyme that govern this phenomenon. The aim of this study was to
use an expression cloning strategy to obtain the gene for
- N-acetylglucosaminidase and to investigate the
interaction between the enzyme and its N-linked carbohydrate
substrates at the molecular level. By the expression of truncated
portions of the
- N-acetylglucosaminidase gene, we have
been able to examine defined regions of the protein that determine
glycosidase specificity.
Materials
Oligonucleotides were synthesized by
British Biotechnology (Oxford, United Kingdom). Restriction and
DNA-modifying enzymes were purchased from Boehringer Mannheim and New
England Biolabs. Radionucleotides were obtained from Amersham Corp. and
ICN-Flow (High Wycombe, Bucks, UK). Prime-It random primer DNA labeling
kit was purchased from Stratagene.
Bacterial Strains
XL1-Blue Escherichia coli strain ( recA- ( recA1 , lac-, endA1 ,
gyrA96 , thi, hsdR17 , supE44 , relA1 , ( F' proAB, lacI,
lacZ
M15 , Tn10))) was purchased from Stratagene.
NM554 E. coli strain ( recA13 , araD139 ,
(ara-leu)7696 ,
(lac)17A ,
galU, galK, hsdR, rspL(strr) mcrA, mcrB) was obtained from
Stratagene.
Purification of
A
homogeneous preparation of the cellular form of
-N-Acetylglucosaminidase
- N-acetylglucosaminidase was obtained from S.
pneumoniae (ATTC 12213) using previously published methods
(3) . A limited trypsin digest of
- N-acetylglucosaminidase followed by amino-terminal amino
acid sequencing was used to obtain two tryptic peptide sequences, a
major sequence, EGADIPIIGGMVA, and a minor sequence, LQPMAFND. Samples
for SDS-polyacrylamide gel electrophoresis were prepared by methods
previously described
(3) .
Construction and Screening of Genomic Expression
Library
Genomic DNA was prepared from frozen S. pneumoniae cells (1 ml of packed cells) following a method used for tissue
samples
(8) . Extracted genomic DNA was partially digested with
AluI and separated by aga-rose gel electrophoresis. Fragments
ranging between 1 and 7 kb(
)
were gently
extracted from the agarose by adsorption to glass beads using the
Geneclean II Kit (Bio101, Inc., Stratech Scientific, Luton, UK) to
avoid shearing. The ends of the size-selected pieces were then
blunt-ended using Klenow and T4 DNA polymerase
(8) .
EcoRI linkers were ligated to blunt-ended genomic DNA
fragments with T4 DNA ligase, and these inserts were ligated into
EcoRI-digested
-ZapII vector arms (Stratagene) and
packaged with GigapackII packaging extracts (Stratagene) to generate
a
bacteriophage expression library. The number of primary
recombinants was determined by plating the library on the XLI-Blue
E. coli host strain. The plated expression library was
screened for
- N-acetylglucosaminidase activity by
incorporation of 50 µ
M 4-methylumbelliferyl
N-acetylglucosaminide in the top agarose and visualization of
hydrolysis by UV light. Positive clones were plaque-purified by
successive plating, and pure plaques were subcloned into plasmid
Bluescript SK- by the in vivo excision protocol that
accompanies the vector (Stratagene).
Rescreening of Library for Overlapping Clones
The
library was plated and screened by plaque hybridization (Colony/Plaque
Screen nylon discs, DuPont NEN) using DNA probes that were
[-
P]dATP-labeled using the Prime-It random
primer labeling kit (Stratagene) and purified over Select-D
,
G-25 spin columns (5 Prime
3 Prime, Inc., CP Labs,
Bishop's Stortford, Herts, UK). DNA probes were prepared from the
5` end of pBStrH7 by restriction digesting with BglII and
EcoRI and from the 3` end of pBStrH17 by restriction digesting
with NcoI and EcoRI to generate probes of 570 and 730
bp respectively.
Plasmid DNA Purification
Clones were amplified and
purified using the maxi-prep pZ523 spin column plasmid
purification kit (5 Prime
3 Prime, Inc.). DNA was also purified
using Magic Mini Preps (Promega).
Southern Blot Analysis of Clones
Southern transfer
of DNA to nylon membranes (Hybond, Amersham Corp.) was made according
to published methods
(8, 9) . Oligonucleotide probes
designed from the amino acid sequence of the major tryptic peptide from
the purified protein, GAA GG(T/A) GC(T/A) GAT AT(T/C) CC(A/T)
AT(T/C) AT(T/C) GG(T/A) GG(T/A) ATG GT, were labeled with
[-
P]dATP using polynucleotide kinase
(8) . After hybridization at 56 °C overnight, filters were
washed and subjected to radioautography.
DNA Sequencing
Nucleotide sequences were
determined using the dideoxy chain termination method
(10) using the Sequenase DNA sequencing kit (U. S. Biochemical
Corp. Cambridge BioScience, Cambridge, UK). Both strands of DNA were
sequenced in their entirety by a combination of specific
oligonucleotide primers and the transposon-facilitated DNA sequencing
TN1000 nested set kit (Gold BioTechnology, Inc., St. Louis, MO).
Sequence analysis was accomplished using MacVector and Assembly-Lign
software (IBI, Cambridge, UK) and on-line NCBI data bases.
Northern Blot Analysis
Total RNA was prepared from
S. pneumoniae using guanidinium thiocyanate
(11) . RNA
(10 µg) was separated on a denaturing formaldehyde agarose gel with
RNA molecular sizing fragments ranging from 0.24 to 9.5 kb and Northern
blotted. The nylon filter was probed with a 570-bp
P-labeled BglII- EcoRI fragment used to
rescreen the genomic library.
Subcloning into the pGX Expression Vector
The
vector pGEX-3X (Pharmacia, Milton Keynes, UK) was digested with
EcoRI and dephosphorylated. The inserts from clones pBStrH7,
-8, and -17 were prepared by digestion with EcoRI and gel
purification. The insert and vector were ligated overnight and
transformed into NM554 E. coli by electroporation. Colonies
were ampicillin-selected on LB/amp plates, and 20 were randomly chosen
for analysis. Mini-prepped DNA was digested with EcoRI and
separated on an agarose gel for verification of vector and insert
sizes.
pGEX Expression and Affinity Purification
The
expression and purification were carried out according to published
methods
(12) . 50-ml cultures of E. coli transformed
with pGEX clones were induced by the addition of
isopropyl-1-thio--
D-galactopyranoside (1 m
M),
and the cells were sonicated. Affinity beads were mixed with bacterial
cell lysates for 2 h at 4 °C and washed 3 times with buffer, and
the fusion protein was eluted from the affinity beads by the addition
of fresh 5 m
M reduced glutathione. Where appropriate, factor
Xa cleavage was conducted by incubating factor Xa (Denzyme, Aarhus,
Denmark) at 1 mg/ml with beads coupled to fusion protein at room
temperature overnight. Supernatants were assayed for enzyme activity
after centrifuging the beads.
Substrate Specificity Assays
Tritium-labeled
biantennary, triantennary, tetraantennary, bisected-biantennary, and
bisected-hybrid oligosaccharide alditols were obtained from Oxford
GlycoSystems (Abingdon, UK). (GlcNAc1,4GlcNAc)
was
obtained from a partial chitin hydrolysis, and a degalactosylated
biantennary native oligosaccharide was purified from human
asialotransferrin
(13) .
- N-acetylglucosaminidase in 50 m
M citric
acid/sodium phosphate buffer, pH 5.0, containing 1 mg/ml bovine serum
albumin at 37 °C for 1 h. The reactants were desalted, and
hydrolysis was monitored by Dionex HPAEC (Dionex BioLC system) using a
CarboPac PA-1 column eluted at 1 ml/min with 150 m
M NaOH, 30
m
M NaOAc, and the reaction products were detected using
triple-pulsed amperometric detection with the following pulse
potentials and durations: E
= 0.01 V
( t
= 120 ms), E
= 0.6 V ( t
= 120 ms), and
E
= -0.93 V ( t
= 130 ms). The extent of hydrolysis was calculated from
empirically derived response factors for substrate and reaction
products, and the data were plotted using a weighted nonlinear
regression analysis (Multifit 2.0, Day Computing, Cambridge, UK).
Radiolabeled oligosaccharide alditols were separated using an isocratic
eluant of 200 m
M NaOH and the fractions taken for
radioactivity determination by scintillation counting. Bio-Gel P-4
chromatography (Oxford GlycoSystems) was also used to separate the
reaction products after enzyme digestion, and the radioactivity in each
fraction was determined as above.
Expression Cloning of StrH
An expression cloning
strategy was adapted from published procedures
(14) to obtain
the gene encoding S. pneumoniae - N-acetylglucosaminidase. A S. pneumoniae expression library of 5
10
primary
recombinants, generated in the vector
-ZapII (Stratagene), was
amplified in the E. coli host strain XL1-blue, and 300,000
clones were screened for
- N-acetylglucosaminidase
activity using the substrate 4-methylumbelliferyl
N-acetylglucosaminide (4-MU-GlcNAc). 32 positive clones were
identified by fluorescent halos encircling each plaque when visualized
under 366-nm ultraviolet light. Hydrolysis was confirmed to be specific
for N-acetylglucosaminide by the inclusion of a control
substrate 4-methylumbelliferyl xyloside in the screening protocol. No
hydrolysis of this substrate was seen. From the group of positive
clones, 20 were selected at random and subcloned into pBluescript
plasmids via in vivo excision according to the
manufacturer's procedures and analyzed by restriction digestion
to determine the sizes of the genomic DNA inserts. Restriction-digested
DNA of the four largest inserts ranging in size from 1584 to 2504 bp
were Southern blotted and probed with a degenerate
P-labeled oligonucleotide designed from the amino acid
sequence of the major tryptic peptide (Fig. 1). Hybridization of
the radiolabeled probe to each clone selected by the enzyme activity of
its translated product confirmed that they encoded the same protein
that had been biochemical purified from the S. pneumoniae cells (Fig. 1). These clones (Fig. 2 a) were
nucleotide sequenced and were found to compose a 2.7-kb continuous open
reading frame with no start or stop codons, indicating that further
screening of the genomic library was required to locate the missing
gene sequences.
P-Labeled DNA probes from the 5` and 3`
ends of this 2.7-kb partial gene were used to identify two clones that
contained the 5` and 3` ends of the strH gene
(Fig. 2 b). Complete sequence analysis of these three
contiguous clones revealed a single, continuous open reading frame of
3933 bp designated strH (str, streptococcus; H,
hexosaminidase) (Fig. 3). The ATG (methionine) start codon,
designated +1, was identified by the positions of three consensus
sequences: the AGGAGG Shine-Dalgarno ribosome binding sequence located
7 bases upstream from the ATG translation initiation codon, and the two
putative promoter sequences TTGACT resembling a
``[minus]35'' transcription initiation sequence
beginning at -87 and TATAAT ``[minus]10''
transcription initiation sequence beginning at -43. The ochre
stop codon located at base 3934 was followed by a hairpin-loop sequence
resembling a rho independent transcription termination sequence.
Figure 1:
Southern blot of strH clones.
Clones 6, 7, 9, and 17 were restriction-digested with the following
panel of enzymes: EcoRI, EcoRV, HindIII,
ClaI, SacI. DNA (1 µg) was applied to a 0.7% Tris
borate/EDTA buffer agarose gel in the order of enzymes listed above.
Panel a shows the EthBr-stained gel. Lane 1, BRL 1-kb DNA ladder; lanes 2-6, pBStrH6; lanes 7-11,
pBStrH7; lanes 12-16, pBStrH9; and lanes 17-21, pBStrH17. In panel b the
gel was transferred to a nylon membrane by Southern blotting and
hybridized with a P-labeled degenerate oligonucleotide
probe designed from the amino acid sequence of the major tryptic
peptide. Filters were then washed twice in 6
SSC at 37 °C,
once in 4
SSC at 37, 42, 47, and 52 °C, and once in 2
SSC at 52 °C with cautious monitoring before subjecting to
autoradiography.
Figure 2:
Alignment of overlapping clones.
a, the clones, selected by screening for
- N-acetylglucosaminidase activity, were sequenced and
aligned using MacVector and Assembly-lign software. The relative
position of the 90-bp tandem repeating regions encoding 30-amino acid
consensus sequences to other hexosaminidases (Table II) is
shaded. b, the relation is illustrated between the
contiguous 2701-bp partial gene composed of pBStrH6, -7, -8, and -17
obtained by activity screening of the genomic library and the pBStrH5`
and 3` clones identified by screening the library with DNA probes
derived from the ends of the contiguous
sequence.
Figure 3:
strH DNA and amino acid sequence and
flanking regions. EcoRI and AluI sites flank both
ends of the genomic sequence. Both tryptic peptide sequences are marked
in boldface. Transcription initiation and termination
sequences are double underlined. Shine-Dalgarno
sequence is underlined. The start and stop codons are marked
by a dotted underline, and the A of the ATG start
codon is designated +1. The two 30-amino acid tandem
repeating regions homologous with other hexosaminidases (Table II) are
underlined. This sequence has been deposited in the GenBank
data base (accession number L36923).
Protein Sequence of
The strH DNA was computer
translated to give a protein of 1311 amino acids with a predicted
molecular mass of 144,210 Da. Both tryptic peptide sequences obtained
from a purified preparation of -N-Acetylglucosaminidase
- N-acetylglucosaminidase
were found (Fig. 3), and a comparison with all other published
sequences in computer data bases did not reveal significant homology,
confirming that the strH gene was unique. However, two amino
acid sequence motifs common to other Gram-positive surface proteins
were discovered
(15) . The first was a putative secretion signal
peptide at the amino terminus revealed by Kyte-Doolittle hydrophobicity
analysis (Fig. 4 a). This sequence motif is characterized by a
positively charged amino terminus followed by a hydrophobic core and a
string of polar residues
(16) . The proposed cleavage site of
this signal peptide is between the two alanine residues at sites 33 and
34 in strH between the hydrophobic core region and the polar residues
(17) . Within this region is a KQQRFSIRKXXXGAASLIG consensus
sequence that appears to be highly conserved in many of the cell
surface proteins of Streptococcus and Staphylococcus (). The carboxyl terminus contained a sequence
homologous to a membrane spanning/cell wall anchorage sorting signal
found in more than 20 Gram-positive surface proteins to date
(18) (), identified by the consensus amino acid
sequence LPXTGX where LPT and G are the conserved
amino acids. In the translated sequence, we obtained LPETGT, followed
by a hydrophobic stretch of amino acids proposed to be the membrane
spanning region, and a short charged tail (Fig. 4 b). The
LPETGT sequence is directly preceded by two lysine residues that may be
of importance in anchoring the enzyme to the cell wall. It has been
suggested
(18) that the two lysines preceding the
LPXTGX motif of protein A may be covalently attached
via a transpeptide linkage to the peptidoglycan of the cell wall of
Staphylococcus aureus. The authors based this proposal on the
finding that the murein of the peptidoglycan in E. coli is
covalently attached to lipoprotein via lysine and arginine residues
(19) . Another striking feature found within the amino acid
sequence of the
- N-acetylglucosaminidase is a tandem
repeat of approximately 335 amino acids from site 180 to 522 and again
between sites 625 and 956. This repeat is apparent in both nucleotide
sequence and amino acid sequence self-alignments, although greater
homology is conserved when comparing the amino acid sequences than is
found between the nucleotide sequences in these repeat regions. Within
each of these tandem repeat regions lies a sequence spanning 30 amino
acids which, when compared to each other, are 67% identical and 93%
similar when considering conservative amino acid substitutions.
Interestingly, both of these 30 amino acid sequences have considerable
homology to protein sequences found in six other hexosaminidases
isolated from a wide variety of species including bacteria and humans
(Table II).
Figure 4:
a, hydrophilicity plot of the amino
terminus of strH. The strH translation product was
analyzed by a Kyte-Doolittle hydrophilicity plot. The first 50 amino
acids of the amino terminus were analyzed for features of a signal
peptide motif found in other prokaryotes. The consensus motifs are
labeled. b, hydrophilicity plot of the carboxyl terminus of
strH. The last 35 amino acids of the strH carboxyl
terminus were scanned for features of a cytoplasmic membrane/cell wall
anchorage motif common in over 20 other Gram-positive bacterial surface
proteins. The features of the sorting signal are
labeled.
Northern Blot Analysis
A total RNA preparation
from S. pneumoniae was used to identify the transcript of
- N-acetylglucosaminidase. A major band hybridizing at 4.0
kb (results not shown) was consistent with the size of the 4.0-kb DNA
obtained from sequencing.
Kinetic Analysis of Recombinant and Wild-type
Clones pBStrH8 and -17, as
expected, showed arylglycoside hydrolytic activity, but to determine
that these had the same activity as wild type
-N-Acetylglucosaminidase
- N-acetylglucosaminidase using a native biantennary
N-glycan, they were subcloned into the pGEX-3X expression
vector (Pharmacia Biotech Inc.) for the high-level production of
recombinant enzyme. The K
and
V
values were determined for the two recombinant
enzymes from clones pGXStrH17, the longest clone, and pGXStrH8, the
most truncated clone. Wild-type enzyme demonstrated the highest
affinity for the natural oligosaccharide with a
K
of 132 µ
M compared with
the K
values of the smaller length
enzymes from clones pGXStrH17 and pGXStrH8, which were 2-2.5
times greater (Table III). The rates of hydrolysis, however, followed
an opposite trend, where the V
measured for the
smallest enzyme clone pGXStrH8, was 4 times faster than the wild-type
enzyme and 3 times faster than the largest recombinant enzyme from
clone pGXStrH17.
and
V
was also determined for the factor Xa cleaved
enzymes from clones pGXStrH8 and pGXStrH17 (I). The
K
of the fusion protein, from clone
pGXStrH17, and the Xa-cleaved enzyme, StrH17, were very similar. By
contrast, the K
values of the clone
pGXStrH8 and StrH8 recombinant enzymes differed by a factor of two.
However, the standard error of the K
of
the StrH8 enzyme was quite large (I).
Aglycon Specificity of Recombinant
Affinity-purified fusion proteins
were assayed for their ability to hydrolyze a panel of radiolabeled
oligosaccharide alditol substrates to compare aglycon specificity with
the wild-type purified -N-Acetylglucosaminidase
- N-acetylglucosaminidase (Fig. 5).
The reaction products from enzyme digestions were separated by HPAEC.
Because this technique shows a shift in retention times between the
substrate and hydrolysis product indicating cleavage has occurred, the
structures of hydrolysis products were verified using Bio-Gel P-4
analysis, which gave actual sizes of the reaction products in glucose
units. The minimum enzyme concentration (units/ml, using 4-MU-GlcNAc)
for each enzyme that was chosen showed no hydrolysis of
(GlcNAc
1-4GlcNAc)3 and thus retained exclusive
1-2 GlcNAc hydrolysis. Recombinant pGXStrH8 enzyme, from the
most truncated clone, at 0.025 units/ml and wild-type
- N-acetylglucosaminidase at 0.012 units/ml, hydrolyzed
the biantennary alditol to completion (Fig. 5 a). Only the
1-2-linked GlcNAc residues were removed from a triantennary
glycan (Fig. 5 b). However, only partial hydrolysis of a
tetraantennary alditol was observed using the recombinant pGXStrH8
enzyme compared with wild-type enzyme (Fig. 5 c), and no
GlcNAcs were removed from either bisected-biantennary or
bisected-hybrid oligosaccharide substrates (Figs. 5, d and
e). Concentrations of 0.25 units/ml of recombinant pGXStrH8
enzyme were required to hydrolyze completely the susceptible
1-2-linked GlcNAc of tetraantennary structures, but even at
45 units/ml, bisected oligosaccharides remained refractory to
digestion. By contrast, the recombinant enzymes from the longest and
intermediate clones pGXStrH17 and pGXStrH7, respectively, at low
concentrations (0.025 units/ml) showed the same specificity as the
wild-type enzyme, but only partially hydrolyzed those oligosaccharides
with a bisecting GlcNAc. At a concentration of 1.5 units/ml,
recombinant enzyme from pGXStrH17 exhibited the same activity as the
wild-type enzyme when assayed using both bisected-biantennary and
bisected-hybrid substrates. At higher enzyme concentrations (5
units/ml), this recombinant enzyme removed both terminal GlcNAc
residues and the bisecting GlcNAc from bisected-biantennary substrates,
an activity also achievable at high concentrations (0.1 units/ml) of
the wild-type enzyme. The recombinant enzyme from clone pGXStrH7
exhibited partial activity against the bisected substrates at the
highest enzyme concentration tested (17 units/ml). Thus the recombinant
enzyme with the greatest number of amino acids, pGXStrH17
(Fig. 2, 1067 amino acids including the fusion tag, see
I) shared the same aglycon specificity as the wild-type
- N-acetylglucosaminidase, whereas the smallest length
clone, pGXStrH8 (612 amino acids, I) was completely unable
to hydrolyze
1-2-linked GlcNAc residues from bisected
oligosaccharide alditols at extremely high enzyme concentrations (the
maximum tested was 120 units/ml of factor Xa-cleaved enzyme). The
intermediate length clone pGXStrH7 (641 amino acids, see Fig. 2)
was partially active in hydrolyzing bisected substrates. A summary of
these results is shown in Fig. 6.
Figure 5:
Aglycon Specificity of
- N-acetylglucosaminidase from S. pneumoniae.
Both substrate and hydrolysis product elution positions are shown for
each oligosaccharide. N, GlcNAc; M, Man. The
H-labeled oligosaccharide alditol substrates (2.5
10
cpm,
) were incubated with 12.5 milliunits/ml
purified wild-type
- N-acetylglucosaminidase (
) and
25 milliunits/ml recombinant enzyme from clone pGXStrH8 (
) in 50
m
M citric acid/sodium phosphate buffer, pH 5.0, containing 1
mg/ml bovine serum albumin at 37 °C for 18 h. Samples were
desalted, and the hydrolysis products were separated by HPAEC using an
eluant of 200 m
M NaOH. Fractions (1 ml) were collected and
scintillation counted for radioactivity.
Figure 6:
Oligosaccharide structures hydrolyzed by
S. pneumoniae - N-acetylglucosaminidase. A
summary of the data presented on the aglycon specificity of wild-type
and recombinant
- N-acetylglucosaminidase. Solid arrows, complete hydrolysis at appropriate enzyme
concentration; dashed arrows, partial hydrolysis;
crossed arrows, no hydrolysis at all enzyme
concentrations tested. See text and Fig. 5 for full details.
N, GlcNAc; M, Man.
Recombinant enzymes that had
the glutathione S-transferase fusion tag removed by cleavage
with factor Xa were checked for activity against the five substrates
used in Fig. 5to confirm that the fusion tag did not affect the
activity of the recombinant fusion proteins assayed above. At the same
enzyme concentration, the factor Xa-cleaved recombinant enzymes showed
identical aglycon specificities to the uncleaved enzymes (results not
shown), demonstrating that the fusion tag did not change the substrate
specificity of the recombinant enzymes.
- N-acetylglucosaminidase from S. pneumoniae not
only makes it a useful tool for oligosaccharide sequencing, it also
makes it an interesting candidate for the investigation of
substrate-enzyme interactions. At low enzyme concentrations, this
enzyme will only hydrolyze
1-2-linked GlcNAc residues and is
restricted by further N-acetylglucosaminidase substitutions of
the
1-6 mannose arm of N-glycans and by bisecting
GlcNAc residues of the core mannose. Little is known about the factors
that might govern this restricted activity, though it might partly be
explained by the potential steric hindrance created by the flexibility
of 1-6-linked sugars, which have the additional
angle of
rotation. However, because these restricted
1-2-linkages are
cleaved by higher concentrations of
- N-acetylglucosaminidase as well as by other
hexosaminidases, the intrinsic properties of the enzyme itself, which
correlate with this narrow substrate specificity, remain to be clearly
defined. In this paper we have shown that S. pneumoniae
- N-acetylglucosaminidase is encoded by a unique
3933-bp gene, strH, which terminates in an ochre stop codon
and possesses typical Gram-positive bacterial transcription initiation
and termination sequences. The computer-translated amino acid sequence
revealed two consensus motifs at both the amino and carboxyl terminus
that are common to other Gram-positive surface proteins. A tandem
repeat was identified in the strH gene, and within each repeat
lies a stretch of 30 amino acids homologous to sequences in six other
hexosaminidases found in a wide variety of species, suggesting that
perhaps these amino acids may be important for the catalytic function
of the enzyme. The tandem repeat regions of protein may be correlated
with the ability of the clones to hydrolyze substrate. Substrate
specificity experiments revealed that the longest enzyme clone,
pGXStrH17, was able to hydrolyze
1-2-linked GlcNAc residues
of bisected oligosaccharides similar to the wild-type enzyme. The
intermediate sized cloned enzyme from pGXStrH7 demonstrated only a
partial ability to hydrolyze these substrates, whereas the enzyme from
shortest clone, pGXStrH8, was completely deficient in this activity. At
the protein level, the differences between these clones are clearly
defined by the presence or absence of the second tandem repeat region.
Recombinant enzyme from pGXStrH17 contains both tandem repeats;
pGXStrH7 contains all of the first and 95 amino acids (of 330) of the
second tandem repeat region; and pGXStrH8 contains only the first
tandem repeat. Sequence alignment revealed that these tandem repeat
regions each contain a 30-amino acid consensus sequence present in six
other hexosaminidases from divergent species, which leads us to
speculate that this region may be part of an extended site of the
enzyme, which is required for substrate orientation around the active
site. In situations where this portion is partially missing,
as with the shortest clone, efficient association with the some
substrates may be affected and hydrolysis impaired. Further insights
into the function of these tandem repeat regions may be gained by the
separate expression of the second tandem repeat region followed by
experiments addressing the substrate specificity. However, it is
noteworthy that expression cloning failed to identify a clone that only
contained the second repeat region and possibly indicates that the
presence of this domain alone is not sufficient to maintain catalytic
activity. The weaker affinity and increased rate of hydrolysis of
biantennary oligosaccharides, together with changes in aglycon
specificity, correlate with an amino acid-specific extension of the
polypeptide carboxyl terminus. This suggests that there is either a
minimum amino acid residue requirement for active site conformation, or
a requirement for protein domains outside the active site to recognize
and direct substrates into the enzymes catalytic center.
- N-acetylglucosaminidase can be derived from the
sequence information of the gene. The identification of a
characteristic Streptococcal secretion signal at the amino
terminus and a carboxyl-terminal sorting motif found in a variety of
Gram-positive cell-surface proteins predicts a membrane-bound enzyme
with an active site extending into the extracellular space. This
structural model explains the origin of the
- N-acetylglucosaminidase purified from the media. Several
lines of evidence support the hypothesis that this form of the enzyme
originates from the cell surface and is released during
protease-assisted autolysis. First, the cell-associated enzyme was
found to be present in much greater quantities than the enzyme found in
the medium over an 8-h growth period
(3) . Purification of the
wild-type enzyme from Streptococcal cells revealed multiple
amino termini, and the presence of a number of isoforms when examined
by native-polyacrylamide gel electrophoresis
(3) . The major
protein and activity stained band of this preparation analyzed by
SDS-polyacrylamide gel electrophoresis migrates with a molecular mass
of 120 kDa, 24 kDa smaller than the 144-kDa translated gene, providing
further evidence that proteolysis, additional to signal peptide
cleavage, occurs during the release of the cell-associated enzyme.
- N-acetylglucosaminidase was expressed by
this organism, then more than one gene might have been isolated by
enzyme activity. Although 32 clones were selected by their
- N-acetylglucosaminidase activity, further analysis of
five of these selected at random revealed that they were overlapping
fragments of the same gene.
- N-acetylglucosaminidase are
derived from the same protein located at the cell-surface.
Table:
Aligned consensus sequences at the amino and
carboxy termini of proteins from Streptococcus and Staphylococcus
species
-GlcNAc`ase is the abbreviation for
- N-acetylglucosaminidase. Residues in other
Streptococcal and Staphylococcal proteins identical
to
- N-acetylglucosaminidase are in boldface and
conservative amino acid substitutions are underlined.
Table:
Hexosaminidase consensus sequence comparison
between six different species
- N-acetylglucosaminidase. Underlined residues
indicate conservative amino acid substitutions relative to the S.
pneumoniae
- N-acetylglucosaminidase.
Table:
Kinetic analysis of wild-type and
recombinant S. pneumoniae -N-acetylglucosaminidase
- N-acetylglucosaminidase. Kinetic measurements were made
as described in the text. The length of the pGX constructs includes 232
amino acids from the 26-kDa glutathione S-transferase fusion
protein tag.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.